Molecular Approaches to Receptors as Targets for Drug Discovery

J . OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH, 17(5). 671-776 (1997)

MOLECULAR APPROACHES TO RECEPTORS AS TARGETS FOR DRUG DISCOVERY

Jeffrey M. Herz', William J. Thornsen', and George G. Yarbrough3

'Applied Receptor Sciences, 14427 12th Dr., S.E., Mill Creek, WA 98012, USA;

2Lasure and Crawford, 130 Fifth Ave., North, Seattle, WA, 98109, USA;

3Panlabs, Inc., 11804 N. Creek Parkway S., Bothell, WA 9801 1, USA

ABSTRACT

The cloning of a great number of receptors and channels has revealed that many of these targets for drug discovery can be grouped into superfamilies based on sequence and structural similarities. This review presents an overview of how molecular biological approaches have revealed a plethora of receptor subtypes, led to new definitions of subtypes and isoforms, and played a role in the development of highly selective drugs. Moreover, the diversity of subtypes has molded current views of the structure and function of receptor families. Practical difficulties and limitations inherent in the characterization of the ligand binding and signaling properties of expressed recombinant receptors are discussed. The importance of evaluating drug-receptor interactions that differ with temporally transient and distinct receptor conformational states is emphasized. Structural motifs and signal transduction features are presented for the following major receptor superfamilies: ligand-gated ion channel, voltage-dependent ion channel, G-protein coupled, receptor tyrosine-kinase, receptor protein tyrosine-phosphatase, cytokine and nuclear hormone. In addition, a prototypic receptor is analyzed to illustrate functional properties of a given family. The review concludes with a discussion of future directions in receptor research that will impact drug discovery, with a specific focus on orphan receptors as targets for drug discovery. Methods for classifying orphan receptors based upon homologies with members of existing superfamilies are presented together with molecular approaches to the greater challenge of defining their physiological roles. Besides revealing new orphan

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672 HERZ, THOMSEN, AND YARBROUGH

receptors, the human genome sequencing project will i-esult in the identification of an abundance of novel receptors that will be molecular targets for the development of highly selective drugs. These findings will spur the discovery and development of an exciting new generation of receptor-subtype specific drugs with enhanced therapeutic specificity.

INTRODUCTION

The Concept and Definition of Receptors

To paraphrase Voltaire, if receptors did not exist it would be necessary for

pharmacologists to invent them. In fact, this is precisely what J.N. Langley did

around the turn of the century when he suggested the existence of "receptive

substances" through which tissues were able to selectively recognize agonist and

antagonist chemicals (i.e. ligands) and, importantly, also provide a mechanism for

the translation of this recognition event into a physiological response (1). Thus, a

basic and enduring operational definition of a receptor is that it "must recognize a

distinct chemical entity and translate information from that entity into a form that the

cell can read to alter its state accordingly, e.g. b j a change in membrane

permeability, activation of a guanine nucleotide regulatory protein, or an alteration

in the transcription of DNA" (2). Hence, the attribute:; of ligand recognition and

signal transduction are both fundamentally necessary to define receptors. The

transduction process may be mediated through an integral part of the receptor

structure or involve receptor interactions with additional non-receptor proteins.

This definition has been the basis for an erormous body of scientific

investigation into the function and regulation of receptors and mechanisms of drug

action. However, final proof of the existence of receptors did not occur until

relatively recent applications of modern biochemistry and molecular biology to

purify, sequence, clone and express pure receptor proteins. This lack of proof

notwithstanding, the therapeutic basis of many modern. and not so modern drugs,

resides in their specific interactions with receptor molecules located in the plasma

membrane or cytosol of target cells. In fact, these specific interactions have

provided the experimental basis for their discovery and development. No doubt, the

ongoing sequencing of the human genome and ensuing characterization of orphan

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RECEPTORS AS TARGETS FOR DRUG DISCOVERY 673

receptors coupled with the precise molecular analysis of receptor structure and

function will continue to reveal new drug receptor targets that will serve as a

platform for much of the drug discovery enterprise.

CRITERIA FOR RECEPTOR CLASSIFICATION AND NOMENCLATURE

Primacy of Molecular Structure

Historically, pharmacologists classified receptors based on the concept that

a single receptor mediated a pharmacological response to a single endogenous

agonist and that receptor subtypes could be defined primarily by pharmacological

properties. Receptor subclassification systems were dependent upon the

availability of selective and potent natural substances (toxins and alkaloids) or

synthetic ligands which could selectively elicit or inhibit biochemical and

physiological responses from receptors. In some cases, subtypes could be

distinguished by different signal transduction mechanisms associated with each

receptor subtype, As an illustration, two types of acetylcholine receptors,

muscarinic and nicotinic, were originally identified based on agonist activity of

muscarine and nicotine and from the antagonist selectivity of atropine and

tubocurarine. The snake venom toxin, a-bungarotoxin, was found to be useful for

differentiating between certain neuronal and muscle subtypes of nicotinic

acetylcholine receptors. While this approach led to understanding the molecular

and cellular mechanisms of many drugs and to significant therapeutic advances, it

did not provide evidence of the numerous, diverse subtypes that comprise the

muscarinic and nicotinic acetylcholine receptor families.

The enormous molecular diversity and multiplicity of receptor subtypes for

a given neurotransmitter or hormone was not fully appreciated until the application

of modern molecular biology techniques. Within each receptor family, the

multiplicity of receptor subtypes has greatly exceeded the numbers that could be

predicted on the basis of pharmacological data alone. The now frequent cloning

of many receptor genes and the study of the encoded recombinant proteins has

both revolutionized the criteria necessary for classification of receptors which

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674 HERZ, T -IOMSEN, AND YARBROUGH

othetwise could not be distinguished pharmacologically and provided fundamental

insight into mechanisms of drug action at the molecular level.

Current classification criteria for receptors developed by the Committee for

Receptor Nomenclature and Drug Classification of the International Union of

Pharmacology (NC-IUPHAR) is based on a combination of information derived from

molecular structure (structural), pharmacological characteristics of the receptor

(operational or recognitory), and on signal transduction mechanisms used by the

receptor (transductional) (2). Of these three essential criteria, the amino acid

sequence of the receptor provides a definitive identificalion of a distinct protein and

thus serves as an unambiguous primary basis for classification. Identification of the

endogenous ligand or ligands provides a secondary means to group receptors into

families. Integration of pharmacological evidence obtained from radioligand binding

studies of selective agonists, antagonists, and alloster ic (modulatory ligands) and

their characterization in functional assays (second messengers such as

intracellular CAMP levels) enables a comprehensive assessment for classification

based upon all three essential criteria. Receptors can be most reliably

subclassified and defined on the basis of antagon st affinities, whereas data

obtained with agonists is considered much less usefLl since intrinsic activity and

potency have been found to be cell and tissue dependent. Information on receptor-

effector mechanisms that reflect the molecular signaling properties of the receptor

provide another tier for receptor classification, although transduction mechanisms

for novel receptors may be unclear and subject to controversy. Furthermore, the

use of heterologous cellular expression systems for the study of recombinant

receptors has revealed the potential for receptor subtypes to initiate signaling

events not normally associated with their physiological role. These studies have

also demonstrated that the ultimate response to receptor activation is cell-type

specific. Hence, the potential ambiguity that may arise in defining receptor

transductional characteristics has led to the recognitiorl that this characteristic has

more limited value among classification criteria. The question of how to integrate

receptor structural information, pharmacological characieristics, and transductional

properties into a rational scheme of receptor classification and nomenclature is a

subject of continuing discussion amongst pharmacologists (2, 3).

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Defining Receptor Superfamilies, Families and Subtvpes

From the vast number of amino acid sequences deduced from cloned

receptor proteins, strong primary amino acid and structural similarities have been

identified that are common to numerous receptor types that possess distinct

pharmacology. This has enabled receptor classification into superfamilies based

on sequence similarities which encompass many receptor proteins that differ

pharmacologically, but are functionally similar since they share a single molecular

signal transductional mechanism. The “common structure-common function”

concept is not unique to receptors, but arises out of the observation that families of

proteins are probably derived from a single ancestral gene. Receptor subtypes

within a superfamily are generally considered to have arisen through evolutionary

mechanisms of gene duplication and genetic drift leading to divergence from a

common progenitor receptor gene rather than reconstruction of new genes de novo.

Indeed, the genomic organization of highly homologous receptors (and receptor

subunits) often reveals identical structures consisting of the same number of

protein-encoding exons. For many members of the G-protein coupled receptor

superfamily, the entire protein is encoded by only a single exon.

Examples of receptor superfamilies are the G-protein coupled receptors

(GPCRs), and ligand gated-ion channel receptors (LGCRs). Other major

recognized superfamilies include the receptor tyrosine kinase, receptor protein

tyrosine phosphatase, hematopoietic cytokine receptor, and intracellular hormone

receptor superfamilies. Detailed discussion of the structural motifs and signaling

characteristics for each of these superfamilies of receptors follows. Undoubtedly,

as many novel sequences of receptors become known that cannot be

accommodated within the existing superfamily framework, and their signal

transduction properties are defined, new superfamilies of receptors will be

proposed.

Receptors may be further subclassified into receptor families and subfamily

groups on the basis of relative sequence homologies, operational and signal

transduction mechanisms. The nomenclature system frequently employed has

been to name families with reference to their endogenous ligands, e.g. glutamate,

serotonergic (5-HT), dopaminergic, epidermal growth factor. Each family typically

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consists of multiple receptors subtypes that share similarities in their molecular

biological, pharmacological, biochemical and physiological properties. Within a

receptor family, members of one subfamily are rnore closely structurally

homologous to one another (on average between 5C-80%) than to members of

other subfamilies (in the range of 25%). However, the current nomenclature system

carries forward several notable idiosyncrasies with regard to ligand-receptor

systems in which a single endogenous ligand serves as the neurotransmitter for

receptors belonging to two structurally and functionally distinct superfamilies.

Hence, acetylcholine is the transmitter for muscarinic acetylcholine receptors which

are G-protein coupled receptors (GPCRs) with seven transmembrane domains (4)

and the unrelated nicotinic acetylcholine receptors which are multi-subunit ligand-

gated ion channel receptors (LGCRs) (5). Similarly, several neurotransmitters

interact with receptors that belong to these two different superfamilies. Glutamate

receptors are divided into metabotropic (GPCRs) and the ionotropic which include

the NMDA, AMPA and kainate families (LGCRs). GABA receptors are classified as

GAB& (GPCRs) and GABA, (LGCRs), and purinergic P2 receptors have subtypes

in both superfamilies. All serotonergic receptors (5HT,-:5HT7 ) belong to the GPCR

superfamily with the exception of the 5HT, receptor, wiich is a LGCR (6,7).

It is also well recognized that all superfamilies contain examples of receptor

subtypes that interact with more than one species cf endogenous ligand. As

selected examples, in the case of each of the following receptors, interleukin-8 (IL-

8 ) A and B, fibroblast growth factor (FGF), transforming growth factor (TGF)-

P/activin/inhibin receptor systems, receptor binding and tissue-culture experiments

have shown that a structurally related family of ligands bind and activate a single

receptor. The complexities in devising a uniform rational scheme that incorporates

endogenous multiligand-receptor interactions and instances in which a cell surface

receptor can simultaneously function as both a ligand for a receptor on another cell

and as an independent receptor entity, are substantial

The central tenet of the current classification system is to define a unique

receptor subtype for each receptor that is composed of only single polypeptide

encoded by distinct gene. As an example, the dopamire receptor family is known

to contain at least 5 distinct subtypes, dopamine D,-D, receptors, each encoded by

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separate genes. A more complex system arises in the case of multi-subunit

oligomeric receptors, as in the case of the LGCR superfamily. These multi-subunit

receptors are known to exist as pentameric heterooligomers with many subunit

combinations that comprise distinct subtypes. For several families, homologous

genes encoding highly related receptor subunits have been designated by a

combination of greek letters and numbers. Molecular cloning studies of GABA,

receptors have identified five different subunit types named a, p, y, 6, and p which

share significant amino acid sequence identity with each other (30-40%) and with

other members of the ligand-gated ion channel superfamily. Nearly all of these

subunits also have a number of different isoforms (a1 -6, D l -4, yl-3, 6, and pl-2)

and all sequences within each isoform family are highly homologous (70-80%

identity). Individual GABA, receptor subtypes are thus defined by the distinct

combination of subunits and stoichiometry that compose the oligomeric complexes,

such as a2a3P3y2, aIa3P2y2, and aIa3p2y2. However, subunits of the NMDA,

AMPA and kainate receptor-ion channel complexes have been classified using an

alternate system; NMDAI and NMDA2A-D and glul-7 and kal-2. The combinatorial

mechanism of receptor assembly has the potential for producing a large number of

receptor subtypes and is a common feature of the multi-gene families that comprise

the LGCR superfamily.

Molecular Mechanisms Producins a Diversity of ReceDtor lsoforms

Additional variation in receptor sequences occurs through naturally arising

mutations, naturally occurring allelic variants, RNA-splicing and RNA editing. For

example, the human dopamine D, receptor subtype contains a direct repeat of a 16

amino acid segment in the putative third intracellular loop of the receptor. Naturally

occurring aflelic variants of this receptor subtype contain variable numbers of direct

repeat units (e.g. 2, 4, 7 repeats; dopamine D,,,, D,,, D4,,) producing a substantial

number of receptor variants in the human with different primary amino acid

sequence but as yet indistinguishable receptor binding and signal transduction

pharmacology for a wide range of ligands (8 ) . Other subtype variants occur at the

level of RNA splicing giving rise to length variants of the receptor subtype. For

example, differential splicing results in the long and short isoforms of the dopamine

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678 H E R Z , TkIOMSEN, A N D YARBROlJCH

D, receptor, D,, (DZA) and D,, (D,,) ( 8 ) and produces two proteins differing by 29

amino acids. R N A editing is yet another mechanim producing sequence and

functional diversity and has been found to produce seben different edited forms of

the kainate GluR6 subunit, all encoded by only one gene. While many receptors

are subject to posttranslational modifications, such as phosphorylation, which

modulate receptor properties, these differences are ?ot considered to define a

receptor subtype.

Species homologues of the same receptor subtype display a high-degree of

amino acid sequence homology, typically showing 85-95% sequence identity

between species. This is a greater sequence similarity than is usually found

between distinct receptor subtypes in the same species. The introduction of cloning

has enabled identification of species homologues of recsptors that otherwise might

be defined as distinct receptor subtypes due to their species-specific pharmacology,

as was found in the case of the rat S-HT,, and human 5-4T,, (5HT,,) receptors (9).

Since a change in as little as a single amino acid within the binding site domain can

markedly alter ligand affinity, some receptor species homologues exhibit

substantially distinctive pharmacology. A notable example is the comparative

binding of the nonpeptide antagonist ligands, CP96345 and RP67580, to the human

and rat neurokinin NK, receptors. While CP96345 binds with nanomolar affinity to

the human NK, receptor, it exhibits two orders of magnitude lower affinity for the rat

receptor, whereas RP67580 shows selectivity for the rat receptor. The magnitude

of the selectivity difference is noteworthy given that the rat and human receptors

are 95% identical. Construction of single residue substitutions in recombinant NK,

receptors demonstrated that an exchange of two residues was sufficient to reverse

the species selectivity of the two ligands. Due to the number of cases in which

species homlogs differ from human receptor pharmacology, the use of cloned

human receptors for drug discovery screening programs is increasingly favored.

Putative receptors identified by gene cloning with exhibit homology to known

receptors in the existing superfamilies but for which na known ligands have been

identified are referred to as orphan receptors. Initially, these novel receptors may

be classified based upon the level of sequence homolo$ly. When the endogenous

ligand for an orphan receptor remains unknown, the receptor may be provisionally

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named based on the binding of a synthetic ligand to the receptor until the

endogenous ligand is identified. As the functions of an orphan receptor are

uncovered, the novel receptor may become a target for the development of new

therapeutics.

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS BASED UPON RECEPTOR

CONFORMATIONAL STATES

Effect of Receptor States on Liaand Affinitv and Classification

One of the major criteria in receptor subtype classification is the

pharmacological selectivity profile as defined by ligand affinities (equilibrium

dissociation constant, &) and activation constants. Since binding data overcome

many of the limitations of studies of biological responses, ligand affinities have

provided a practical method to define and classify related subtypes. Nevertheless,

it has been difficult to obtain accurate comparisons of agonist affinities of cloned

receptors with those characteristic of the native cell type or tissue. Agonist affinities

determined for cloned receptors expressed in distinct heterologous cell types have

frequently varied substantially and differed from that of the native cell type (10).

These findings underscore the need to verify ligand affinities and function of an

expressed recombinant receptor in several host cell lines and for related receptor

subtypes to be expressed in a common host cell line to allow accurate

pharmacological comparisons (1 1 ). Receptor classification schemes will need to

take into account the host cell environment when evaluating ligand affinity and

functional data.

In contrast to traditional receptor theory, it is now evident from molecular

pharmacological analysis that transitions between receptor states can be induced

by interaction of a receptor with a full spectrum of ligands which have very different

capacities to activate the receptor. Ligand-induced changes in the population

distribution of receptor molecules between these states govern the binding and

functional properties of the receptor. As discussed in detail below, measurements

in vitro and in situ have revealed the importance of temporal responses in

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680 HERZ. THOMSEN. AND YARBROUGH

characterizing multiphasic ligand-receptor interactiorls for the ligand-gated ion

channel and G-protein coupled receptor superfamilies

An agonist, by virtue of the molecular "information" it contains (e.g. size, 3-D

configuration, charge distribution, hydrogen- or ionic bonding residues, chirality),

selectively binds to a complementary three-dimensicnal surface or binding site

domain formed by amino acid residues of a receptor protein and initiates a cellular

response. Drugs classified as 7ull agonists elicit the maximal response while

compounds which only elicit a fractional response are referred to as partial

agonists. Regardless of whether the agonist is an endogenous physiological

ligand, a natural product or a synthetic compound, agonist binding is coupled to

conformational changes in the receptor protein that involve a molecular transition

to a common, active receptor state. As an example, the binding of a variety of

strong, weak and partial agonists to the nicotinic acety choline receptor induces a

transition to a open channel state which is characterized by the same unitary

conductance, although the frequency and duration of the open state differs (12).

Hence, studies of different agonists provide evidence for a single, active

conformation of the receptor. The simplest schemes .:or receptor activation take

into account that occupation of a receptor (R) by an agonist (L) results in a

conformational change in the receptor to create an agonist -activated state (LR*)

which can bring about an effect or response, and can be represented in scheme

1 as:

L + R + LR + LR* - Response K A E

where L is the agonist, R represents the unoccupied (inactive) receptor, and R* is

the active state of the receptor and where KA is the dissociation constant for

agonist-receptor binding and E is the constant describing the equilibrium between

the LR and the LR* states of the receptor.

Contemporary models attempt to incorporate increasingly detailed

knowledge of the conformational states of proteins. A fundamental component of

these molecular activation schemes for receptors is that receptors exist in a

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RECEPTORS AS TARGETS FOR DRUG DISCOVERY 68 1

dynamic equilibrium between two states, an inactive (R) and active conformation

(R*); and that the biological response to a given ligand is governed by its ability to

change the equilibrium (or its relative preference for binding) between the two

states. Continued exposure of LGCRs and GPCRs to agonists promotes a further

multiphasic conversion to desensitized receptor states which develop on times

scales ranging from milliseconds to minutes.

To illustrate one such model, a two-state cyclic scheme for receptor

desensitization (including only one desensitized state) was initially deduced from

extensive studies of the kinetics of nicotinic acetylcholine receptor (AChR) state

transitions induced by ligand binding (13,15). This model was expanded to a

general coupled-equilibria model which describes the molecular species and

influence of agonists and noncompetitive inhibitors (noncompetitive antagonists)

on receptor states (15). In this model, the AChR contains two agonist (L) binding

sites (RR) and a topographically distinct binding site for an allosteric,

noncompetitive inhibitor (A). As indicated in Scheme 2 below, the AChR exists in

at least three distinct states, which are: resting (RR), activated or open channel

(R*R*), and desensitized (R'R'). The sequential binding of two agonists (L) to the

receptor in the resting state leads to the rapid activation of cation permeability via

the LR*R*L species. In the continued presence of agonist, the receptor is

converted to the desensitized state (LR'R'L) in which the channel is refractory to

opening. Noncompetitive inhibitors, such as PCP, affect receptor funciton by shifting

the equilibrium between preexisting receptor conformations, influencing the duration

of the open state and by stabilizing the desensitized state. The allosteric constant, M, defines the ratio of receptor in the desensitized state to the resting state in the

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682 HERZ, THOMSEN. AND YARBROUGH

absence of agonist (RRIRR). Similarly, an analogous allosteric or “cubic” ternary

complex model describing ligand activation of receptors belonging to the GPCR

superfamily has been developed and extensively studied.

For many receptors, the agonist binding site of the receptor protein can

interconvert between high and low affinity binding states or receptor conformations.

Moreover, differences in affinity of the agonists for the same site in different

receptor states may be dramatic. In the case of LGCRs, the desensitized states

display higher agonist binding affinity and no receptor-mediated ion permeability.

It is the preference for ligand binding to the desensitized receptor which is the

driving force underlying conversion to the desensitized state. The binding affinity

(KJ of acetylcholine to the muscle subtype of the niccltinic acetylcholine receptor

measured at equilibrium is about 10 nM (desensitized state), 4-5 orders of

magnitude below the apparent dissociation constant fo- the permeability response

(activatable state) mediated through the same binding site (5).

Multiple agonist-affinity receptor states have als:, been described for many

GPCR receptor families. GPCRs may adopt either a high-affinity or low-affinity

state for agonists (K,, and & ), which may differ by 1030-fold when characterized

in native membranes purified from brain tissue or derived from cell lines. Lou

affinity agonist states for receptors, which cannot be dstermined by direct binding

of a radiolabeled agonist, have been measured by employing radiolabeled

antagonists and examining the pattern of competition over a wide range of agonist

concentrations. For recombinant GPCRs, agonist-induced changes in affinity

depend upon efficient coupling of the expressed recombinant receptor to

endogenous G-proteins present in the host cell line, and coupling is highly

dependent on the combination of receptor and clon,sl cell line. The literature

reports document the lack of agonist-stimulated D3 dopamine receptor-mediated

effects and inability to demonstrate significant affinity shifts for agonist ligands in

the presence of guanine nucleotides in COS or CHO cells (16). However,

receptor coupling to inhibition of adenylyl cyclase was readily obtained for closely

related subtypes in the D2 receptor subfamily, recombinant D2 and D4 receptors,

expressed in a variety of cell lines (CHO-K1 , HEK-293, C6-glioma, Ltk-) (1 7-1 9).

Studies found a guanine nucleotide mediated shift of about 100-200 fold for the

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dopamine dissociation constant (K,, vs. &) and associated changes from a two-site

to one-site competition curve for dopamine (20). While the observed absolute

agonist affinities varied significantly in different studies (1 00-fold), the magnitude

of the guanine nucleotide-induced shift (K+,/ &) was nearly similar for D2 and D4

receptors expressed in several host cell lines.

Antaqonist Modulation of Receptor Conformational States

According to classical models for drug-receptor interaction, full competitive

antagonists and agonists share the ability to bind to a common site on the receptor

molecule, but differ in that antagonists are devoid of intrinsic activity. While a

competitive antagonist occupies the binding site of the receptor, it does not promote

conversion of the receptor to the active conformation. Indeed, at the biochemical

level, nicotinic acetylcholine receptor agonists and antagonists which differ in size

and structure (acetylcholine, tubocurarine, and snake venom cx-toxins) have been

mapped to the same molecular site on the receptor a-subunit and their binding is

mutually exclusive. However, recent data suggest that no overlap in the binding

site for competitive ligands is required if they bind in a mutually exclusive manner

to different binding sites that are present only in different receptor conformations.

Competitive antagonists may be subclassified into at least two categories; 1 )

neutral antagonists which do not exhibit a preference for an inactive or active

receptor conformation and have no effect on basal receptor activity, and 2) negative

antagonists, also termed inverse agonists, which exhibit the defining property of

inhibiting agonist-independent receptor activity and possess negative intrinsic

activity. Furthermore, negative antagonists stabilize a different receptor

conformation (state) than neutral antagonists.

For both LGCRs and GPCRs, whose activity is governed by distributions

between inactive and active states, negative antagonists promote conversion of the

receptor to an inactive conformation and stabilize a conformation of the receptor

that is distinct from the unliganded receptor (resting state). For G-protein coupled

receptors, studies of receptors expressed at high levels using the baculovirus

expression system in Sf9 cells or of receptors that have been rendered

constitutively active by site-directed mutagenesis have provided an experimental

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system in which agonist-independent, spontaneous atlenylyl cyclase activity can

be measured. This phenomenon has been best characterized for the P,-adrenergic

receptor, but has also been observed for the bradykinin, dopamine D,, and 5-HT2,

receptors, suggesting it is likely to be a general feature 0" the superfamily. A group

of well-characterized P,-adrenergic receptor antagonists were found to inhibit, to

varying degrees, receptor dependent spontaneous activity (21 ). Negative

antagonists have been shown to promote the dissociation- of spontaneously

occurring receptor-G-protein interactions using constitutively active mutant

receptors (21). As an example, the ligand ICI 118551 has been shown to inhibit the

basal signaling activity of the P2-adrenergic receptor, thus acting as a negative

antagonist (22). Inverse agonist drugs may also span a range of activities, and thus

current ligand and drug classification schemes recognize full agonists, partial

agonists, neutral antagonists, partial and full inverse agonists.

For the ligand-gated ion channels, pharmacological studies of the interaction

of the nicotinic acetylcholine receptor with a series of: N-substituted analogs of

decamethonium identified several compounds as antagonists, but unique in their

capacity to antagonize receptors which had been previously exposed to agonists

(21 ). Through an extensive series of equilibrium and kinetic binding experiments,

it was shown that these antagonists preferentially bound to the desensitized

receptor state and had the ability to promote the conversion ot the nicotinic

acetylcholine receptor to this inactive state. In contrast, c:lassical antagonists, such

as tubocurarine, demonstrated the same affinity for both resting and desensitized

receptor states. Antagonists which demonstrated enhanced affinity for the

desensitized receptor state were termed metaphilic antagonists.

Noncompetitive antagonists interact with a spatially distinct site (NCI site)

which may be allosterically regulated by the agonist binding site(s) and block

receptor function through an allosteric mechanism (Scheme I). In the absence of

agonist, the allosteric constant, M, is increased by binding of these inhibitors, and

in the presence of agonist, the rate of agonist-elicited conversion to the

desensitized state is accelerated. For the LGCRs, the allosterically coupled NCI

site is fundamentally different from the agonist site in its pharmacology. A study of

the interaction of a phenylphenanthridium ligand, ethidiurr , with the high affinity NCI

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site of the nicotinic acetylcholine receptor determined that it bound with extremely

high selectivity to the desensitized state relative to a state stabilized by interaction

with snake a-toxin at equilibrium, showing a ratio greater than 2800 (24).

Conversely, ethidium binding at the NCI site and its occupation converted the

receptor to a state of higher agonist affinity. Similarly, association rates for the

binding of radiolabeled phencyclidine (PCP) to the NCI site increased by a factor

of 1000-10,000 for the transient open-channel state relative to the toxin stabilized

receptor state (25). Potential explanations for these findings are that the binding

site location within the transmembrane ion channel is sterically inaccessible in a

toxin-stabilized receptor conformation and that binding occurs only to selected

conformational states, such as the open channel state (25, 26). Numerous ligands

act through allosteric mechanisms to regulate receptors in the LGCR superfamily.

As in the case of the AChR, drugs such as MK-801 and PCP bind within the ion

channel domain of the NMDA receptor, blocking agonist-dependent activation.

GABA, receptor function is also regulated by the binding of benzodiazepines,

barbiturates, and steroids at distinct allosteric sites on the receptor.

The concept of efficacy provides an explanation for observations that different

agonists may bind to the same receptor but produce maximal responses of differing

magnitudes. It is defined as the relative capacity of a agonist or drug to elicit a

response by each drug-receptor complex once it has formed (27). In terms of

allosteric receptor models, affinity and efficacy are separable attributes that reflect

ligand preferences for distinct receptor conformations. Thus, agonists may possess

high affinity and low efficacy at a receptor and vice-versa. At a molecular level,

differences in agonist efficacy at similar receptor occupancy levels may reflect the

ability of a agonist to promote the conversion of the receptor into an active

conformation (27, 28).

Methods to Study Drua-Receptor Interactions with Transient Conformational States

Drug-receptor interactions may quantitatively differ among rapidly converting

multiple receptor states that are induced by agonist binding. While most studies

commonly focus on analysis of ligand-receptor interactions and cellular responses

under presumed steady-state conditions, experimental techniques available for the

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quantitative analysis of drug-receptor interactions with transient receptor

conformational states are more limited. Since the actions of many receptors

regulate rapid (within seconds) and transient changes in intracellular calcium and

protein kinase activity, the magnitude and the duration of the response to either

transient or sustained agonist stimuiation will determine the integrated cellular

response. Thus, the effects of drugs on the temporal integration of individual

receptor responses is critical in determining the response of a cell to extracellular

stimuli. Below, we briefly highlight several areas where molecular pharmacological

techniques have been applied to study rapid, real-time analysis of ligand-receptor

conformational states and cellular signaling.

A variety of rapid mixing techniques have prodded sensitive and rapid

measurements capable of defining receptor conformational transitions induced by

agonist or drug binding to membrane bound receptors. 1 he earliest ligand-receptor

kinetic studies measured radioligand binding of agonists and functional responses

using rapid filtration, but were technically limited in kinetic resolution. Direct

spectroscopic measurements extending into the milliseccnd time domain have been

made of conformational transitions for the nicotinic ace:ylcholine receptor and the

fMLP receptor because the affinity of fluorescent agonist ligands is greatly altered

by interaction with these LGCR and GPCRs (25, 28). Real time analysis of

fluorescent formyl peptide ligand-binding to intact human neutrophils by both

spectrofluorometric and flow cytometric methods ha: led to a model of signal

transduction dynamics and ligand-receptor-G-protein ternary complex interactions

(28, 29). Extrinsic fluorescence labeling of receptors Iby covalent modification of

the receptors with reporter groups has also provided evidence for conformational

changes in LGCRs and GPCRs. Spectroscopic signals originating from

incorporation of an environmentally sensitive fluorescent probe into the purified,

human p2- adrenergic receptor provided kinetic information indicative of both

agonist and negative antagonist mediated receptor conformational changes (30).

Changes in intrinsic fluorescence of receptor tryptophan residues has also been

used to measure receptor activation, but this approach has the disadvantage of

only being applicable to purified proteins since all proteins have a certain amount

of intrinsic fluorescence.

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Electrophysiological analysis of drug interactions with single receptor ligand-

gated ion channels and voltage-dependent channels has provided another

invaluable approach to analyze the activated open-channel states and closed

states of LGCRs and voltage-activated ion channels with time resolution unmatched

by other techniques. Using the methods of patch clamping, these high-resistance

seals (gigaohm) attached to membrane patches or intact cells have recorded

changes in conductance arising from the opening of individual transmembrane

channels. Studies of this nature have been crucial in defining functional

characteristics of many subtypes of receptor-ion channels and allowed the

investigation of the mechanism of interactions of drugs with open channel states of

these receptors, which are otherwise not amenable to study. In a classic study, the

blocking interaction of the local anesthetic QX-222 with the nicotinic acetylcholine

receptor was studied (59 ). Rapid and repeating flickering changes in conductance

of one channel were observed as single drug molecules stochastically entered,

blocked and left the receptor ion channel. Kinetic constants derived from such

measurements can define state-dependent blockade of receptor-channels.

Flow cytometry has been utilized as a powerful approach to collect

multiparameter kinetic data to evaluate multiple activation parameters

simultaneously in individual cells and to correlate these parameters with the ligand

occupancy of each cell. Cellular functional parameters that can be measured

employing this technique include intracellular calcium, magnesium, pH, and

membrane potential. Choices of functional parameters depend only upon the

availability and utilization of a wide range of specific fluorescent probes which can

act as reporters of the desired cellular parameter. A now common procedure is to

employ a fluorescent probe for calcium to measure continuous changes in

intracellular calcium in populations of cells in suspension or in cellular monolayers

of immobilized cells using conventional spectrofluorometers. Utilized in

combination with fluorescence digital imaging microscopy, spatial gradients

reflecting changes in ion concentrations within individual living cells in the

millisecond time domain have been visualized. New instrumentation has enabled

adaption of this technique to a 96-well plate format which is suitable for high-

throughput screening in drug discovery programs. Progress in defining the spatial

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688 HERZ. TlIOMSEN, A N D YARBROUGH

and temporal cellular location of receptors in living cell!; during signal transduction

has advanced through the use of confocal fluorescence microscopy coupled with

the development of receptor-specific fluorescent probes. Use of receptor

fluorescent probes has allowed measurements of rapid changes in receptor cellular

distribution in the membrane and the subsequent internalization of ligand and

receptor. While receptor studies employing fluorescent ligands are still few, this

nascent area will likely expand in the near future as additional specific fluorescent

ligands and recombinant receptors become more available.

LIGAND-GATED ION CHANNEL SUPERFAMILY

Structural and Functional Features

The iigand-gated ion channel superfamily comprises a group of receptors with

shared structural features and similar functions. The prototypic member of the

family is the nicotinic acetylcholine receptor, and other members include the

serotonin 5-HT3 (31), GAB% (32), glycine (33), purinergic P,, (34), and the

ionotropic glutamate receptors (35). Subfamilies of the gutamate excitatory amino

acid receptors include NMDA, kainate and AMPA. Interaction of the

neurotransmitter with the receptor-channel directly mediates rapid changes in the

ionic permeability of the intrinsic ion channel component of the receptor, allowing

the selective movement of ions down their electrochemical gradients. The transient

open periods of an individual receptor-channel may only be several milliseconds

in duration. An essential feature of the receptor-channel is the gate, which controls

the flow of ions through the channel, and is located ,at some distance from the

neurotransmitter binding sites. The molecular mechanism linking binding of

agonists to conformational changes in the gate, which is presumably part of the ion

channel domain, remains to be elucidated.

Recent advances in molecular genetic approaches have facilitated the

elucidation of the functional architecture of this superfamily (31 -39). Furthermore,

these techniques have shown an unexpected level of complexity among these

receptors since each multi-gene family contains multiple highly homologous

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subunits. The ligand-gated ion channels are composed of multiple subunits that

are distinct, yet related integral membrane proteins. Within the superfamily, the

homology between subunits of different family members is typically 20-40%,

whereas the homology between the subunits of a given family that form individual

channels is much greater.

The receptors are pentameric complexes in which the subunits are arranged

in a ring and the central axis forms the ion channel. The functional oligomeric

receptors may either be homooligomers or heteroligomers (34, 35, 39). Based

upon amino acid sequence similarities, the individual polypeptides ?hat form a

receptor complex share a common structural organization (Fig. 1). Among the

subunits of the superfamily that function in ligand binding, a combination of affinity

labeling and sitedirected mutagenesis studies has identified a portion of the large

extracellular amino-terminal domain as the neurotransmitter binding pocket. Within

the superfamily, the glutamate receptor subunits have a much larger extracellular

N-terminal domain, making them about twice the size of the acetylcholine receptor

family (Fig. 1). Hydropathy plots and proteolysis experiments suggest that all

subunit polypeptides span the membrane four times (sequential transmembrane

segments termed M1 -M4), although alternative folding patterns have been

proposed.

A combination of sitedirected mutagenesis and affinity labeling experiments

have also provided significant knowledge of the structural principles and the

molecular basis of the ion permeation mechanism for ligand-gated ion channels.

The M2 transmembrane segment has been identified as the transmembrane portion

of each subunit that lines the central channel of the receptor (Fig. 2). Functional

analysis of chimeric nicotinic acetylcholine receptors constructed from Torpedo and

bovine &subunits demonstrated that the residues in M2 and residues lying between

M2 and M3 influence ion permeation (40). Charged residues at either end of the

M2 sequence that are positioned at both entrances to the pore play an important

role in determining cationic vs. anionic selectivity (Fig.2). In an elegant experiment

delineating the role of these charged residues, Changeux and coworkers

demonstrated that conversion of three amino acid residues of the M2 region of the

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690 HERZ, TIJOMSEN, A N D YARBROUGH

A c h R a , N P -C I

N A c h R c y : I

5 H i 3 R

G I u R - A ---

K A - 1

N M D A R - 1 1-

M1 M 2 M3 M4

-4- - -

/ \

Figure 1. Subunit organization of the receptor families comprising the ligand-gated

channel receptor superfamily. The polypeptide chains of a-subunits in the

acetylcholine (AChR muscle and neuronal subtypes), 5HT,, GABA,,, and glycine

families and in the glutamate receptor family (AMPA, GluR-A; kainate, KA-1; and

NMDA, NMDAR-1 receptor subfamilies) are shown. The length of each subunit is

proportional to the number of amino acids in each polypeptide. The position of the

four putative membrane spanning segments, M1 -M4, are represented by solid

rectangles. Glutamate receptor subunits have substantially longer extracellular N-

terminal domains (450-500 amino acids instead of 200), making them an average

of about twice the size of the acetylcholine receptor a-subunit.

neuronal nicotinic a, receptor to residues of the glycine receptor converted the

calcium permeable nicotinic receptor to an anion selective receptor (41). In

addition, the charged residues flanking M2 control the rate of ion transport and

influence ion selectivity among similarly charged ions 1:42).

Continued exposure of ligand-gated ion channels to agonist rapidly leads to

loss of responsiveness (50 - 100 msec), termed homologous desensitization

(reviewed in 43). This process is an intrinsic feature of receptors in the superfamily

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Figure 2. Comparison of amino acid sequences for the M2 transmembrane

segment of receptor families in the ligand-gated channel receptor superfamily.

Subunits comprising the families have been grouped into cation-selective (1 -5, 9-

14) and anion selective (6-8) ion channels to illustrate the highly conserved

sequences which are important for ion permeation and ionic selectivity. Residues

that are in bold at either end of the transmembrane segment are determinants of

cationic or anionic selectivity for the receptor heteroligomers and likely form the

binding site for ions in the permeation pathway. The stippled column in the

glutamate receptor family indicates the positions of residues that determine

monovalent vs. divalent cation selectivity. Residues identified by the boxed column

are a highly conserved leucine in the transmembrane segment of the acetylcholine

receptor family and which is present as phenylalanine in the glutamate receptor

family. Mutations in the leucine ring dramatically alter desensitization of the

acetylcholine receptor and allow ion conductance in the desensitized state.

Residues within M2 also contribute to the binding sites for many noncompetitive

inhibitors of the nicotinic acetylcholine receptor, such as QX-222, chlorpromazine,

and meproadifen.

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and does not 'require phosphorylation to mediate its effect, although

phosphorylation states of the receptor may influence the kinetics of the process.

Conversion to the inactive state reflects an isomerization of the liganded receptor

to a desensitized conformation (43). The large intracellular loop between the third

and fourth transmembrane segments often possess a variety of sites that can be

phosphorylated by CAMP-dependent kinase, protein kinase C (PKC) or tyrosine

kinases (44-46). Phosphorylation of these sites has been shown to modulate the

activity of several members of the superfamily and this may represent a mechanism

for integrating the function of these receptor-channels in cellular signaling and/or

provide a mechanism to enable localization of receptors at synaptic junctions.

Nicotinic Acetylcholine Receptor Family

Nicotinic acetylcholine receptors (AChR) exist as distinct forms at the

neuromuscular junction and within the central nervoLs system. These receptors

form ligand-gated ion channels that mediate synaptic transmission between nerve

and muscle and between neurons upon interaction with the neurotransmitter

acetylcholine. Upon binding the ligand, the AChR is transiently converted to an

open channel state allowing cation influx and subsequent depolarization of the

postsynaptic cell.

The structure of the muscle AChR is similar in diverse organisms, including the

marine electric ray, Torpedo, and mammals. Since the Torpedo receptor occurs at

high density in the electric organ, this receptor has been most intensively studied

and has served as a model for understanding the strmture and function of other

members of the ligand-gated ion channel superfamily. The receptor complex is

composed of five subunits which are present with a stoichiometry of 2a, 18, l y or

c, and 18 (reviewed in Ref. 5). All four types of subunits span the membrane and

possess considerable amino acid sequence homology as deduced from cloned

cDNA sequences (47-50). The receptor has been purified by affinity

chromatography and functionally reconstituted in lipid vesicles. In addition,

expression of cDNA clones for the four subunits in Xenopus oocytes or in fibroblast

cell lines results in the formation of active receptor-channels (50). Thus, these

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complementary techniques established that the ligand binding sites and channel

are part of the same macromolecular complex.

The general quaternary structure of the muscle AChR has been determined

by electron microscopy of tubular crystals of AChR grown from native Torpedo

membranes (51-53). These studies revealed that the five subunits are arranged

with pentagonal symmetry around a central opening that forms the permeation

pathway for cations (Figure 3). The cobra a-toxins associate with the synaptic

surfaces of the two a-subunits (54, 55). The agonist binding sites have been

localized to the a-subunits by affinity labeling (56, 57) and insight into the structure

of these sites has come from affinity labeling and mutagenesis experiments. These

studies have shown that residues Cys-I92 and Cys-I93 comprise part of the

binding site. As shown in Fig. 3, the location of the two acetylcholine binding sites

has been mapped within the three-dimensional structure of the AChR, and are

located 20-30 A from the membrane surface as shown by fluorescence energy

transfer experiments using agonist and noncompetitive inhibitor ligands (58 - 60).

Ligands binding to the acetylcholine recognition sites include: agonists such

as acetylcholine and its nonhydrolyzable analog, carbamylcholine, as well as the

alkaloid, epibatidine and synthetic analogs; antagonists such as d-tubocurarine,

and the snake a-neurotoxins. Occupation of both agonist sites, one per a subunit,

is necessary for channel opening whereas the competitive binding of only one

antagonist or snake venom a-toxins results in channel blockade. Binding of agonist

ligands and the AChR response to agonists exhibit positive cooperativity (43) while

negative cooperativity has been observed for the binding of certain antagonists.

Since the two a- subunits are non-adjacent, conformational changes upon ligand

binding at one a-subunit must be translated through the intervening subunit(s). A

combination of electrophysiological studies (61 ) and rapid mixing techniques (62 -

64) have been used to measure the kinetics and define conformational transitions

induced by agonist binding to the AChR. These studies have shown that the

receptor can exist in at least three interconvertible receptor states: resting (or

activatable), open and desensitized (Scheme 2). These distinct receptor states

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694 HERZ, THOMSEN. A N D YARBROUGH

Snake a-Toxin

Synaptic Cleft

Neurotransmitter Binding Sites

Noncompetitive Cytoplasm Inhibitor Site

Figure 3. Structure and ligand binding sites of the muscle subtype of the nicotinic

acetylcholine receptor. The structure is derived from electron microscopy of two-

dimensional crystals of receptors in Torpedo membranes. The five subunits

together form a cylindrical shape approximately 120A long with a 70A diameter

which spans the membrane. The receptor on the right shows a section along the

axis of the receptor and shows the cation conducting pathway in profile. A 60A

diameter central hydrophilic tube is present on the synaptic side which is the

entrance to the transmembrane ion channel and narrows at the level of the

membrane. Current resolution of the receptor cannot resolve dimensions of the ion

channel within the membrane bilayer which permeability studies have defined as

an aqueous pore of about 7 A diameter. Locations of the ligand binding sites have

been mapped within the receptor structure by fluorescence energy transfer

techniques (58, 60). The two agonist binding sites, one on each a-subunit, are 20-

30A from the membrane surface and the single allosterically-coupled

noncompetitive inhibitor site is located in the channel. Positions of two snake

venom a-toxin molecules (peptides of 7,500 kD, peptide backbone shown) bound

to the synaptic surfaces of the a-subunits are shown for the receptor on the left.

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RECEPTORS AS TARGFFS FOR DRUG DISCOVERY 695

differ substantially in their agonist binding properties as well in their interaction with

drugs which bind within the ion channel.

Another important class of ligands which can regulate AChR function are

noncompetitive antagonists or inhibitors (NCls) which are potent blockers of the

AChR both in vivo and in vitro. Examples of ligands which function is this manner

include the tertiary and quaternary amine local anesthetics (QX-222, QX-314), the

hallucinogen phencyclidine, histrionicotoxin, and phenothiazines, and other organic

cations such as ethidium (65). Radioligand binding and fluorescence studies have

established that these drugs interact with a single high affinity NCI site per receptor

which is allosterically coupled to agonist binding (58, 65). Many of the local

anesthetic drugs also interact with additional lower affinity sites that are not

allosterically coupled to agonist binding. Electrophysiological, mutagenesis and

biochemical data support the hypothesis that NCI ligands bind to a site within the

ion channel and that amino acid residues from all four subunits contribute to the site

(66 - 69). Indeed, channel permeant cations compete selectively with bound NCI

ligands with the same dissociation constants determined from electrophysiological

ion permeation data (70). The ion channel domain of other ligand-gated ion

channels also appears to be an important site for noncompetitive drug interaction.

In neural tissue, recognition of the molecular diversity of neuronal AChRs

emerged from the application of recombinant DNA techniques which identified the

existence of a multi-gene family containing 12 members (for review, see 71).

Molecular and biochemical approaches have allowed neuronal AChR subunits to

be classified as either subunits involved in binding of acetylcholine (a-subunits) or

structural subunits (termed either non-a or D). The acetylcholine binding subunits

have been defined on the basis of adjacent cysteine residues (Cys 192 and 193)

in the primary sequences that are known to be part of the agonist binding site and

by reactivity to acetylcholine affinity alkylating agents. There are nine neuronal a-

subunits (a,-a,) which can be divided into two classes on the basis of their ability

to bind a-bungarotoxin and four neuronal p subunits (lj2- ps). Subunits a, and a8

have been shown to bind a-bungarotoxin on the basis of expression studies in

oocytes and purification of native receptors from chick brain and the human

neuroblastoma SH-SY5Y cell line (72).

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To date, seven different functional neuronal AChRs have been constructed in

heterologous expression studies. Pairwise coexpressiorl of either a*, ag, or a, with

p2 or p4 subunits has produced active acetylcholine-gated ion channels. These

expressed receptor subtypes differ in their pharmacological profiles with respect to

both agonist and antagonist sensitivities, as well as blockade by K-bungarotoxin

and are thereby pharmacologically distinguishable. F urthermore, single-channel

conductances and gating properties were found to be dependent on subunit

composition (73). In contrast to other nAChR subunits, a, has been shown to form

homoligomer channels when expressed in Xenopus ooctyes. a7 homooligomer

receptors are characterized by high Ca2+ conductance and rapid desensitization.

Clearly, there is a multitude of possible neuronal AChR functional variations based

upon combinations of five subunits (74) and identification of subtypes found in vivo

is an ongoing process.

Purification of neuronal nAChRs, electrophysiolcgical recordings from brain

tissues, and pharmacological characteristics have demonstrated a diversity of

neuronal AChRs within the CNS (71). Some of the pharmacological profiles for the

expressed receptor subunit combinations are correlated with properties of

endogenously expressed receptors found in ganglia, the CNS and in cell lines. On

the basis of immunopurification, it is thought that AChRs comprised of two a4 and

three p2 (2a43p2) subunits represent the majority of neLronal CNS receptors in the

mammalian brain. In addition, the majority of the a-bungarotoxin binding subtypes

contain a7 and a, subunits (72).

The existence of distinct receptor subtypes corriposed of multiple subunits

allows a functional diversity of subtypes based upon selective subunit

combinations. The challenge for the future will be to correlate the diversity of

subtypes with specific functional roles within the CNS and to identify novel drugs

which can distinguish among the numerous subtypes. The recent description of

novel and highly potent nicotinic ligands (picomolar affinity) with improved subtype

selectivity suggests that agonists for the treatment of A zheimer’s Disease may be

developed.

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Extracellular II 111 IV

nn

lntracellular I Figure 4. Schematic diagram of a Voltage-Sensitive Ion Channel Primary Structure.

The sodium channel and calcium channel a-subunit is a large polypeptide which

contains four internally homologous domains (brackets labeled as I, II, Ill and IV).

Each domain has multiple stretches of predominantly hydrophobic residues that are

assumed to be membrane spanning (Sl-S6). One sequence, S4, has many

positively charged residues at every third position, and this sequence functions as

the voltage-sensor for channel.

VOLTAGE-GATED ION CHANNEL SUPERFAMILY


Voltage-gated ion channels exist as a superfamily of structurally related

proteins since they share a high level of primary sequence homology and similar

predicted structure based on hydropathy profiles (75-77). Within the superfamily,

the larger members of the voltage-gated Na'and Ca2' channels families are more

closely related in structure than the smaller K' family. Subtypes of the Na' and

Ca2' channels are all large polypeptides, termed the a or a, polypeptide (approx.

240-260 kDa), containing four homologous repeating domains (I-IV in Figure 4).

Each domain is characterized by six hydrophobic, putative a-helical transmembrane

spans (Sl-S6) including one (S4) which also contains a large number of positively

charged basic residues. The S4 segment, which is particularly conserved, is

thought to be the channel's voltage sensor (78, 79). Designation of S4 as the

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698 HERZ. THOMSEN. A N D YARBROUGH

voltage-sensor is supported by site-directed mutagenesis experiments which show

that neutralization of charged residues in the S4 segment reduces channel gating

charge (86). The a polypeptide is believed to fold its four domains into a

transmembrane array that surrounds a central water-fiIl*?d pore. Expression of only

the large a-subunit of either the sodium or calcium charinel is sufficient to form fully

functional channels,- although association of other smaller subunits with the a-

subunit modulate channel function (81 ).

In contrast, K'channels are thought to have a sinilar molecular architecture

but to consist of oligomeric tetramers with each smaller a-subunit functioning as a

structural homologue to one of the four domains within the large a-subunit of the

Na' and Ca2' channels (82). Each subunit contains 6 iydrophobic segments ( S I -

S6). Functional voltage-gated K' channels have been demonstrated to be

tetrameric structures which may contain a combination of homologous or heterologous a-subunits (83, 84). The diversity of possible subunit compositions

for a given K' channel is thought to account for the diversity of channels that are

prevalent in different tissues and further suggests a high degree of developmental

regulation (85). Mutagenesis experiments have been used to identify multiple ion

binding sites and structural elements involved in ion f1o.N and channel gating for all

members. A region involved in channel inactivation has been mapped to the

intracellular surface of the a-subunits.

In the past few years, a startling diversity of subtypes within the sodium,

calcium and potassium families of voltage-gated ion chz nnels has been discovered

by a combination of molecular biological methods, electrophysiological and

biophysical studies. Differences in the primary sequences which arise from

expression of different genes or by alternative splicinc_ have been shown to result

in important functional and pharmacological differences. As an example,

mammalian Na' channels are encoded by a multi-gene family with at least six

structurally distinct members which share a high level 2f homology (86). Isolation

of separate cDNA clones for the a-subunit has led to identification of at least three

distinct channel isoforms in rat brain, two isoforms iri rat skeletal muscle and a

unique isoform derived from cardiac tissue (87). Sodium channel isoforms in

mammalian cardiac membranes exhibit pharmacologicai properties which are quite

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distinct from channels in nerve and muscle. While channels in nerve and muscle

are blocked by nanomolar concentrations of the neurotoxins, tetrodotoxin and

saxitoxin, about 1-5 VM tetrodotoxin is required for half-maximal block of

mammalian cardiac muscle sodium currents K' selective charinels also comprise

a family of proteins that are extraordinarily diverse with regard to gating mechanism,

biophysical characteristics, and regulation (86). Important pharmacological

differences among subtypes have also been described for members of the calcium

channel family (see below).

At a molecular level, the function of voltage-gated channels is governed by

interconvertible states of the channel which may be simply characterized as:

closed, open and inactivated (review, see 88). An inactivated channel state is a

closed state which cannot react to form open channels upon depolarization of the

membrane. Channels at rest are distributed between resting and inactivated

conformations, depending on the resting potential. Changes in electrical potential

exert a force on the charges in the S4 segment and cause a conformational change

to an activated, ion conducting state (76). Formation of the inactivated state from

the open channel state is a time dependent process which varies for individual

channel subtypes. More complex kinetic models containing multiple channel states

have been formulated to describe drug interactions with channels.

In its original form, the "modulated receptor hypothesis" postulated a single

site located within the pore of the sodium channel to which local anesthetics bound

and whose affinity changed (i.e. was modulated) as the channel cycled through

open, closed and inactivated states (89). Many drugs which bind to voltage-gated

channels are now recognized to preferentially interact with specific channel

conformational states (80, 89) and this has enabled a molecular understanding of

their macroscopic properties. Evidence supports a mechanism in which some local

anesthetic and antiarrhythmic drugs selectively interact with open states of Na' channels to inhibit ion permeation. Na' channel blockers include the clinically

useful local anesthetics (e.g. lidocaine) and related Class I antiarrhythmic agents

which are widely used in the therapy of ventricular tachy-arrhythmias.

A variety of neurotoxins have been extremely valuable as molecular probes

Use of these toxins has been instrumentat in of voltage-gated ion channels.

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700 HERZ, TH3MSEN, AND YARBROUGH

defining distinct channel subtypes and has assisted if correlating structural and

functional features of the channels. Toxins bind to at least four separate receptor

sites on the Na channels (76). Tetrodotoxin (TTX), saxitoxin (STX), and the small

peptide p-conotoxins inhibit ion transport by binding at neurotoxin site 1. The

alkaloids batrachotoxin, veratridine and aconitine act a: site 2 to cause persistent

activation. The polypeptide sea anemone toxins and the 3-scorpion toxins slow the

inactivation of the channel (site 3 ) and &scorpion toxins act at site 4 to enhance

activation. Allosteric interactions occur between batrachotoxin at site 2 and the

scorpion or sea anemone toxins at site 3. The binding of either sea-anemone toxin

of a-scorpion toxin enhances the affinity of batrachoto> in.

Similarly, numerous peptides isolated from scor3ion venoms have proven

valuable in studying the K' channel family, and such structures could provide a

basis for the design of novel drugs. Charybdotoxin (ChTX), a 37 amino acid

peptide purified from scorpion venom has been characterized biochemically and

electrophysiologically. While originally described as a selective blocker of Ca2+-

activated K+ channels, it also blocks voltage-gated K+ channels (KJ (90, 91).

Iberiotoxin, a toxin isolated from the scorpion, Buthus, shares 68% sequence

identity with ChTX. Magratoxin, another recently described component of scorpion

venom from Centruroides margaritatus, appears to be a selective inhibitor of &,,3

channels. Other toxins which are homologous to CiTX have been identified

(kalliotoxin, noxiustoxin) and their channel specificity hEs not yet been completely

defined. High affinity probes (6 =pM) such as these tcxins will undoubtedly play

an important role in elucidating the biochemical properties and physiological

functions of these target ion channels.

Characteristics of the Ca2+ Channel Family

Multiple classes of Ca2+ channels have been classified on the basis of their

biophysical and pharmacological properties (92, 93). Four major types of Ca2'

channels have been described: T, L, N and P types (76, 94). T-type channels are

activated at relatively negative membrane potentials (low threshold), have a small

single channel conductance and mediate brief Ca2+ currents which regulate the

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frequency of action potential generation in neurons and cardiac muscle cells. In

contrast, L, N and P channel types require a more positive membrane potential for activation (high threshold) and are distinguished by their sensitivity to various

pharmacological agents. L-type channels are sensitive to dihydropyridines and

phenylalkylamines, N-type channels are blocked by small o-conotoxin peptides

isolated from Conus geographus, and P-type channels are blocked by funnel web

spider toxin (FTX) and w-agatoxin IVA (95). This channel diversity is in part due

to the expression of unique a,-subunits which are known to encode both the

voltage-sensor and Ca2+ selective pore (95). The primary sequences of cDNAs

encoding functional calcium channels exhibit homologies of 41 -70% and give

predicted masses of 212-273 kDa. Furthermore, distinct isoforms of certain a,

subunits are generated as a result of alternate splicing (96).

The L-type channels are the predominant type in muscle cells and are

responsible for the influx of Ca2+ that initiates contraction in cardiac and smooth

muscle cells. L-type Ca2+ channels are a heterooligomeric complex of five subunits,

a,, az, p, y and 5 . The three genes encoding L-type a, subunits are differentially

expressed in different tissues: als (skeletal), alC (cardiac, smooth muscle,

neuronal) and alD (neuroendocrine) (97). The large 212 kDa transmembrane a,

subunit is associated with an intracellular p subunit of 55 kDa, a glycosylated

transmembrane y subunit of 25-30 kDa, and a disulfide-linked glycoprotein complex

of a2 and 6 subunits of 125 kDa (76, 94).

More recently, two additional types of Ca2' channels have been

characterized which occur in the central nervous system. The Q- and R-type Ca2+

channels appear to be generated by the alA and alE subunits, respectively. The

a,A subunit has been coexpressed in Xenopus oocytes along with other channel

subunits a2, and y and shown to confer distinct electrophysiological and

pharmacological properties from L-, N-, and P-type channels (98). Specifically, Q-

type channels containing a,, subunits activate and inactivate more rapidly and

display steeper voltage-dependent gating than channels containing the L-type

channel subunit ale. Unlike alC channels, the a,, channels are largely insensitive

to dihydropyridines (98). In contrast to P-type Ca2+ channels in rat cerebellar

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Purkinje cells, the a,A channels in oocytes are approximately 100-fold less sensitive

to w-Agatoxin-IVA toxin and approximately 1 O-fold more sensitive to w-Conotoxin-

MVllC toxin (98). R-type channels are little affected by either of these treatments.

Studies exploiting specific pharmacological btockades 2f N- and Q- channels have

established that both channel types play roles in synaptic transmission in

hippocampal neurons (99). These results suggest ihat multiple Ca2+ channels

participate in the control of transmitter release and this may allow fine regulation of

the strength- or frequency-dependence of synaptic function.

Three groups of pharmacological agents are viidely used to target L-type

channels in the therapy of cardiovascular diseases. The phenylalkylamines, such

as verapamil, apparently enter the transmembrane pore from the intracellular

mouth, bind to their site and occlude the pore. Dihydropyridines can act either as

activators (agonists) or inhibitors (antagonists) of the Loltage-dependent gating of

L-type channels (I 00). The widely employed dihydrop}*ridine antagonists, such as

nisoldipine or nifedipine, preferentially bind with high affinity to the inactivated

channel state. The receptor site for dihydropyridines is located in a hydrophobic

region of the channel near the outer surface of the membrane (93). The

benzothiazepines have been shown to label the a1 polypeptide (1 01 ).

The binding sites for phenylalkylamines and c ihydropyridines have been

localized to two highly conserved regions of the a1 subLnit (93). These regions are

highly conserved among t-type Ca2+ channels from skeletal muscle, heart and

neurons and in other neuronal Ca2' channel types. Despite these similarities in

channel structure, phenylalkylamines and dihydropyridines have been identified

which are specific for L-type channels in cardiac arid smooth muscle. These

findings suggest that subtle modifications of these basic drug structures will lead

to selective drugs for other Ca2+ channel subtypes.

Inward-Rectifier Potassium Channels

As a group, the inwardly-rectifying K' channels, such as the ATP-sensitive

K' channel and the G-protein activated muscarinic K' channels are clearly distinct

from the previously described members of the voltage-gated channel superfamily.

These channels retain the pore and cytoplasmic gate that are the hallmarks of the

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voltage-gated K' channels However, the activity of these channels is controlled

by the electrochemical driving force for K' and intracellular signals (e.g. ATP or a

G-protein), but are not gated by the membrane potential per se Recently, the

cloning and expression of the rat heart K,,, channel was achieved (103). The

amino acid sequence showed strong homology to other potassium channels in the

inward rectifier familysuch as GlRK (73%), IRK1 (70%) and ROMK (64%). These

channels have only two putative transmernbrane spanning domains in each

sequence, S5 and S6, which are separated by a H5 segment 1-hey do not contain

any other hydrophobic segments that correspond to S1, S2 and S3 of the voltage-

gated channels, It has been suggested that these channels may belong to a new

superfamily that IS related to, but distinct from the voltage-gated superfamily (1 04).

G-PROTEIN COUPLED RECEPTOR SUPERFAMILY


GTP-binding protein coupled receptors (GPCRs) are a large multigenic

superfamily of integral membrane proteins which transduce the binding of

extracellular ligands into intracellular signalling events via guanine nucleotide

regulatory proteins (G-proteins). Agonist occupation of GPCRs subtypes may result

in some combination of the following direct cellular responses stimulation or

inhibition of adenylyl cylcase, activation of phospholipase C (PLC) and the

generation of the IP, as second messenger, activation of PKC and an increase in

intracellular calcium, activation of phospholipase A, (PLA,) and the generation of

the arachidonic acid as second messenger, or a direct G-protein activation of

inwardly-rectifying K' channels or voltage-dependent N-type i3S well as PIQ type

calcium channels. The specificity of the interaction between the receptor and

agonist on the extracellular surface as well as between the receptor and the G-

protein transducers on the intracellular surface defines the particular effector

pathway(s) for signal transduction in a given cell type.

Members of the superfamily bind a wide variety of ligands and mediate

recognition of diverse signals such as biogenic amines, neuropeptides, protein

hormones, odorants, calcium, as well as responding to photons. Application of

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M J A C h

M V ~ J L G N A V R S L L M H L I C L L V U Q F D I S I S P V A A I V T D T F N S S D C C R L F Q F ~ so H E P P G A Q C A P P P P x c s Z T W V P Q A N L S S A P S Q N C S A K O Y I Y Q D S I s 4 5

u 2 ' 1 L s P G Q C N N T T s ? P A P F E T G G N T T C I S D V T 32 ~ D H Q D P Y S 8

~ D P L N L S W Y D D D L E R Q N W S R P F N C S D G K A D 30 M Y N S T N S S N N S L A L T S P Y X 13 ....................... r ....................... ................... XI ..............

..... ...................

..... ................... .................. I V ................... .................... ....

.................. N ................... .................... v .... ...............

............

I T Y P L T I Y V

L V Y L R I Y L I

A L Y C R I Y V E V L Y G R I F R A

L V Y I K I Y I v .... V L Y W H O n I S R A

T L M L L R C H T - - E E E L A N M S L N F L N C C C K K N C C E - - - - - - - 276 L K Q T P N R - T C X R L T R A Q L I T D S ? C S T S S V T S I N - - - - - - - 271 R K T V K E V E S T C X O T R H G A S P A P Q P K K S V N G E S C S R N W R L C 166 R R G P R A X C G ? C P C E S K Q P R P D ~ C C A L A S A K L P A L A S V ~ S ~ 1 4 3 K R V N T K R S S ~ A F R A H L R A P L K , N C T H P E D ~ K L C T V I ~ K S N 260 K K D K K E P V A N P D P V S P S L V Q G ~ I V K P N N N N ~ P S S D D G L E H 237

............................................................ 9fL _. - ............................................................ 271

R E V N G ~ S K S T G E X E E C E T P E D T C T R A L P P S W A ~ L P N S C Q C Q ( E C V C G A S P E D E A E E E E P E 303

N K I Q N C K A P R D P V T E N C V Q C E E K E S S N D S T S V S A V A S N U R D ) E I T Q D E N T V S T S L G H S K D 317

V E S K X ~ G X L C A N G A V ~ Q C D D ~ A A L E ~ ~ E ~ H R ~ ~ N ~ - - - - - - . - - - - - - - - - - - - - - - - - - 3 0 ,

G S F P V N S R R V E A A R R A Q E L E ~ E M L ~ ~ T ~ P P E R T R Y ~ P I P P ~ ~ H Q L T L P ~ P ~ H H ~ L H ~ T P ~ 320

.................................

............... ............ E E N A P N P N ' D Q K P R R K K K E K R P R G T M Q 303

.................... VI .....................

.................... V I ..................... .................

.................

I H C D T F C A

V R Q I

D A C - . W S S C - - H K H C - - I C N I( *$I" - - - c ' I P C I - - -

P R V A A T 390 421 450 4 4 3 466

...........................

A L S C R E L N V N I Y R H T N E hSHT1D I c1-e ml hSHT1A M R U l S hD1 hYPACh

R V A R K A N D P E P G I E U Q V E N L E L P V N P S N V V S E R I S S V 460 rSHT1C

molecular biological and pharmacological techniques ha: revealed a functional and

structural similarity underlying the extraordinary diversity of the numerous members

of the superfamily. A characteristic feature of the GPCR:, is the presence of seven

stretches of hydrophobic amino acid residues which are span the membrane (Fig.

5). Comparison of the primary amino acid sequences for GPCR superfamily shows

that individual receptors share the greatest degree of homology in the seven

transmembrane domain region, and demonstrate high levels of sequence identity

between receptor families (Fig. 5).

363 361 393 419 419 4 3 3

413

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To date, a high resolution three-dimensional structure for' a GPCR has not

been obtained. Current models of the membrane topology of G-protein coupled

receptors are based for a large part on the low-resolution structure of

bacteriorhodopsin, although it itself is not a GPCR. Electron microscopy studies

showed that this largely integral membrane protein is composed of a bundle of

seven transmembrane a-helices which lie perpendicular to the membrane. Direct

structural studies of rhodopsin and the P-adrenergic receptor (proteolysis and

chemical modification) combined with structural predictions from hydrophobicity

analyses of the primary sequence support a seven transmembrane a-helical model.

According to this model, all GPCRs are composed of seven transmembrane

segments of 22-28 hydrophobic amino acids each, and three intervening

extracellular and cytoplasmic loops (Fig. 6 and 7). All G-protein coupled receptors

are composed of a single polypeptide containing between 31 7 and 1198 residues.

The extracellularly located N-terminus consistently contains sites for N-linked

glycosylation.

Figure 5. Primary amino acid sequence comparison between some 5HT1 serotonin

receptors and selected other G protein-coupled receptors that inhibit adenylyl

cyclase. The represented sequences are: rSHT1 C, rat serotonin 5HT,, receptor ;

hSHT1 D (clone S12), human serotonin 5HT1, receptor; 5HTl A, human serotonin

5HT,, receptor (G-21); ha2B, human a,,-adrenergic receptor ; hD2, human

dopamine D,-receptor; hM2ACh, human M,-muscarinic acetylcholine receptor.

Gaps introduced in the sequences to optimize the alignments are represented with

dashes. The boxes indicate amino acids that are identical in at least four of the six

compared receptors. The seven putative transmembrane domains of the human

5-HT1 receptor are indicated with stars above and below the sequences and have

been derived by a combination of hydropathy analysis and comparison with the

suggested transmembrane domains of the other G protein-coupled receptors,

indicated in bold italics for each receptor according to the above references. The

high level of homology among the members of the 5HT1 receptor family in the seven

membrane spanning domains is shared by other biogenic amine receptors which

couple to inhibition of adenylyl cyclase. Adapted from Levy, et. al. (274).

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0 h i n o a c ~ s (general) @!3 h l n o acids Involved In Ilgand blndlng

@ h l n o mclds Involved In 0 pmtelrrcoupllng Amlno aclds Involved In derensllllzatlon

MI Palmltoylatlon

Figure 6. Seven transmembrane spanning model of the human P,-adrenergic

receptor G-protein coupled receptor. The circles represent amino acids identical

to the corresponding position in all amino acids residues. The core of about 175

amino acid residues that forms the transmembrane domains (defined by

hydrophobicity analysis) are highly conserved among family members. The extra-

and intracellular loop regions and C-terminus are much more divergent, even

among closely related receptors. Sites for glycosylation are indicated on the amino

terminus and a palmitolylated Cys on residue 341 in the C-terminal domain is

shown. lntracellular residues involved in G-protein coupling and interaction with

receptor specific-kinases (PKA and PARK ) which mediate desensitization are

indicated.

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Figure 7. A molecular model of the dopamine D2 receptor with a ligand docked in

the binding site. The model of the D2 receptor transmembrane helices was

constructed from the coordinates of the bacteriorhodopsin structure derived from

two-dimensional electron diffraction experiments and is consistent with the

projection structure for rhodopsin. The transmembrane helices are represented by

a solid ribbon and the drug, apomorphine, is a space filling representation. The top

view looking down the helical axis of the receptor clearly delineates the seven

transmembrane helices which are the key structural motif for the GPCR superfamily.

Some of the helices are inclined relative to the perpendicular to the membrane

plane. The bottom view is in the plane of the membrane with the extracellular

space at the top of the figure. Figure adapted from Teeter, et. al. (275).

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708 HERZ, THOMSEN. AND YARBROUGH

Regulation of GPCR signaling occurs t y phosphorylation and

dephosphorylation of sites on the intracellularly located C-terminus by

serinekhreonine kinases and a serinelthreonine phosphatase of the PP2A family.

Rapid desensitization of GPCRs is promoted by phospk,orylation of these sites by

kinases belonging to a family of G-protein coupled rece?tor kinases (GRKs). The

GRK family consists of six members, divided into three zubfamilies on the basis of

structural and functional similarities. Membrane associz ted GRKs have the ability

to recognize and phosphorylate their receptor substrates only when they are in their

active conformations, i.e., when they have been stimulated and/or are occupied by

their cognate agonist ligands. For example, P-adrenerg*c receptor kinase (PARK)

and rhodopsin kinase have been identified as the kinases responsible for agonist-

specific phosphorylation of the P,-adrenergic receptor an 1 rhodopsin, respectively.

Desensitization also occurs through phosphorylation by protein kinase A. This

heterologous desensitization mechanism provides a feedback pathway whereby

other receptor signalling events may be integrated at the cellular level and regulate

GPCR signalling.

A combination of molecular, biological and biopqysical approaches have

been utilized to develop a picture of the ligand binding sites for GPCRs. Valuable

information concerning the structure of peptide-recepto- ligand binding sites has

been obtained through binding studies of receptor chimeras which has allowed the

contributions of large receptor domains to be rapidly assessed. The precise role of

individual amino acid residues has been elucidated through the introduction of site-

specific mutations in GPCRs expressed in mammalian cells (Fig. 6). Comparison

of data derived from this method for closely related members of the GPCR

superfamily has identified common residues forming a b'nding pocket.

Given the phenomenal variation in the structure, size and chemical nature

of ligands that serve as endogenous agonists of the GPCR superfamily, it should

not be surprising that multiple and different binding modes exist for different

classes of ligand-receptor pairs. As shown in Fig. 7, for receptors which are

activated by small, nonpeptidic ligands which include the rr onoamines, nucleotides,

and lipids (including retinal), substantial data support a loc3tion for a ligand binding

site which resides entirely within a hydrophobic pocket created by the

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transmembrane domains (105). A structurally more complex ligand binding site for

neuropeptide and peptide hormone receptors is composed of multiple extracellular

sequences as well as transmembrane regions of these receptors (106).

Unexpectedly, an additional level of complexity in ligand-binding site interactions

has emerged from differences in receptor domains involved in molecular recognition

of agonist ligands versus competitive antagonists. For example, in response to

site-directed mutagenesis of Tyr-129 (to Ala) in transmembrane region 2 of the endothelin A (ET,) receptor, the affinity of the ETA selective antagonist, BQ123,

decreased 2-3 orders of magnitude while having virtually no effect on binding

affinities for endothelin 1 (106). The ability to define the structure of ligand binding

sites will be very useful in the design of more specific and potent drugs targeted

towards individual G-protein coupled receptor subtypes (1 07).

Another important functional domain of GPCRs consists of regions of the

receptor that contribute to receptor-G-protein coupling. These domains have been

mapped by site-directed and deletion mutagenesis studies, generation of receptor

chimeras, and through the use of synthetic peptides and antibodies. A number of

studies based upon both the a- and P-adrenergic receptors and muscarinic

receptors indicate that the third intracellular loop plays a dominant role in mediating

receptor G-protein coupling (Fig. 6). In different GPCRs, this segment is of variable

length, ranging from as little as 15 amino acids (fMLP) to 240 amino acids

(muscarinic M3). In contrast to the adrenergic and muscarinic. receptors, a large

number of GPCRs contain small intracellular loops ranging from 16-30 amino acids

and C-termini consisting of only 16-50 amino acids. Examples of receptors in this

group include the N-formyl peptide (fMLP), interleukin-8 (also CXCl and CXC2),

bradykinin, VIP and neurokinin receptors. Deletion and mutagenesis studies of the

fMLP receptor found that the third intracellular loop did not play a critical role in

coupling to G-proteins. Despite sequence variation in the intracellular loops, local

homology in these domains is observed between receptors subtypes that interact

with the same type(s) of G-proteins.

In the C-terminal region, a cysteine residue is a potential palmitoylation site

that may be necessary for formation of a fourth intraceltular loop required for G

protein interaction. While the extracellular recognition site has' been the locus for

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710 HERZ. THOMSEN, AND YARBROUGH

targeting drugs that compete with the endogenous agoiist or mimic its action, an

alternative locus is the binding site that provides the interface between the receptor

and its cognate G-protein. The identification of synthe:ic peptides that mimic this

interaction is the first step towards the development of drugs which specifically

disrupt or mimic the interaction of a given receptor and its cognate G-protein.

In Table I , the currently established receptc~r subtypes are listed as

members of major GPCR families. For each receptcir subtype, the size of the

human receptor protein deduced from its amino acid sequence, the effector systems

activated, and typical radioligands that may be employed in binding studies (not all

are subtype selective) are listed. Studies currently in progress will undoubtedly

lead to the characterization of new subtypes to be added to many GPCR families.

Features of Receptor-G-Protein Couplinq

This review will not cover the detailed characterist cs of individual G-proteins

and numerous reviews provide specific information on t 7ese very important signal

transduction molecules (1 08-1 10). However, discussiorl of some general features

of the mechanism G-protein activation of effector proteins is provided to facilitate

an understanding of some of the specific features of GPCR signaling.

All G-proteins are heterotrimeric proteins consisting of a-, p-, and y-subunits.

Genes and cDNAs encoding G-proteins which have been cloned, couple to more

than 100 different GPCRs and together these G-prot .ins mediate activation of

diverse effector proteins including adenylyl cyclase, phospholipase C,

phospholipase A,, phospholipase D, and various ior channels. Specificity of

effector protein activation is determined by the particular isoform of G-protein

activated by a given receptor. Currently, there are at kast 16 a-subunit genes, 4

P-subunit genes and 5 y-subunit genes.

Agonist binding to G-protein coupled receptors promotes a conformational

change in the receptor that facilitates physical coupling between receptor and G-

protein in a GDP bound inactive form. This coupling event results in a transition of

the receptor from a low affinity state to a high affinity state, which consists of a

ternary complex between agonist, receptor and G-protein. Agonists promote

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Receptor Subtvpe

A1

A,

A20

A,

a l A

a l E

a 2A

a20

a2c

P1

P2

P 3

AT,

AT*

ANP,

ANP,

BE,

BB2

TABLE I Receptor Families and Subtypes of the Human

G-Protein Coupled Receptor Superfamily

Family

Adenosine

Adrenergic

Angiotensin

Atrial Natriuretic Peptide

Bornbesin

- Size Effectors -

326 Decrease in CAMP Activate K+ channel Inhibit Ca'2 channel

409 Increase in CAMP


318 Decrease in CAMP

466 Increase in IP, and DG

515 Increase in IP, and DG

560' Increase in IP, and DG

450 Decrease in CAMP Activate K' channel Inhibit Ca" channel

450 Decrease in cAMP Inhibit Ca'2 channel

461 Decrease in cAMP

477 Increase in cAMP

41 3 Increase in cAMP


359 Increase in lP,and DG

363 Protein tyrosine phosphatase regulation

1061 Increase in cGMP

1047 Increase in cGMP

390 Increase in IP,and DG


Radioliqands

[3H]CGS21 680

['H]CGS21680

['251]IABA

[3H]prazosin

[3H]prazosin

[3H]prazosin

13H]yohirnbine

[3H]b~sopropol

[3H]ICB 118551

['251]~odocyanopindolol

[3H] DUP-753

[ 1 2 5 1 ] CGP 421 12

[1251] ANP

[ 1 2 5 1 ] ANP

['251] BH-NMB

['251]-[D-Tyr6] bornbesin 6-13

methyl ester

(continued)

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712 HERZ, TqOMSEN, AND YARBROUGH

TABLE 1. Continued

Bl

B2

CGRP

CB1

CB2

CCRl

CCR2

CCR3

CCR4

CCR5

CXCRl

CXCR2

CXCR3

CXCR4

CCK,

CCK,

C5a

CRF,

CRF2,

CRF,,

Dl

D2

D3

D4

Bradykinin

CGRP

Cannibinoid

CC Chemokine

CXC Chemokine

Cholecystokinin

Complement Factor 5a Corticotropic Releasing Factor

Doparnine




472 Decrease in CAMP Activate K+ channel Inhibit Ca2' channel

360 NA

355 Increase in IP,and DC;

360 Increase in IP3and DC;


360 Increase in IP3and DCi

360 Increase in IP,and DCi



NA Increase in IP, and DCi

NA Increase in IP,and DC;

428 Increase in IP3and DCi

447 Increase in IP,and DCI

350 Increase in IP3and DG

41 5 Increase in CAMP

41 1 Increase in CAMP



443 Decrease in CAMP Activate K' channel Inhibit Ca" channel

400 Inhibit Ca2+ channel

[1251] Bradykinin

[1251] -[Ty$]BK

[1251]-BH amylin

[,HI WIN5521 2-2

[,HIWIN 55212-2

['"IJMIP-la

['251]MCP-1

['251]eotaxin

[1251]MlP-l a

[1251]MlP-la

[ 1251] I L8

[' 251]1L8

[1251]eotaxin

['251]SDF-1

[3H]devazepide

[3H] PD 140376

[ 1251]C5a

[3H]CRF

[3H]CRF

[3H]CRF

[,H]SCH 23390

[3H]YM-091 51-2

['Hlspiperone

387 Decrease in CAMP [3H]YM-09151-2

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D5

ETA

ET,

FPLl

FPL2

mGlu,

mGlu,

mGlu,

mGlu,

mGlu,

mGlu,

mGlu,

mGlu,

GABA,

Galanin

Hl

H2

H3

5-HT1,

5-HT1,

5-HT9,

~-HT,DI,

5-HT7,

Endothelin

FMetLeuPhe

Metabotropic Glutamate

GABA

Galanin

Histamine

Serotonin

TABLE I. Continued









91 2 Decrease in cAMP

1171 Increase in iP,and DG




960 Decrease in cAMP Activate K' channel inhibit Ca" channel

349 Decrease in cAMP Activate K' channel Inhibit Ca2+ channel



NA NA

421 Decrease in CAMP Activate K' channel





[3H]SCH 23390

['251]BQ123

[ '251]BQ3020

13H]fMLP

['HIfNILP

[3H]APDC

[3H]APDC

[3H]AIJDC

[3H]L-AP4

[3H]APDC

[3H]APDC

[3H]L-AP4

['H]L-AP4

[ ''51jCGP6421 3

3-[1251'I-[Tyr26] g a I an ii n

[3H]mepyramine

[3H]tic~tidine

[3H]N--a-methyl- histamine

[3H]8-OH-DPAT

[ 'Z51]G~TI

[3H]s~~rnatriptan

[3H]sumatriptan

(,H]5-HT

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714 HERZ. THOMSEN. AND YARBROUGH

TABLE 1. Continued

5-HT1,

5-HT2,

5-HT2,

5-HT2,

5-HT4

5-HTSA

5-HT5,

5-HTe

5-HT7

LTB,

LTC,

LTD,

MLIA

ML,,

Ml

M2

M,

M4

M5

Yl

y2

y4

Y5

Leukotriene

Melatonin

Muscarinic Acetylcholine

366

47 1

479

458

378

357

370

440

445

NA

NA

NA

350

362

460

466

590

479

590

Neuropeptide Y 384

38 1

375

445

Decrease in cAMP

Increase in IP,and DG



Increase in cAMP

Increase in CAMP

NA

Increase in CAMP

Increase in CAMP

Increase in IP, and DG


Increase in lP,and DG

Decrease in CAMP

Decrease in CAMP


Decrease in CAMP Activate K' channel Inhibit Ca2+ channel


Decrease in CAMP


Decrease in CAMP

Decrease in CAMP

Decrease in CAMP

Decrease in CAMP

['2SI]LSD

[,H]ketanserin

[3H]5-HT

[3H]mesulergine

[3H]GR 11 3808

[3H]5-CT

[,H]5-CT

[,H]5-CT

['H]5-CT

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RECEPTORS AS TARGETS FOR DRUG DISCOVERY

TABLE I . Continued

Y6 290 Decrease in CAMP

PPl 375 Decrease in CAMP

Neurotensin Neurotensin 41 8 Increase in IP, and DG Decrease in CAMP

CI

6

K

ORLI

PAF

DP

FP

IP

TP

EP,

EP2

E p3

EP4

P2Y,

P2Y2

P2Y4

P2Y,

SSt,

Opioid 400 Decrease in CAMP Activate K' channel Inhibit Ca" channel

372 Decrease in CAMP Activate K+ channel Inhibit Ca2+ channel

380 Decrease in CAMP Activate K' channel Inhibit Ca2' channel


PAF 342 Increase in IP,and DG

Prostanoid 359 Increase in CAMP






390 Decrease in CAMP Increase in IP,and DG


Purinoceptor 373 Increase in IP,and DG




Somatostatin 391 Decrease in CAMP

715

['HIDAMGO

[3H]DP DPE

[3H]noc:iceptin

[,HIWEB 2086

[3H]PGD2

[3H]PGF2a

[3H]iloprost

[3H]SQm 29548

[,H]PGE,

[3H]PGE,

[3H]PGE2

[3H]PGE2

["SIADP-p-S

NA

[ 35S]AD P-p-S

[35S]ADP-P-S

['251]SRIF-1 4

(continued)

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TABLE 1. Continued

sst,

SSt,

SSt,

SSt,

N Kl

N K2

N K,

TRH

VlA

Vl B

v2

OT

VIP,

VIP,

GLP-1

GRF

PACAP

Secretin

369

41 8

388

363

Tachykinin 407

398

468

Thyrotropin- 398 Releasing Hormone

Vasopressin 395

424

37 1

Oxytocin 388

Decrease in CAMP

Decrease in CAMP

Decrease in CAMP

Decrease in CAMP




Increase in IP,and Df3



Increase in CAMP


Vasoactive 457 Increase in cAMP intestinal peptide


463* Increase in CAMP




['251]SRIF-14

['251]SRIF-14

['2SI]SRIF-1 4

['251]SRIF-1 4

[1251]BH-SP

[' 251]NKA

[1251]-[MePhe7]NKB

[,H]MeTRH

[,H]d( CH,),[Tyr( Me2]AVP

[,H]AVP

[,H]d( Va14)AVP

['H]d( CH,),Tyr( Me2)- ThS, OM8-Ty$-NH2)]OT

['251]VIP

['251]VIP

[1251]GLP-l

['251]GRF

[1251]PACAP

[ 1251]secretin

size of non-human receptor listed and size for cloned from ral species indicated NA indicates that receptors have not been cloned to date

receptor G-protein coupling upon binding. Neutral antagonists do not exhibit a

preference for binding to high and low affinity states of GPCRs, evidence suggests

that inverse agonists will stabilize the receptor in the unsoupled state (1 11). While

the unoccupied receptor is not usually coupled to Cl-protein in the absence of

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agonist, there are examples where certain receptor populations exist in a high

affinity precoupled state in the absence of agonist (1 12). This precoupling may be

due to a much higher stoichiometry of G-protein to receptor in the plasma

membrane. Formation of the high affinity agonist-receptor-G-protein ternary

complex in turn alters the conformation of the heterotrimeric G-protein which

promotes the release of bound GDP and facilitates the binding of GTP. Binding of

GTP in turn promotes the dissociation of the trimeric G-protein complex into a free

GTP bound a- (G,-GTP) and p-y subunits.

GPCR activation is mediated through the direct action of either the

dissociated G,-GTP subunit or GP." complex, or a combination of both, which may

interact with multiple effector proteins, such as adenylyl cyclase, phospholipase C, or a particular ion channel. While initially considered controversial, a direct role for

the G,, complex as a mediator of the signal transduction pathway is now well

established for a number of effector molecules. These effectors include certain

forms of adenylyl cyclase, phospholipase Cp, 0- adrenergic receptor kinase, a G-

protein modulated inwardly rectifying K' channel, and voltage-dependent N-type as

well as P/Q type calcium channels. The G-protein cycle is terminated by the

hydrolysis of a-subunit bound GTP to GDP. This turnoff switch terminates the

activation of the effector protein and allows the GDP-bound a and G,, subunits to

reassociate into the heterotrimeric complex. In the case where GP.y subunits are the

direct activator, GDP-bound G,, with a high affinity for G,, , acts a sink by favoring

heterotrimer formation.

Recent findings also indicate that a family of RGS proteins (regulators of G-

protein signalling) bind specifically to certain G,-GTP subunits to regulate the rate

of deactivation of G,-GTP subunits. Biochemical, genetic and physiological

evidence has demonstrated that RGS proteins act to stimulate GTP hydrolysis by

G, subunits, thereby controlling the sensitivity of the signaling pathway and the

duration of the cellular response. As a result of G-protein cycling, the receptor

reassumes a low affinity state until agonist binds and initiates the reformation of the

high affinity ternary complex. The high affinity ternary complex is short-lived in the

presence of cellular levels of GTP (1 13).

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Although GPCRs initiate a characteristic response, a single receptor is

frequently coupled to multiple effectors and generates multiple second messengers

(114). Such divergence in signaling occurs either zt the level of the receptor

interfacing with multiple G-proteins or at the G-protein-effector interface (1 15). In

addition to the intracellular domains of a given G ' X R , receptor specificity in

coupling is also determined by specific domains on the G, subunits. Receptor

activation of G proteins has been inhibited by experimentally introduced mutations

in the C-terminus of the G, subunit, peptide specific antibodies directed against it,

and peptides mimicking C-terminal sequences. Furthermore, elegant experiments

have found that substitution of only three amino acids in the C-terminus of a, or ai

is sufficient to switch receptor specificity from receptors that employ either G, or Gi

to stimulate PLC or inhibit adenylyl cyclase, respecti\fely. Thus, the C-terminus of

the a-subunit plays a signficant role in defining receptcr specificity and leads to the

prediction that it should be possible to engineer a rec,lmbinant system expressing

a GPCR-Ga pair in which the receptor is coupled to any particular chosen signaling

pathway. The complexities inherent in receptor-effeztor signalling for the GPCR

superfamily are the subject of numerous ongoing stu jies.

Characteristics of the Adreneraic Receptor Familv

Among the most intensively studied and perhaps the best characterized of

the numberous GPCR families is the adrenergic receptx family. For each receptor

subtype, important structural domains and effector systl?ms activated by receptor-G-

protein interactions are presented. A limited descripi ion of therapeutic relevance

and distribution of receptor subtypes are also highligited.

Receptors for the adrenergic amines are sirbclassified into three major

distinct types subfamilies, namely al, a2 , and p. The adrenergic receptor family

is composed of a diversity of receptor subtypes since each subfamily also contains

at least three subtypes, thereby comprising a famil!/ of at least nine receptors.

Among adrenergic receptors in different subfamilies ( a1 vs. a2 vs. p ), amino acid

identities in the membrane spanning domain rancie from 36-73%. However,

between members of the same subfamily (a,* vz. aIB) the identity between

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membrane domains is usually 70-80%. This homology relationship appears to be

a general rule that holds for other GPCR families. Together, these distinct receptor

subtypes mediate the effects of two physiological agonists, epinephrine and

norepinephrine.

Distinct adrenergic receptor types couple to unique sets of G-proteins and

are thereby capable of activating different signal transduction effectors. The

classification of a,, a2 , and p types not only defines the receptors with regard to

signal transduction mechanisms, but also accounts for their ability to differentially

recognize various natural and synthetic adrenergic arnines. In this regard, a

number of highly selective radioligands have been developed and utilized to

characterize the pharmacological properties of each of these receptor types.

Functional responses of a,-receptors have been shown in certain systems to

stimulate phosphatidylinositol turnover and promote the release of intracellular

calcium (via Gq), while stimulation of a,-receptors inhibits adenylyl cyclase (via Gi).

In contrast, functional responses of 13-receptors are coupled to increases in adenylyl

cyclase activity and increases in intracellular calcium (via G,)

In light of recent studies, it is now accepted that there are three different a,

receptor subtypes which all demonstrate a high affinity (subnanomolar) for the

antagonist, prazosin (1 17). The subdivision of a,- adrenoreceptors into three

different subtypes, designated a,,, aIB, and alD (1 18, 11 9) is based on extensive

ligand binding studies of endogenous receptors and cloned, expressed receptors.

Pharmacological characterization of the recombinant receptors led to revisions in

the original classifications. The cDNA clone initially termed the ale, based upon

apparently novel binding characteristics, corresponds to the pharmacologically

defined a,* receptor (1 20). Agonist occupation of receptor subtypes results

in activation of phospholipase C, stimulation of PI breakdown, generation of the IP,

as second messenger, and an increase in intracellular calcium. In addition, PKC

activation may occur.

Tissues expressing predominantly alA receptors in the rat include

submaxillary gland, renal and mesenteric vascular beds. The rat aIB receptor has

been reported to be present in liver and spleen. Other tissues including

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hippocampus, cerebral cortex, kidney, and heart have mixtures of a,, and a,B

receptors (121). The aID receptor has been found in rat lung, heart and cerebral

cortex (121). The most widespread therapeutic applications for drugs that block a, receptors are hypertension and urinary bladder contro .

Pharmacologically, a,-adrenergic receptors are defined by exhibiting high

affinity for the antagonists yohimbine, rauwolscine, and idazoxan, as well as

selective agonists such as UK14,304 and clonidine, and low binding affinity for

prazosin (1 22). Three different human a,-receptor s~ btypes have been cloned,

sequenced, and expressed in mammalian cells (123, 124). These subtypes not

only differ in their amino acid composition but also in their pharmacological profiles

and distributions. An additional a,-receptor subtype, aZD (rat gene rg20), was

originally proposed based on radioligand binding stucies of rodent tissues. It is

now recognized that RG20 represents a species hDmolog to the human a% receptor (sequence CIO) which shows species variation in its pharmacology. This

illustrates the difficulty that may be faced in correlating subtypes proposed from

molecular biology classification with binding studies a 7d functional experiments,

and the confounding effects of differences in species pharmacology.

Functionally, all three a, subtypes are couple3 to inhibition of adenylyl

cyclase by specific coupling to Gi, In addition, the a2+, and azB receptor receptors

have also been reported to mediate stimulation of a G-xotein coupled potassium

channel as well as inhibition of a G-protein associated calcium channel. Inspection

of the sequences of the different a,-receptor subtypes indicates that they have

relatively large third cytoplasmic loops compared to o!her adrenergic receptors.

There is relatively little homology between the third cytoplasmic loops of the

different subtypes, and highest homology is observed in transmembrane spanning

domains which contribute to agonist binding.

Central a,-receptors play a role in diverse physiological processes including

central cardiovascular control, affect, arousal, pain and hormone release.

Peripheral receptors regulate vascular smooth muscle contraction, renal function,

platelet aggregation, pancreatic function, urogenital function, endothelial function,

and lipoprotein metabolism. The a, receptor has been suggested to be a good

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therapeutic target for new drugs to treat hypertension, attention deficit disorder,

spasticity, and pain.

Radioligand binding studies using agonists, such as isoproterenol,

epinephrine, norepinephrine, and numerous antagonists originally suggested that

two pharmacologically distinct P-adrenergic receptors (PAR) exist in humans, the

P,AR and PAR subtypes. The P,-adrenergic receptor (P,AR) was found to be very

important in control of the cardiovascular system and has been an important target

for new cardioselective antihypertensive drugs. The existence of the &AR was

confirmed by isolation of cDNA encoding the P,AR receptor (1 25). The P,AR bears

54% amino acid similarity in overall amino acid sequence and 71% similarity in

transmembrane sequences with the P,AR (1 25). A newly cloned and sequenced

PAR from turkey erythrocytes has been shown to contain the greatest similarity to

the P,AR compared to other PARS (126). In common with all other PARS, agonist

occupation of the P,AR results in activation of a specific G-protein, G,, which in turn

activates adenylyl cyclase.

Genes encoding the human D,ARs have been cloned and sequenced.

Mutagenesis studies have revealed a number of functional domains important for

ligand binding and receptor-G-protein coupling (127-1 31 f . Conclusions from these

studies have also been extended to related families of GPCRs. Deletion of N-

terminal N-linked glycosylation sites on the receptor has been reported to alter the

transport of the PAR to the plasma membrane (132). As illustrated in Fig. 6, amino

acid mutation studies have revealed that aspartate and serine residues located

within the hydrophobic transmembrane spanning regions of the receptor are critical

for ligand binding (133-135). Studies involving amino acid substitutions in the third

intracellular loop indicated that this region is important for receptor G-protein

coupling and determines receptor-G-protein coupling selectivity (1 36). Agonist

occupation of the P,AR in both membrane preparations and cells expressing the

PAR typically results in G,-mediated stimulation of adenylyl cyclase. P,AR/a,AR

chimeras containing the third cytoplasmic loop of the a,AR are observed to inhibit

rather than stimulate adenylyl cyclase upon agonist binding, providing additional

evidence that this region participates in receptor-G-protein coupling (1 33).

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The molecular mechanism underlying desensitization of the P,AR in

response to prolonged exposure to high concentrations of agonist has been

elucidated at the molecular level (1 37). A specific serindthreonine kinase referred

to as j3-adrenergic receptor kinase or PARK, has been iss,lated, cloned, and shown

to phosphorylate agonist occupied P,AR resulting in receptor desensitization.

PARK has also been shown to phosphorylate and desensitize other G-protein

coupled receptors.

Epinephrine and norepinephrine binding to the P,-<adrenergic receptor (P,AR)

results in a number of physiological responses including increased heart rate,

increased cardiac output, and relaxation of certain types of smooth muscle,

including bronchial smooth muscle. Drugs specifically interacting with the j3,AR

have been used therapeutically to treat hypertension, asthma, and to control ocular

tone in glaucoma.

The j3AR was first reported to be present in browri adipose tissue of rats and

is distinguished from other PARS in radioligand binding studies by a lower affinity

for typical PAR agonists and antagonists. Physiological studies of this P,AR

subtype indicate it may play an important role in adipose tissue metabolism and

gastrointestinal function (1 38). Responses mediated by this receptor subtype

include increased lipolysis and metabolic rate and decreased gastric motility. For

this reason, this human subtype is of interest as a target for drug discovery to

enable the identification of selective agonists for the treatment of obesity and type

I1 diabetes.

Mouse, rat, and human PARS have been cloned, sequenced, and expressed

in mammalian cells (139-142). The PAR contains the hisjhest homology with other

PARS in the seven transmembrane domains (86% in TMCl and 6), while showing 51

and 46 percent overall identity with the P,AR and J3,AR, respectively. Little

homology between the PAR and the other PAR subtypes is observed in the N- and

C-terminal regions or in the third cytoplasmic loop. The rnRNA for the human P3AR

has been detected in white and brown adipose tissue, heart, gall bladder, and

colon. The PAR, like other PAR subtypes, is positively cfwpled to adenylyl cyclase

and this receptor is unique among the other PARS because it is not subject to

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agonist induced desensitization. Furthermore, consensus sites for phosphorylation

present in the other two PARS are not found within the C-terminus of the P,AR.

Mammalian cell lines (CHO) expressing the human BAR have been used to identify

selective P,AR agonists through use of a primary functional screen based upon

stimulation of adenylyl cyclase activity. These efforts have led to the initial

identification of specific and potent PAR agonist ligands (subnanomolar and 1000-

fold selective), such as .L-755,507, This benzenesulfonamide ligand evokes

lipolysis in isolated human adipocytes, but is much less potent in rat adipocytes.

L-755,507 stimulates lipolysis and causes an increase in me.tabolic rate in the

rhesus monkey. Thus, the use of all three recombinant receptor subtypes has

enabled the identification of a receptor-subtype specific agonist ligand with potential

therapeutic activity in a subfamily of highly homologous receptors.

RECEPTOR TYROSINE KINASE SUPERFAMILY


A wide variety of polypeptide growth factor receptors that possess intrinsic

tyrosine kinase activity have now been characterized. Activated receptor tyrosine-

kinases (RTKs) undergo dimerization and initiate signaling through tyrosine-specific

phosphorylation of diverse intermediates, activating a cascade of intracellular

pathways that regulate phospholipid and arachidonate metabolism, calcium

mobilization, protein phosphorylation (involving other protein kinases), and

transcriptional regulation. The growth-factor-dependent tyrosine kinase activity of

the RTK cytoplasmic domain is the primary mechanism for generation of intracellular signals that initiate multiple cellular responses. Cellular responses

mediated by RTKs include alterations in gene expression, cell proliferation,

cytoskeletal architecture, cell metabolism, differentiation and CCAI survival.

Many of the RTK subfamilies are recognizable on the basis of architectural

similarities in the catalytic domain as well as distinctive motifs in the extracellular

ligand binding regions. Among the RTK subfamilies, residues in the catalytic

domain share greater than 50% identity. The extracellular domain of the RTKs

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typically contains discrete structural units that are derived from a limited group of

biochemical domains (Figure 8). These domains include: cysteine rich regions,

immunoglobulin-like loops (IgLs), or fibronectin type Ill (FN-Ill) domains. Base upon

these structural considerations, a nomenclature defining several subfamilies of

RTKs has been proposed (142,143). The six receptor subfamilies referred to on the

basis of their prototypic members include: EGF-receptor, insulin receptor, PDGF-

receptor, the fibroblast growth factor receptor (FGFR), TRK receptor and EPH/ECK

receptors. Members of a given subfamily share common structural features that are

distinct from those found in other subfamilies.

A common structural feature shared by a group of three subfamilies, the

EGF, insulin and EPH/ECK receptors, is the presence of cysteine-rich regions in

the extracellular domain (142). The EPH recepfor extracellular region is

characterized by a single cysteine-rich box which is related to the two tandem

cysteine-rich boxes found in members of the EGF-receptor subfamily. Also

containing a single cysteine-rich region, the insulin receptor is the prototypic

receptor for a subfamily whose distinctive structural feature is its organization as

a heterotetrameric species of two a and two p subunit:;. The extracellular ligand-

binding subunit, a, is disulfide-linked to the transmembrane P-subunit, which

contains the tyrosine kinase domain.

A second major structural category is represented by a group of three

subfamilies, fibroblast-growth factor (FGFR), platc?let-derived growth factor

(PDGFR), and Fl t l NEGF receptors, which are characterized by extracellular

domains consisting of three, five, or seven IgLs. Cytoplasmic regions of these

receptors contain a tyrosine kinase domain that is interrupted by what is termed the

“kinase insert” (1 42). The kinase insert contains sites of autophosphorylation and

has been proposed to function in binding of signal mollxules through interactions

with phosphotyrosine (1 44, and section below). Reciptors containing five lgLs

include two PDGFRs (a and p), the macrophage colon)! stimulating factor-I (CSF-

1 R) receptor, the c-kit protein (a receptor for the steel ligmd) and the product of the

FLT3/FLK2 gene. The FGF receptors, which have three IgLs, constitute a separate

subfamily. Currently, there are at least seven FGFR members which mediate a

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RECEPTORS AS TARGETS FOR DRlJG DISCOVERY 725

Receptor Protein Tyrosine Kinase Subfamilies

I I kFLT

EGF receptor Insulin receptor PDGR receptor FGF receptor TRK subfamily EPHlECK subfamily subfamily 8 u bfam ily subfamily TRK subfamily

EGFR 1NS.R FLG TRK-B EPH a-PDGFR N-FLG ECK

P-PDGFR BEK CEKZ

ERBBZ IGFIR ERBB3 IRR

CSFI-R c-Kit

IgG-like FLT

-Catalytic domain

Cysteine-rich box - Conserved cystetne : Transrnernbrane helix

Figure 8. Structural organization of Receptor Protein-Tyrosine Kinase

Families. The figure illustrates the distinct families of the receptor-tyrosine kinases

showing that all members consist of an amino-terminal extracellular ligand binding

domain, a transmembrane domain, and a carboxyl-terminal intracellular tyrosine

kinase domain. The characteristics of the extracellular receptor domains may

consist of either multiple cysteine-rich domains (striped box), immunoglobulin-like

domains (semi-circles) or conserved cysteine residues. A transmembrane domain

of about 25 hydrophobic amino acids spans the membrane Receptors that are

members of the insulin receptor family are composed of two types of subunits,

forming disulfide bound heterotetramers of a and p chains. The intracellular

tyrosine kinase domains of the PDGF receptor family and the an FGF-receptor

family are interrupted by an insert region of amino acids that are not related to other

protein kinase catalytic domains. Receptor with seven lgLs have been proposed

to represent a separate family. Not shown is the HGF receptor family.

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diverse array of biological responses, including the capacity to induce

angiogenesis. In addition, a group of RTKs with seven lgLs has been proposed to

represent a separate subfamily. Its known members, FLTI, FLKl and FLT4 show

a similarity of structure and expression. Seveial lines of evidence suggest that this

subfamily of growth factor receptors play an important role in the growth and

differentiation of endothelial cells.

One group of receptors that does not fall into either of the above categories

is the Trk subfamily. Recent work on the Trk subfamily has established that these

molecules constitute signal-transducing receptors fo- a family of structurally and

functionally related neurotrophic factors, collectively known as the neurotrophins.

This receptor subfamily contains neither cysteine-rich regions nor lgLs in the

extracellular domain. Instead, cysteines are found throughout the binding domain

and are also clustered near the N-terminus (Figure 5). The cytoplasmic domain of

the trk subfamily also has an equivalent kinase insert.

Although there is a tremendous diversity among the numerous members of

the RTK family, the signalling mechanisms used by ..hese receptors share many

common features. Biochemical and molecular genetic studies have shown that

binding of the ligand to the extracellular domain of the RTK rapidly activates the

intrinsic tyrosine kinase catalytic activity of the intrazellular domain. Enzymatic

activity of the RTK kinase domain is essential for, signal transduction. The

increased activity also leads to phosphorylation of a number of intracellular

substrates and activation of numerous downstream signalling molecules. Examples

of proteins that are frequently activated include: phospholipase-C-y (PLC-y),

phosphatidylinositol 3-kinase (PI-3-kinase), GTPase activating protein, pp60"'"

protein tyrosine kinase, p21 -ras and others.

Many of the intracellular targets of the RTKs possess certain recognition

domains, referred to as Src-homology 2 (SH2) domains, that directly recognize

phosphotyrosine-containing sites on autophosphorylated RTKs and thereby

mediate the activation of biochemical signalling pathways (for review, see 145,

146). While some of the SH2-containing proteins have known enzymatic activities

(e.g. PLC-y and PI-3-kinase), others appear to functiori as "linkers" and "adapters"

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between the RTKs and downstream effector molecules (147). Thus, these SH2- phosphopeptide interactions recruit other signaling molecules to the receptor,

where they are phosphorylated by the RTK Specificity of signlalling is achieved

because SH2 domains recognize not onry the phosphotyrosine residue, but also the

three residues immediately C-terminal to the phosphotyrosine. Thus, the SH2

domains are responsible for coupling the activated growth-factor receptors to

cellular responses which include alterations in gene expression, cell proliferation,

cytoskeletal architecture and cell metabolism.

The last several years has seen a dramatic increase in the number of

described RTKs due to efforts to identify new family members using molecular

cloning approaches. The approaches have included screening cDNA libraries

under low-stringency conditions using probes derived from other genes and

screening libraries with degenerate oligonucleotide probes that correspond to

highly conserved stretches within the catalytic domain. This has led not only to an

increase in the both the number of members within exising subfamilies, but also to

the establishment of new subfamilies and the identification of a number of orphan

receptors.

Epidermal Growth Factor Receptor Family

The human epidermal-growth-factor receptor (EGFR) is expressed on a wide

variety of cell types and is one of the most well-studied mernbers of the RTK

superfamily. A family of ligands (polypeptide growth factors) have been identified

which bind to the receptor with high affinity and elicit mitogenic responses in EGF- sensitive cells. Members of the family include the prototypical polypeptide mitogen,

epidermal growth factor (EGF), transforming growth factor-a (TGF-a), vaccinia

growth factor (1 48), amphiregulinlschwannoma-derived growth factor (AR or SDGF)

(149,150), heparin-binding EGF-like factor (HB-EGF) (1 51), the neu differentiation

factor (NDF) (152), and the heregulins (153). All members of this ligand family are

characterized by the presence of one of more EGF structural units in their

extracellular domains, and all share some degree of amino acid homology,

including the positioning of six conserved cysteines over a sequence of 35-40

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728 IIERZ. TIIOMSEN. A N D YARBROIJGH

amino acids and three disulfide bonds formed by these cysteines (154). 'The

heregulins are a family of molecules that were first isolated as specific ligands, for

HER2. Heregulin also appears to be important in development and maintenance

of the nervous system due to its abundance in cells of neuronal origin and because

alternatively spliced forms of the heregulin gene encode neurotrophic activities.

The human EGFR has been cloned froin a human carcinoma cell line (A-

431) that over-expresses the receptor (1 55). The 170 kDa receptor consists of an

extracellular domain with a high cysteine and N-linked glycosylation content, a

single transmembrane domain, and a cytoplasmic domain with tyrosine kinase

activity (Figure 8) . Binding of EGF to the receptor results in the formation of

receptor dimers, activation of intrinsic tyrosine kinase and concomitant

autophosphorylation of the cytoplasmic domain, as well as phosphorylation of

cellular substrates (1 56,157). Interestingly, expression of the recombinant

intracellular domain of the human EGFR has shown it is a functionally independ'ent

domain (1 58). Not only does the recombinant intracellular domain exhibit

constitutive catalytic activity, but its specific activity and properties are similar to

that reported for the EGFR holoenzyme (159). Wlutations within the kinase domain

of the EGFR which eliminate catalytic activity abolish EGFR signal transduction.

Within the intracellular domain of the EGFR, the sites of tyrosine

autophosphorylation (residues 992, 1068, 1148, 1 173, and 11 86) (160) are

clustered at the C-terminus. These phosphorylated tyrosine residues are critical

sites for interaction with a number of signalling molecules. This can be illustrated

for the interactions of PLC-y1 with the EGFR and can likely be generalized for

interactions with other signalling molecules. Autophosphorylation of the EGFR

creates high affinity sites for the binding of PLC-yl SH2 domain. Tyr-992 and

adjacent residues of the EGFR are part of a sequence recognized by PLC-v 9-12

domain. In fact, peptides containing phosphorylated tyrosine residues and

sequences of 5-10 residue regions of the E!GFR bind to SH2 domains (161). fn

vim, it is likely that recruitment of PLC-yl IS important for activation of PI turnover.

Association of PLC-y1 with the EGFR precedes PLC-yl tyrosine phosphorylation.

Bound PLC-yl is then phosphorylated by the receptor, leading to stimulation of PLC

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activity and is the basis for increased phosphaticlylinositol turnover in EGF-

activated cells. Current evidence indicates that s,ignal transduction involves

activation of several second messenger systems, including protein kinase C,

increases in intracellular free calcium and the arachidonic acid pathway (1 62).

In addition to mediating normal cellular growth, rnembers of the EGFR family

of RTKs are frequently overexpressed in a variety of aggressive epithelial

carcinomas and this is thought to directly contribute to malignant tumor

development. A number of studies have shown that the EGFR is frequently

amplified in certain types of tumors, including glioblastomas, squamous

carcinomas, and brain tumors (163). Additionally, HER2/p18YrbB2 (and in the rat

called neu), HER3/p160erbB2, HER4/180erbB4 (164) are three RTKs which have

extensive amino acid sequence homology to the IEGFR. is

frequently amplified and overexpressed in human breast tumors and ovarian

carcinomas (1 63), and this amplification is correlated with poor patient prognosis.

Simultaneous overexpression of pl85""" and the EGFR synergistically transforms

rodent fibroblasts and is often observed in human cancers Mechanistically, this

transregulatory effect is probably mediated by heterodimerization of the EGFR and

~185"'" , resulting in increased tyrosine phosphorylation of pl85""". Finally, HER3

expression is amplified in a variety of human adenocarcinomas.

HER2/pl 85erbB2

The role of RTKs in cellular proliferation has led to the suggestion that

inhibitors of kinase activity could in principle have therapeutic potential. Several

classes of compounds have been reported to inhibit tyrosine kinases. These

compounds include a number of microbial and plant natural products, such as the

alkaloid staurosporine, flavonoids such as genisteiri and quercitin, herbimycin,

lavendustin, and erbstatin. A recently developed group of cinnamic acid dervatives

that are referred to as tyrphostins also demonstrate inhibitory activity in vitro against

the EGFR and block EGF-dependent cell proliferation (165). Although some of

these inhibitors are potent, many of the inhibitors identified to date lack selectivity

with respect to RTKs and other kinases, including the serine/threonine protein

kinases. This is not surprising since a high degree of homology and conservation

of amin'o acid residues exists among the catalytic domain of tyrosine kinases.

However, since distinct substrate specificities are observed for protein tyrosine

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730 HERZ. T-IOMSEN, AND YARBROUGH

kinases, it should be possible to identify potent and specific inhibitors and examples

of such compounds are now emerging (1 66,167) An alternative approach which

may be fruitful has focused on the development of spmfic monoclonal antibodies

directed against the extracellular domain of the HER; receptor

RECEPTOR PROTEIN-TYROSINE PHOSPHATASE'S


A large number of recent studies have derronstrated the existence of superfamily of receptor-like protein tyrosine phosphzttases in humans which are

defined by sequence homology and function. The known transmembrane PTPases

are the CD45 (also referred to as leukocyte-common a itigen, LCA), LAR (a CD45

homolog), LRP, HPTP, RPTP, and many others (Fic. 9). The overall structural

organization of CD45 and its transmembrane homologiies resemble that of several

receptor protein tyrosine kinases, specifically memb 3rs of the PDGR and FGF

receptor families (Fig. 8). The extracellular segment of LAR is homologous to

neural cell adhesion molecules, suggesting that other cell surface molecules could

serve as ligands regulating LAR activity. The external segments of HPTP molecules

may share some of the cell-adhesion properties of fibronectin and mediate

heterophilic interactions with ligands located on other cc?IIs. The distinctive receptor

structure of the extracellular regions composed of three lgLs or nine FN-Ill domains,

or combinations of these two domains bears marked similarities to the extracellular

domains of several families of the RTKs, such as the FGF (3 IgLs) and PDFR (5 or

7 IgLs) families. Thus, it is widely thought that these receptor-PTPases are capable

of initiating transmembrane signalling in response to external ligands. Most

members of the superfamily consist of a large exti-acellular domain, a single

transmembrane region and a large, highly conssrved cytoplasmic domain

containing two tandem PTPase domains.

Characteristics of the CD45-Rece~tor Family

CD45 is a major cell surface glycoproteir expressed on nucleated

hematopoietic cells. Expression of CD45 has been shown to be important for

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Ig domain am Fibronectin type three repeat

Unique sequences A5 homology domain

4-2

* CAHdomain PTPase catalytic domain

Figure 9. Structural Organization of Receptor Protein-Tyrosine Phosphatases.

The figure illustrates the distinct members of the receptor-tyrosine phosphatases

showing that all members consist ofan amino-terminal extracellular ligand binding

domain, a transmembrane domain, and a carboxyl-terminal intracellular catalytic

tyrosine phosphatase domain. The characteristics of the extracellular receptor

domains vary and may consist of either tandem fibronectin type three repeat

domains (dark boxes), immunoglobulin-like (lg) domains (semi-circles) or an N-

terminal domain with homology with carbonic anhydrase CAH) . A transmembrane

domain of about 25 hydrophobic amino acids spans the membrane. The highly

conserved intracellular tandem PTPase domains (DI and D2) characteristic of most

receptors are interrupted by a short insert region.

activation of both B and T cells via their antigen-specific receptors. The CD45

molecule consists of a large, variable extracellular domain (390-452 amino acids),

a single transmembrane region and a large, highly conserved cytoplasmic domain

containing two tandem PTPase domains (705 amino acids). Alternative splicing

results in at least five different isoforms that vary in the arnino terminus. Cell type

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and developmental stage specific expression of the multiple isoforms occurs, and

genetic evidience suggests the isoforms are functionall distinct in regulating T-cell

receptor signalling. By analogy with the transmembrane receptor tyrosine kinases,

it is widely assumed that CD45 is also a receptor, kut to date the physiological

ligand for CD45 has not been identified.

The PTPase activities of CD45, LAR, and ot ier receptor PTPases have

been examined using either purified native forms or recombinant PTPase generated

in a variety of expression systems. Another reported structure-functional parallel

between CD45 and EGFR is the finding that expression of the recombinant

intracellular domain of human CD45 produces a functionally independent domain

(1 58). A recombinant baculovirus was constructed to encode the entire cytoplasmic

domain of the human CD45 tyrosine phosphatase prctein (CD45-IPD), consisting

of two tandem PTPase domains. The recombinant protein produced in Sf9 insect

cells was isolated, purified, and found to be a high];/ active. The recombinant

intracellular domain exhibited constitutive catalytic acti Aty, and its specific activity

and properties were similar to that reported for the mzymatic properties of the

whole receptor. These studies have shown that th? cytoplasmic domain can

function independently of regulatory constraints thE t may be imposed by the

extracellular and transmembrane domains of CD45.

Studies have shown that CD45 is essential for the T-cell receptor (TCR) to

couple to second messenger pathways, IL-2 production, and the proliferative

response of T-cells in response to specific antigen. In particular, the initial event in

T-cell activation is T-cell receptor mediated activation of p56ICk and ~ 5 9 ~ " tyrosine

kinases. Recent findings indicate CD45 is requirec for induction of the early

tyrosine phosphorylation events and effective coupling of the T-cell receptor. Lck

and fyn, as all src family protein tyrosine kinases, possess a C-terminal tyrosine

which when phosphorylated, inhibits kinase functiori. Models of CD45 action

postulate that it functions to dephosphorylate regulator r/ phosphotyrosine residues

on TCR associated tyrosine kinases, making it possible for the kinases to respond

to T-cell receptor occupancy. Inhibition of protein ty'osine phosphatase activity

has been shown to increase tyrosine phosphorylation levels of many cellular

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proteins, including PLCy This sequence leads to increases in the levels of

intracellular calcium in Jurkat cells and consequent T cell activation The

identification of specific inhibitors of CD45 tyrosine pTosphatase actlvlty are of

therapeutic interest since such compounds would llkely serve as

immunomodulators

CYTOKINE RECEPTOR SUPERFAMILY

IL-1 Cvtokine Receptor Family

I L - l a and IL-113 are polypeptides which have a number of biological

functions which include immunoregulatory, proinflammatory, and hematopoietic

activities. These actions are mediated by one of two IL-1 receptors, type I IL-1 or

type I I IL-1 receptors. The IL-1 receptors are structurally distirict and belong to a

separate superfamily characterized by the presence of immunoglobulin binding

domains. The 1L-1 receptor bears close amino acid homology with other receptors

containing immunoglobulin domains, including CD8 chain II, human IgGyl peptide,

and the T cell receptor C-p peptide, and PDGF, CSF-1, and steel factor receptors

(168). The larger type I IL-I receptor is present on T cells and fibroblasts while the

smaller type I1 IL-I receptor is present on B cells, monocytes, neutrophils, and bone

marrow cells (169). The type I IL-1 receptor has been reported to be rapidly

internalized upon IL-1 binding and receptor-IL-1 complexes have been shown to be

localized in the nucleus of IL- I receptor expressing cells This localization more

than likely is associated with the biological actions of IL- I . Interestingly, the type

II IL-I receptor binds IL-I p with high affinity but IL-I p binding does not initiate IL-1

receptor associated intracellular signal transduction as i t does upon binding to the

type I IL-I receptor. The type II receptor has been shown to be shed from cells and

it has been suggested that soluble receptor may act as a physiological IL-1p

antagonist.

The signal transduction pathway(s) associated with the IL-I receptors is

currently unclear. Production of IL-I associated biological responses does not

appear to require receptor subunits other than the cytokine binding subunit. The

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type I IL-1 receptor has been reported to contain considerable amino acid sequence

homology to the IL-2RP and gp130 subunits both of which mediate signal

transduction of other hematopoietic cytokine receptors. It is possible that the IL-I

and hematopoietic cytokine receptors activate similar signal transduction molecules

but this remains to be supported by experimental evicence. It has been reported

that IL-1 receptor activates the important transcription factor NF-KB (1 70) and may

also activate other transcription factors by a mechanism involving the MAP kinase

serine/threonine kinase cascade (1 71 ).

A secreted IL-I receptor, referred to as the IL-1 antagonist protein (IL-IAP),

has been cloned and sequenced (172). Although this protein binds IL-I with high

affinity, it does not activate the cellular signal transduction machinery activated by

membrane associated IL-1 receptors. Three dimensiorial structural analyses have

indicated that the IL-I antagonist protein and IL-lP are $tructurally similar. The IL-I

antagonist protein has been shown to play a physiolocical role in suppressing the

biological actions of IL-I.

Class I Cytokine ReceDtor Familv- Structural Domains 0. Receptor Bindina Subunits

The large hematopoietic cytokine receptor superfamily consists of EPO, IL-

2, IL-3, IL-4, IL-5, IL6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, EPO, PRL, GH, G-CSF,

and GM-CSF, LIF, CNTF, and thrombopoietin receptors In general, these receptors

mediate hematopoietic cytokine induced growth and differentiation of hematopoietic

cells. While it was once thought that each cytokine exerted a specific effect on its

particular target cell, this is not the case, since most cytskines exhibit a wide range

of biological effects on various tissues and cells.

The receptor binding subunits for hematopoie:ic cytokine receptors have

been characterized at the molecular level and comparison of amino acid sequences

has revealed several shared structural features or regions of sequence homology

(1 73). Class I cytokine receptors are characterized by the presence of one or two

copies of a conserved domain of about 200 aminc acids, which contain two

modules of FN-Ill like motifs located in the extracelluler portion of the receptor. A

second region is characterized by a conserved cysteine motif (four conserved

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cysteines and one tryptophan residue) in the N-terminal half of this homology

region. Also contained in this homology region is a common Trp-Ser-X-Trp-Ser

sequence (where X is a nonconserved amino acid) at the C-terminal end. This

extracellular homology region forms a three-dimensional barrel-like structure in

which the Trp-Ser-X-Trp-Ser sequence contributes to the cytokine binding site.

There are also regions of shared amino acid sequence homology in the intracellular

domains of hematopoietic receptors that are referred to as homology boxes 1 and

2. Mutational studies indicate that. these regions are important for activation and

interaction with specific signal transduction molecules, such as the JAK kinases

(1 74-1 76). The physiological actions of hematopoietic cytokines are often found

to be redundant. The pleiotropic actions of these cytokines can be explained at the

moiecular level by the presence of homologous regions common to the receptor

family and the activation of common cellular signal transductiori molecules.

The Class I cytokine receptor family has been subdivided into four receptor

subfamilies based on common mechanisms of signal transduction resulting from

cytokine binding to the receptor binding subunit. The EPO, G-CSF, PRL and GH

receptors comprise the GH receptor subfamily in which cytokine binding to a single

receptor binding subunit promotes the formation of a functional high affinity receptor

dimer. Receptor homodimerization appears to involve conserved extracellular

domains containing highly conserved cysteine residues (1 76) . The other three

subfamilies of hematopoietic receptors do not form dimers upoln cytokine binding.

Agonist binding to structurally unique cytokine binding subunits for each of

the members in these families of hematopoietic cytokine receptors results in the

formation of a high affinity complex with a shared signal transducing subunit. For

example, within the IL-2R subfamily, the structurally unique binding subunits for

IL-2, IL-4, IL-7, IL-9, IL-13 and IL-15 receptors all form a high affinity functional

complex with a common signal transduction protein referred to as the IL-2y subunit

(177-182). The IL-2 receptor, unlike other receptors in this IL-2y, family also

associates with a third protein subunit, IL-2Ro. In this case, the high affinity IL-2 receptor exists as a heterotrimeric complex. Similarly, the structurally distinct IL-3,

IL-5, and GM-CSF receptor binding subunits (IL-3 subfamily) form a high affinity

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functional receptor complex with a shared signal transducing protein, referred to as

KH97 (1 83).

The IL-6 receptor subfamily of Class I cytokine receptors includes the IL-6,

IL-I 1, CNTF, OSM, and LIF receptors. These all fo-m a high affinity functional

receptor complex upon interaction of cytbkine occupied binding subunit with a

common signal transduction protein called gpl30 (1 70,184). In the case of the IL-6 receptor, IL-6 binding to its binding subunit leads to association with a homodimer

of gp130 instead of a single gp130 monomer (170). Recently, a human IL-12

binding receptor component has been cloned and fcund to be highly related in

primary structure to gpl30. The observation that cytotines within these different

cytokine receptor families produce similar physiological actions can now be explained at the molecular level by the observation that they share similar signal

transduction subunits which in turn activate similar signal transduction pathways.

An important area of future research concerning henatopoietic receptors is to

determine the structural domains important for cytokine binding and the interaction

of cytokine binding subunits with signal transduction subunits.

The naturally-occurring existence of soluble, tiuncated forms of a number

of hematopoietic cytokine receptors have been -eported. Lacking signal

transduction functions, these cytokine binding pro .eins arise as a result of

alternative splicing of the mRNA for the complete receptor sequence or as a result

of proteolytic cleavage and release of the membrane-bound form of the receptor.

Although the in vivo functions of these soluble, truncated "receptors" are not clearly

understood, they may act as physiological antagonists of endogenous cytokines.

This antagonism may occur because scavenging of the ligand through binding to

its soluble receptor would reduce the effective free ccncentration available to the

membrane-bound receptors. Production of such solub e receptors may render the

target cells less sensitive to the activity of their ligands.

Common Features of Class I Cytokine Receptor Sianal Transduction

None of the hematopoietic receptors or their associated signal transduction

subunits have intrinsic tyrosine kinase activity. Howe\!er, the earliest biochemical

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response associated with cytokine binding is a rapid tyrosine phosphorylation of

specific intracellular proteins Recent evidence now suggests that hematopoietic

receptors themselves (EPO and G-CSF receptors) or signal transduction proteins

(KH97, IL-2y, and gpl30) associate with and activate a specific family of tyrosine

kinases referred to as the JAK family of tyrosine kinases (185). Currently, four

members of the JAK kinase family have been identified which include TYK2, JAKI,

JAK2, and JAK3. Members of the IL2-y cytokine receptor family have been reportedl

to activate JAKI and JAK3 whereas members of the KH97 and growth factor

families have been reported to selectively activate JAW kinase. Most members of

the gp130 have been reported to activate both JAKI and JAK2, and some have

been reported to activate TYK2 (1 86). The intracelluiar homology box 1 of the €PO receptor has been reported to be the receptor domain that is critical for JAK kinase

association and activation (174,186,187). The same homology box 1 of other

hematopioetic receptors is also anticipated to be important for JAK association and

activation. The next step in signal transduction appears to be JAK kinase mediated

phosphorylation of certain latent or inactive SH2 and SH3 domain-containing

cytoplasmic transcription factors referred to as STAT proteins. Phosphorylation of

these transcription factors allows them to transverse the nuclear membrane and

participate in the regulation of transcription of certain genes associated with

hematopoietic receptor activation (1 85, 186). Transcription factors specifically

activated by EPO, IL-3 (1 88), and lL-6 receptors (1 89-1 93) have been identified and

characterized. Other tyrosine kinases may also be involved in hematopoietic

receptor signal transduction. A complex between the IL-2RP subunit and certain

members of the src family of tyrosine kinases has been reported but the

physiological importance of this association remains to be elucidated (1 94-1 96).

Evidence suggests that some hematopoietic receptors may also activate the Ras-

dependent MAP kinase pathway leading to activation of other transcription factors

such as cFOS and cJUN (170).

Class II Cvtokine Receptor (Interferon) Family

The interferons (IFN) are responsible for a diverse range of biological

actions. The type I (IFN-a and IFN-P) and type II (IFN-y) IFNs control induction of

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738 HERZ, THOMSEN, A N D YARBROUGH

a number of early response genes associated with tissus repair, inflammation, and

host defense (viral infection). Interferons bind to type II cytokine receptors. Both

IFN-a and IFN-P bind to the same IFN receptor (197) whereas IFNy binds to a

distinct receptor (198). It has been proposed that ligancl binding to the Type I1 IFN

receptor induces dimerization of the receptor and activation of receptor associated

JAK kinases (199). Function of the IFN type I I receptor also requires an accessory

protein that has been recently cloned and denoted as the @-chain (200) or AF-1

(201 ).

The signal transduction pathway associated with the activation of interferon

receptors has been more extensively determined than fcr other cytokine receptors.

Based on studies of mutant cell lines, it appears tha. both interferon receptors

activate specific sets of STAT proteins by a mechanism that appears to involve JAK

tyrosine kinases (202-205). Upon phosphorylation, the STAT proteins enter the cell

nucleus and activate specific IFN promoters.

Tumor Necrosis Factor (TNF) Receptor Family

TNF is a cytokine mainly produced by activated mscrophages that has many

biological act ions including cyt otoxici ty , ant i-vira I activity , immunoreg u la t ory

activities, and transcriptional regulation of several genes that are mediated by

specific TNF receptors (206-208). Originally, two different receptors termed TNF-

R1 and TNF-R2 were cloned and characterized (209,210). Currently, 12 different

TNF-related receptors have been identified (TNFR-1, --NFR-2, TNFR-RP, CD27, CD30, CD40, NGF receptor, PV-T2, PV-A53R, 4-1 BB, OX-40, and Fas) with which

eight different TNF-related cytokines associate (21 1,2' 2). All of these receptors

(except PV-T2 and PV-A53R) are also produced as soluble receptors.

Receptors in this family are single transmembrane proteins with considerable

homology in their extracellular domains whereas their relatively short intracellular

domains bear very little sequence homology. Unlike ott-er growth factor receptors

which form dimers, ligand binding to TNF receptors incuces formation of a trimer

of receptor molecules However, other observations indicate that more complex

oligomers between different receptor subunits may also form in the plasma

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membrane. Most of the ligands for these receptors, including TNF itself, are

retained on the cell membrane and cell-cell contact is a primary means of ligand-

receptor interaction (21 2). The signal transduction pathway(s) employed by the

family of TNF receptors is currently unknown. As with other cytokine families,

ligands to these receptors induce pleiotropic biological responses including

differentiation, proliferation, and cell death. It is interesting to note that the

intracellular domains of these receptors show very little homology suggesting that

they may activate diverse signal transduction pathways. Since cell-cell contact is

the vehicle for ligand-receptor interaction, bipolar signaling may occur.

INTRACELLULAR HORMONE RECEPTOR SUPERFAMILY


Unlike most integral membrane receptors, the intracellular hormone

receptors are located either in the cytoplasm or in the nucleus of the responsive

cell. The hormones and vitamins which bind to these receptors are small lipophilic

compounds which are distributed throughout the body by the hemo-lymphatic

system, freely crossing the cell membrane of the responsive cell. The superfamily

of intracellular receptors mediate the effects of the steroid hormones (reviewed in

21 3,214). The superfamily includes the receptors for cortisol and corticosterone

(glucocorticoid receptor), testosterone (androgen receptor), aldosterone

(metallocorticoid receptor), progesterone (progesterone receptor), estrogen

(estrogen receptor), thyroxin (thyroxin receptor), vitamin D (vitamin D receptor),

and retinoic acid (retinoic acid receptors, RAR). In addition to the receptors for the

known steroids, thyroid hormone, and vitamins A and D, more than three dozen

additional members of the intracellular hormone receptor farnily sharing similar

structure have been identified by molecular cloning. The ligands for these orphan

receptors are at present unknown, however their structure is most closely related

to the thyroid hormone and vitamin receptors. Three of the receptors originally

classified as orphan receptors were subsequently shown to be activated by binding

of 9-cis retinoic acid, and hence became known as RARs (215,216).

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Steroids, and their receptors, are important in a wlde range of diseases. The

role of corticosteroids in Cushing's syndrome and Addison's disease is well known,

and the anti-inflammatory effects of corticosteroics have long been used

therapeutically. Reproductive steroids, which include estrogen, progesterone, and

testosterone, may function in several hormone dependent cancers and

osteoporosis. Estrogen and androgen antagonists are currently used to treat breast

and prostate cancer, respectively. Estrogens, vitamin [I, and thyroid hormone are

used in replacement therapies, and estrogens and progestins are used as

contraceptives.

In addition to the endogenous ligands, several competitive inhibitors of the

reproductive steroids have been developed recently, including inhibitors for

glucocorticoid, progesterone, androgen, estrogen, and metallocorticoid receptors.

Most of the receptor antagonists appear to act by binding competitively to the

receptor, inducing a conformational change leading to dissociation of the receptor

from an intracellular complex with heat shock proteins (2.1 7). Apparently, this event

still allows the receptor to dimerize and associate with DfJA, but the inhibitor-bound

receptor fails to associate with the transcriptional complelc to modulate transcription

of target genes. One antagonist, an anti-progestin ZK98299, binds to the

progesterone receptor but does not promote binding of .he receptor to DNA (21 8).

As shown in Figure 10, members of the intra;ellular hormone receptor

superfamily share in common a three domain structure and range in overall length

from approximately 400 to 1000 amino acids (214). The amino-terminal domain

functions in transcriptional activation (transactivation) lither by direct interaction

with the transcriptional machinery or indirectly through bridging proteins termed co-

activators and co-repressors. This domain is highly i.ariable in length between

family members, ranging from 27 amino acids (vitamin D receptor) to 600 amino

acids (metallocorticoid receptor). Across the intracellular receptor superfamily,

there is little protein sequence similarity within this region ('1 5%). However,

between receptors belonging to the same class, i.e. metallocorticoid, glucocorticoid,

progesterone, and androgen receptors, greater than 50% sequence identity is

observed in this region. The centrally located DNA binding domain is the most

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NH2 Modulator ~ DNA ~ L i g a n ] COOH

- DNA Binding

4 + Ligand Binding

t HSP Complex Binding - - - Transcriptional Activation

c-----* Gene Specifier

U - Dimerization

c,* Nuclear Translocation

Figure 10. Multiple functional domain structure of members of the intracellular

hormone receptor superfamily. Members of the superfamily have a conserved

domain structure that includes DNA-binding, ligand-binding and one or more

transcriptional activation domains. The approximate locations of regions involved

in specific functions of the receptors are depicted below the receptor structure.

highly conserved region, generally between 66 or 68 amino acids in iength with

greater than 40% sequence identity across the family. The DNA binding domain

is responsible for recognition of the specific steroid response elements located

upstream of target genes, and contains conserved cysteine residues which are

believed to form two zinc fingers that are involved in DNA recognition and binding.

This domain also contains sequences involved in nuclear localization. Though

similar in length between receptors, the carboxyl-terminal domain is highly variable

in sequence, with 15 to 57% sequence identity across the family. Ligand binding

occurs within this domain and probably involves ligand contact with several amino

acid residues over a broad region of the molecule. In addition, the carboxyl-

terminal domain is important for transactivation, hetero-homodimerization and is implicated in the binding of a variety of heat shock proteins to the inactive receptor.

Based on certain structural and functional properties, the superfamily can

be divided into two broad subgroups, type I and type II receptors. Type I receptors

(steroid receptors) are localized in the cytoplasm and nucleus. In the absence of

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ligand, the glucocorticoid, metallocorticoid, progesterone, androgen, and estrogen

receptors are found in large macromolecular complexes with the 90 kDa heat shock

protein (hsp90), hsp70, hsp56, and possibly other proteins (21 9). Upon hormone

binding, a conformational change in the receptors occu's and the macromolecular

complex dissociates, releasing monomeric receptor subunits (220). Released

monomeric subunits form homodimers which bind cooperatively to two

palindromically arranged hexanucleotide DNA target seq Aences known as hormone

response element half-sites (221). In contrast, type II receptors such as thyroxin

receptor, vitamin D receptor, retinoic acid receptor, and most orphan receptors are

exclusively localized in the nucleus, can bind DNA also in the absence of ligand,

and bind preferentially as heterodimers with other meribers of the superfamily to

direct or inverted repeat half-sites of hormone responss elements (222).

Target gene specificity is achieved by the orchestration of three major1

processes. First, tissue-specific expression of receptors 2nd co-factors imposes one

level of restriction. Secondly, although many hormones re freely available through

the circulation, some cells are able to regulate the lccal levels of the hormone

through the tissue-specific expression of biosynthetic or degradative enzymes. An

example of the latter is mineralcorticoid action regulation. Thirdly, the precise

composition of the DNA target sequence including sequmces of the half-sites and

the intervening spaces is likely to be a critical factor. Each responsive element is

named for the receptor which binds to it, for example the response element for the

glucocorticoid receptor is known as the glucocorticoid response element (GRE).

However, the steroid response elements have a high degree of sequence similarity

and may bind several distinct activated receptors. For example, a glucocorticoid

response element may also act as a metallocorticoid-, progesterone- and androgen-

response element (224). Steroid response elements are found in both orientations

and at different positions relative to the transcription iniiiation site of target genes,

consistent with their function as transcriptional enhancer sequences. Binding of the

activated steroid receptor to the response element alter:; the transcriptional rate of

downstream genes, either positively or negatively, depending on the context of the

steroid response element.

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It has recently been shown that transcriptional activity mediated by several

of the receptors can be modulated by ligands binding to membrane bound receptors

as well as the levels of nutrients and intermediary metabolites. For example, most

of the nuclear receptors are transcriptionally activated by dopamine (21 3).

Furthermore, a number of growth factors have been shown to modulate the activity

of estrogen receptors. The precise mechanism of transcriptional activation via

these alternative pathways is not yet understood. It is known that steroid receptors

are phosphoproteins, and phosphorylation of the receptors either by ligand induced

events or by alternative pathways may modulate receptor function (225). Because

nuclear receptors can be modulated by other receptor-mediated events, these

receptors may be of importance in signaling by membrane-bound receptors.

Glucocorticoid Receptor

Glucocorticoids are final effectors of the stress response and exert a

negative feedback at multiple levels of the hypothalamic-pituitary-adrenal axis.

RNA encoding receptors for the glucocorticoids are expressed at detectable levels

in virtually all tissues studied including adrenal, kidney, liver, spleen, heart, brain,

and testes (226). Like other intracellular hormone receptors, glucocorticoid

receptors transduce hormone signals directly. Following activation by hormone

binding, activated glucocorticoid receptors bind directly to specific enhancer

sequences in the genome (glucocorticoid response elements) and activate or repress the transcription rates of target genes. These receptors regulate a number

of central nervous system and peripheral target genes, therefore the effects of

glucocorticoids are numerous and widespread. Important actions of glucocorticoids

include effects on carbohydrate metabolism, protein metabolism, lipid metabolism

and distribution, and effects on a wide range of blood cells In this regard, it is the

potent anti-inflammatory properties of glucocorticoids that have made them such

usefu I therapeutic agents .

In cytosolic extracts, glucocorticoid receptors exist in complexes with hsp90,

hsp70, and hsp56, and a number of other low molecular weight proteins (227,228).

The binding of hsp90 to glucocorticoid receptors is hormone-regulated and

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mediated via the hormone-binding domain of the recep:or (229) and is necessary

for the receptor to convert to the high affinity ligaid binding conformation.

Association of the receptor with hsp90 blocks receptor DNA binding activity. Like

hsp90, hsp70 binds to the hormone binding domain of glucocorticoid receptors and

may be required for hsp90 binding to the receptor (230). lSp56 is an iinmunophillin

of the FK506 binding protein class and binds to the glucocorticoid receptor complex

via hsp90 (231).

A current model for the role of hsp90, hsp70, and lisp56 (228) suggests that

following formation of the heterocomplex, hsp70 mediates unfolding of the receptor

to a state stabilized by hsp90. In the unfolded state, the receptor is in the high

affinity glucocorticoid-binding conformation. It is further suggested that hsp70 then

dissociates from the complex, leaving hsp90 and the glucocorticoid receptor in the

high affinity state. The association of the receptor wiih hsp90 is necessary for

transport of the receptor from the cytoplasm to the nucleus. When hormone binds

to the receptor, the association of the glucocorticoid remptor with hsp90 converts

to a weaker interaction, and the receptor is released to interact with target DNA

sequences.

Human, mouse, and rat cDNAs encoding glucocorticoid receptors of 777,

783, and 795 amino acids have been cloned and expressed (232-234). These

receptors have a three-domain structure like other members of the intracellular

hormone receptor superfamily. A region of the N-term nal domain of the human

glucocorticoid receptor between amino acids 77 and 262 known as ~1 is required

for full transcriptional activation by human glucocorticoi receptor and overlaps a

highly basic region of the receptor which is found betwsen amino acids 187 and

285 (235). Disruption of the structure of the N-terminal domain of human

glucocorticoid receptor by insertion of three or four amiio acids at positions 120,

204, or 214 inhibits transcriptional activity, but duplication of the full TI domain

increases transcriptional activity two- to four-fold. It is bE lieved that the N-terminal

domain may be important in formation of the g1ucocortic:oid receptor dimer and in

interactions of the glucocorticoid receptor with specific: subsets of transcription

factors (236).

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The DNA-binding domain determines the specificity of transcriptional

activation by glucocorticoid receptors and contains nine cysteines which are

conserved in all steroid receptors. The solution structure of this domain determined

by two-dimensional nuclear magnetic resonance reveals two zinc fingers formed by

tetrahedral coordination of two zinc atoms by four pairs of cysteines (237). a-

helices are located immediately adjacent to the distal side of each zinc finger. The

importance of this domain for determining specificity of interaction with the

response element was demonstrated by substitution of the glucocorticoid receptor

DNA-binding domain for the estrogen receptor DNA-binding domain in the estrogen

receptor which created a chimeric receptor that activates transcription through

glucocorticoid response elements rather than via estrogen response elements.

Mutational analysis has identified some of the specific residues which determine

specificity of DNA binding. For example, converting the gly-ser between the distal

cys-cys pair in the first zinc finger of glucocorticoid receptor to glu-gly (as found in

estrogen receptor) results in a receptor which activates transcription at estrogen

response elements (238-240). Whereas the first zinc-finger is important for DNA-

binding specificity, the second zinc-finger is also required for DNA binding, since

deletions, insertions, and point mutations in this region give rise to inactive

receptors (233, 238, 239, 241). Substitution of the second zinc-finger with the

estrogen receptor second zinc-finger yields a receptor which still binds

glucocorticoid response elements (228).

The DNA sequence recognized by the giucocorticclid receptor, is a

palindrome with a consensus sequence of GGTACAnnnTGTTCT. In the current

model of glucocorticoid receptor binding to DNA (237), one glucocorticoid receptor

binds to each half of the glucocorticoid response element palindirome. The a-helix

distal to the first zinc finger of each receptor binds to the major groove of the

glucocorticoid response element and the two halves of the receptor dimer interact

through the first pair of cysteines in the second zinc finger and the amino-acids of

the second finger involved in protein-protein interactions face away from the DNA

to allow interaction with transcriptional factors.

Immediately distal to the DNA-binding domain of glucocorticoid receptor is

the COOH-domain of the receptor which contains a highly basic region which

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appears to be involved in hormone-dependent nuclear localization of the receptor

after synthesis (242). An additional hormone-dependent nuclear localization

sequence has been identified in the COOH-domain of gI xocorticoid receptor (242).

Deletion analysis, linker scanning mutagenesis, and isolation of C-terminal

truncation mutants has demonstrated that nearly the entire COOH-domain is required to form the glucocorticoid binding site (232, 241, 242-244). The

involvement of this region in hormone binding has 3een confirmed by affinity

labeling in several studies.

In addition to hormone binding, the COOH-domain contains a second

transcriptional activation domain known as ~2 (235, 245) Both TI and ~2 are acidic

regions which may function similarly to the yeast transcription factors GAL4 and

GCN4 which depend on negatively charged residues on the surface of the DNA-

bound protein (235). The glucocorticoid receptor contains several sites which

become phosphorylated in metabolic labeling experiments (246), with most of the

phosphorylation occurring in the N-terminal domain. Receptor activation by

glucocorticoid treatment of cells results in increased receptor phosphorylation.

However, the process of receptor activation does not depend on phosphorylation

or dephosphorylation of the receptor, and the role of receptor phosphorylation

remains unknown.

FUTURE DIRECTIONS IN RECEPTOR RESEARCH FOR DRUG OiSCOVERY

Functional Roles for a Multiplicity of Receptor Sibtypes

Over the past several years, the approach of mc lecular cloning has greatly

expanded our knowledge of many receptor subtypes and has revealed an

impressive heterogeneity of receptors not previously en Jisioned. The discovery of

multiple receptor subtypes that are highly homologous has provoked examination

of several key questions related to their physiological functional significance and

importance as individual drug discovery targets. Foremost among these is what

physiological role and advantage is conferred to the xganism by expression of

multiple receptor subtypes that have "apparently" identical endogenous ligand

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binding and signal transduction properties? To answer the above query, studies

must ascertain whether any group of related receptor subtypes or isoforms differ in

their functional properties in a manner that has important physiological

consequences. Despite indistinguishable binding and functional coupling

properties, closely related receptor subtypes and isoforms may exhibit differences

in properties such as the time course of activation, ionic selectivity, desensitization,

phosphorylation by kinases or efficacy in coupling to second messenger systems,

as well as displaying differential regulation of expression in particular cell types and

tissues.

Small differences in receptor structure (one to several amino acids) have

been found to endow important pharmacological and functional differences to

receptor subtypes and species homologues. Within the GPCR superfamily, for

example, a change in a single amino acid residue was found to underlie the major

pharmacological differences between the rat 5-HT1 B and the 5-HT,,, receptors

(247). Essential differences in coupling of splice variants of the dopamine D2

receptor, mGlu-I receptor, somatostatin-2 receptor, and prostaglandin EP3 receptor

have been observed. Within the ligand-gated ion channel superfamily, it has been

found that the charge and size of a single amino acid residue in a critical channel

site (the Q/R site of the GluR-B subunit) determines the particular ionic selectivity

and permeability properties that characterize subtypes of AMPA-kainate-activated

channels (248). For the muscle subtype of the nicotinic acetylcholine receptor, the

functional properties of the receptor (channel conductance and open time) are

altered during development by substitution of one of the subunits in the pentameric

complex; from (al),f3ly6 in embryonic muscle to ( ~ ( 1 ) ~ p I €6 in the adult muscle.

Cellular and tissue expression patterns for related receptor subtypes may

overlap, but are often highly specific and tightly regulated. For example, there are

striking differences in the cellular distribution of CXCRI (IL-8R1) and CXCR2 (IL- 8R2) receptors (77% sequence identity overall). CXCRl expression is restricted

to neutrophils, monocytes and a few myeloid cell lines, whereas CXCR2 is widely

distributed in myeloid cell lines, but also in lymphocytes, melanoma cells,

melanocytes and fibroblasts (249). These specific cellular distributions suggest that

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748 HERZ, TlIOMSEN, AND YARBROUGH

the main function of the CXCRI receptor is neutrophil activation and mobilization

of phagocytes in host defense while the CXCRZ receptor serves to mediate

migration and growth of cells not involved in defense. ' n situ hybridization studies

of mRNAs encoding subunits of the neuronal nicotinic acetylcholine receptor gene

family ( LGCRs) and subtypes of GPCRs have documented that transcripts

corresponding to homologous receptor subunits and subtypes within a receptor

family exhibit distinct anatomical distributions in the mammalian brain (250).

Among the family of human dopamine receptor sulitypes, D2 is most highly

expressed in the striatum, D3 and D5 receptors exhibit a preferential localization

in the limbic area of the brain and low expression in motor areas such as the basal

ganglia, while D1 and D4 receptors are both abundant in the cerebral cortex.

Thus, the operative paradigm is that functional diversification and heterogeneity in

expression patterns for receptor subtypes allows each subtype and isoform to

perform a unique physiological role and the associated corollary is that orphan

receptors must have a function.

The process of defining receptor subtypes associated with cloned sequences

and their associated physiological and pharmacological properties remains a major

challenge in the field, particularly for the ligand-gated i o i channel superfamily. As

described above, the molecular diversity of subunits {hat comprise the nicotinic

acetylcholine, GABA,, and glutamate LGCRs familie; allows for a plethora of

subtypes composed of unique heteropentameric subunit combinations. Many of the

subunits do not in themselves directly bind ligands, but nevertheless dictate binding

properties for the receptor complex. Expression of distinc:t combinations of subunits

will be required to understand the individual roles each subunit plays in creating

pharmacologically distinct receptor subtypes when expressed in heterologous

cells. Such expression studies have shown that reconbinant GABA, receptors

composed of different a-subunit isoforms in combinat on with invariant p and y

subunits display distinct benzodiazepine pharmacolociy, GABA-benzodiazepine

interactions and steroid modulation (25). The existence of multiple receptor

subtypes with unique pharmacological characteristics and differential

subanatomical localization provides the rationale for their use as molecular targets

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for the development of novel subtype-selective drugs that are targeted to specific

tissues and are thus devoid of the side effects associated with existing non-

selective drugs.

Cloned Human Receptors as Druq Discovery Tarqets

An important application of recombinant DNA technology has been the

capability to provide human, cloned receptor subtypes as expressed functional

proteins for drug discovery efforts. Prior to the availability of the cloned receptor

targets, screening programs relied upon utilization of receptors obtained from

animal tissue homogenates which had the inherent disadvantages of receptor

heterogeneity, nonhuman pharmacology, and low receptor expression levels. The

development and utilization of cell lines stably expressing high levels of a single

human receptor subtype for drug screening allows for a more accurate and

selective pharmacological screening method compared to conventional methods,

and has enabled the discovery of highly selective drugs which discriminate between

various receptor subtypes. Furthermore, in cell lines that express a recombinant

receptor linked to a second messenger or other downstream biochlemical response,

the functional consequences of receptor occupancy can be detected. Thus, not

only can drug binding affinity of novel lead compounds be quantitatively defined,

but the use of such recombinant cell lines permits rapid characterization of agonist

or antagonist activity, and measurements of compound efficacy at the cellular level.

Many human recombinant receptor subtypes expressed in a wide variety of human and other mammalian host cell backgrounds (e.g., CHO, HeLa, L-cells) have

been found to exhibit ligand binding properties indistinguishable from their non-

recombinant "native" receptor counterpart. However, the use of recombinant

receptor systems for receptor classification and pharmacological characterization

can generate misleading data if heterologous host cell-specific: properties have

been imposed on the receptor. Ligand binding and signal transduction properties

of many GPCRs have been found to depend upon the host cell as well as the

expression level of the receptor, and thus agonist affinities and coupling

mechanisms determined in different recombinant expression systems may not be

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identical. In the case of the expression of the humzn 5HT1, in HeLa cell lines,

certain compounds acted as agonists in a cell line with the greater number of

receptors but acted as pure antagonists in a cell line with only a 6-fold lower density

of receptors. Similarly, data for other receptors has been obtained showing the

dependence of agonist efficacy and pharmacological pnperties on receptor density.

Insight into the complexities of analyzing the responses of recombinant

receptors in heterologous cells is provided by studies of the signal transduction

properties resulting from the activation of the D2 dopamine receptor stably

expressed in two different cell lines. Activation of D, rxeptors in either GH,C, rat

pituitary cells or mouse Ltk- fibroblasts produced in iibition of adenylyl cyclase

activity (1 16). However, while D, receptor activation cwsed a rapid stimulation of

PI hydrolysis and an increase in intracellular calcium iri Ltk- cells, it failed to effect

PI hydrolysis and induced a decrease in intracellular cilcium in GH,C, cells (1 16).

Similarly, activation of the 5HTl, receptor expressed in Ltk- cells was coupled to

an increase in intracellular calcium, but resulted in a decrease in calcium influx

when expressed in GH,C, cells. Since the cell-specific differences in the signaling

pathways for the D, and 5HT,, receptors were the ssme, these effects may most

likely be attributed to the complement of G-proteins present in each host cell type.

Thus, the effector systems and responses of a transfect ?d receptor depend not only

on the receptor, but upon the particular cell type in which it is expressed.

The high level of membrane expression for recombinant GPCRs in insect

cells (1-40 pmol/mg in Sf9 or Sf21 cells) using the baculovirus system has

demonstrated appropriate receptor pharmacology for antagonist ligands (affinities

and rank order potencies), whereas anomalous agonist binding properties and lack

of functional coupling has been observed for some receptors. The GPCRs

expressed using baculovirus typically show only a sins le, low affinity binding state

for agonists, whereas the same receptors demonstr2te high (coupled) and low

affinity (uncoupled) agonist binding states in mamrialian cells. Similarly, the

pharmacological properties of GPCRs expressed in yeast (M, muscarinic and D, dopamine receptors) were not comparable to mammalian cells since only a single

low affinity agonist binding state was detected, indicating that the recombinant

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receptors do not to couple to the endogenous G-proteins present in these cells

(251,252). Coexpression of the recombinant receptor and the appropriate G-

protein (if known) may provide an approach to overcome such limitations. Toward

this end, it was recently found that heterologous coexpression of the G protein a-

subunit G,, with a variety of GPCRs in mammalian cells enabled coupling to PLCP

activity. In yeast, functional coupling was only achieved using a mutant strain

lacking one yeast G-protein a-subunit and including a cDNA encoding an

appropriate G-protein (252).

The integration of molecular biological approaches into receptor

pharmacology has been useful in elucidating receptor structure-function

relationships. Construction of chimeric receptors (hybrid polypeptides composed

of adjacent portions of two related receptor subtypes) has helped to identify

functional domains of many individual receptors. Such studies have successfully

i dent if i ed I i g a nd b i nd i ng d om a i n s and c y t o p I as n i i c do m a i ns med i a t i ng si g na I

transduction of many receptors Using in vitro niutagenesis to change single amino

acids to create point-mutated receptors has defined critical residues involved in

receptor-ligand interactions, conformational transitions, ion channel selectivity,

signal transduction, and sites of receptor phosphorylation. With limited direct

structural information available, this approach often requiTes making the assumption

that overall receptor conformation and the equilibrium distribution between receptor

states (resting, active and desensitized) has not been altered in the mutant

receptor. Nevertheless, this experimental approach has been the basis for a large

body of molecular modeling aimed at understanding the specific functional groups

on receptors that interact with drug ligands. The combination of such molecular

pharmacological and modeling approaches can be expected to reveal the structural

determinants of drug binding and novel allosteric sites on the receptor protein for

drug interaction. Ultimately, the ability to produce large amounts of purified

receptor protein from recombinant expression systems will enable the use of

physical methods (NMR, electron microscopy, X-ray crystallography) to obtain high

resolution receptor structural data that will delineate ligand binding sites and

precisely define drug-receptor interactions

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Druq Discoverv Using Adopted Orphan Receptors

There are now numerous examples of orphan -eceptors which have been

identified as novel types or subtypes of existing receptor families for which

functional roles have been characterized as proven therapeutic drug targets. Many

of these orphan receptors are of immediate interest as potential drug discovery

targets since they represent novel receptor subtypes tt-at extend existing receptor

families which have members which are therapeutica ly important drug receptor

targets. The close structural relatedness that is the molecular basis for organization

of the receptor superfamilies has allowed a rational-based search for new members

of the ligand-gated ion channel, G-protein coupled, receptor tyrosine-kinase, and

intracellular receptor superfamilies. In general, discclvery of new receptors (or

subunits) has been accomplished by screening cDNA libraries using sequences

conserved among the receptor family at low stringency (homology screening). An

alternative method has been to employ PCR amplification of human genomic DNA

with degenerate oligonucleotides encoding conserved receptor domains.

Libert and coworkers were the first to exploit t i e PCR methodology and

clone several novel orphan receptors belonging to the (;-protein coupled receptor

superfamily (253). These orphan receptors were subsequently shown to include

the adenosine A, and receptors (254,255), a 5-HT,, receptor (257,258), and a

central cannabinoid receptor (CB,). PCR using degenerate primers was

subsequently used to identify cDNAs encoding the NK-1, NK-2, dopamine D, and

D, (259), histamine H,, adenosine A, (260), olfactory receptors, and numerous

other G-protein coupled receptors. The application of thsse approaches has been

highly successful and has led to the cloning of a series of orphan receptors (or

novel subunits) in each superfamily for which functions hzve not yet been assigned.

Within the last two years, more than 100 publications have described orphan

receptors. To date, more than 40 additional members of the intracellular receptor

superfamily have been cloned for which ligands habe not been identified. In

addition, the ligands for the many orphan members of them receptor-protein tyrosine

phosphatase superfamily have also not yet been established. Numerous orphan

receptor-tyrosine kinases belonging to the EPH family have been identified which

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likely function by transducing signals initiated by direct cell-cell interaction (261).

Many of the EPH receptors are specifically expressed in the nervous system (262) ,

and these receptors have been implicated in the control of axon guidance, in

regulating cell migration, and in defining compartments in the developing embryo.

The identification of gene sequences is only the beginning of the process for

the development of the useful small molecule therapeutic agent Analysis of a

stretch of only 10-25 amino acids from an orphan receptor sequence may be

sufficient to identify whether the orphan of interest is homologous to either a known

protein or a recognizable motif that corresponds to a ligand recogriition or functional

domain characteristic of a receptor scrperfamily Based upon the level of observed

sequence homology with known receptors, it may be possible to either deduce the

class of ligand bound by the orphan receptor and/or postulate a mechanism of

signal transduction. The systematic search for natural cognate ligands of orphan

receptors is more difficult when the properties of such receptors are not well

predicted by sequence comparisons to known receptors and/or the orphan

receptors exhibit novel pharmacology.

As an example of the first strategy, the process leading to the

characterization of the orphan ORLI (Opioid Receptor-Like I ) receptor, which has

become known as the orphanin or nociceptin receptor (263-265), i s of interest since

it represents one model that can be readiiy adapted to other orphian receptors. The

ORLl receptor is a novel G-protein coupled receptor which is most closely related

to the opioid receptors. Based upon substantial sequence identity of the ORLI

receptor with opioid receptors (50% overall, 65% within transmernbrane domains),

it was reasonable to hypothesize that the related orphan receptor would share

signal transduction properties in common with the p, 6, and I< opioid receptor

subtypes. Since the opioid receptors are all negatively coupled to adenylyl cyclase

(Table II), a stable recombinant CHO cell line expressing ORLl was constructed for

use in a functional screen using crntransfected CHO cells as a (control. A survey

of opiate ligands identified etorphine, a nonselective opiate agonist, as mediating

inhibition of forskolin-induced accumulation of CAMP, although its potency was

found to be about three orders of magnitude less compared to other opioid

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754 HERZ, 'IHOMSEN, AND YARBROUGH

receptors. However, additional efforts to characterize the pharmacology of the

ORLI receptor by analysis of agonist effects indLced by endogenous opioid

peptides; endorphins, enkephalins and dynorphin!;, or other synthetic opioid

ligands, were not successful.

Based upon the structural homology of ORI-1 with opioid receptors, in

particular, the acidic extracellular loop 2 of the K-opioid receptor, it was

hypothesized that the endogenous ligand might be a peptide that resembled

dynorphin. A biochemical fractionation procedure w3s used to isolate a pituitary

peptide whose structure was identified as a 17 amino azid neuropeptide that shares

many features in common with other opioid peptides and is now known as

nociceptin. Isolation of the endogenous ligand was a zhieved through a functional

assay on the basis of its ability to inhibit adenylyl cyclase in a stable recombinant

cell line. The identity of nociceptin was confirrred through synthesis of a

radiolabeled derivative which was found to bind in a saturable manner with high

affinity and to be a potent and specific activator of .he ORLI receptor. In vivo

activity of nociceptin induced hyperalgesia when administered

intracerebroventricularly to mice, indicating the ag mist of the ORLI receptor

appears to possess pro-nociceptive properties. The unique pharmacology,

physiology and brain distribution of this novel receptor make it an important target

for drug discovery.

For the full value of the receptor gene sequence to be realized, the function

of the expressed orphan receptor, as well as its regulat on and expression will need

to be elucidated. In many cases, exemplified by the nociceptin receptor, the orphan

receptor will be activated by an as yet unknown endDgenous ligand (transmitter,

peptide, or hormone) or the endogenous ligand may be identified as a previously

characterized molecule whose function has not been assigned. Strategies for

identification of a ligand for an orphan receptor will likely be based upon utilization

of high- and ultra-high throughput "agonist screening" assays in which ligand

binding is coupled to a cellular functional response. FLnctional assays based upon

measurements of common cellular signal transductioi pathways (ie., mobilization

of intracellular calcium, CAMP, transcription response elements) have been

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employed (266). Since many types of receptors (LGCRs, GPCRs and RTKs) mediate cellular responses through elevations in intracellular calcium, real time

measurements of intracellular calcium using fluorescent probles has provided a

generic assay for receptor activation. In addition, cell-based transcription activation

assays represent a functional screening method which requires engineering of a

cell line(s) stably expressing the recombinant orphan linked to a luminescent or

fluorescent-based reporter gene (ie., luciferase or green fluores,cent protein) under

the transcriptional control of a promoter element (ie , CAMP response element

(CRE), NFAT or STAT binding elements) (267, 268) The advantages inherent in

this one-step screening approach include high detection sensitivity and

compatibility with automation

An alternative approach may be to employ an immobilized orphan receptor

to capture the cognate ligand from biological extracts, fractions and other

combinatorial chemistry libraries Orphan receptor screening efforts will be able to

take advantage of large-scale synthetically produced random peptide,

peptidomimetic and combinatorial chemical libraries, as well as phage display

libraries which are available as novel sources of chemical and structural diversity

for discovery of potent and selective chemical entities for each of the receptors

(269). Finding new natural product-based lead compounds by screening

fermentation broths, and extracts from plant and marine organisms may yield

ligands with novel structures, as in the case of the subtype-selective endothelin

receptor pe pt i d ic ant ag on is t s ,

Among the GPCR superfamily, the discovery and characterization of the

large number of novel receptor subtypes belonging to the chemokine,

dopaminergic, serotoninergic, somatostatin and opioid receptor families provide

examples of the tremendous impact of gene cloning. Recent estimates suggest that

there are currently over one-hundred more orphan receptors in this superfamily.

As a consequence of the original cloning of the orphan receptor sequence

corresponding to the 5-HT,, receptor (dog RDC4 gene) in 1989 (253), the human

receptor sites involved in the action of acute anti-migraine drugs were subsequently

identified. Studies of these receptors have led to significant insights into the

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pathophysiology of migraine and the development of ne\v anti-migraine drugs, such

as naratriptan and zolmitriptan, which were selected fcr development based upon

their high affinity and selectivity for the human recombinant 5-HT,, receptors.

Clearly, the value inherent in orphan receptor characterization for drug discovery

has been proven and will continue to lead to the discoLery of substantial numbers

of novel, human receptors designated as "orphans" which will provide novel targets

for drug development.

Human Genome Sequencinq -New Orphan Receptors and Subtypes

Among the most intensive research efforts in biomedicine currently underway

is the immense task of sequencing the human genome and identifying all of the

expressed human genes. The tremendous impact of the ;e developments is driving

a paradigm shift in the fundamental approach to drug discovery as genomic data

becomes the initiation point for the drug discovery process. Considerable progress

has been made in the first several years of the Human Senome Project. Through

a combination of government and industry efforts, more than 465,000 human

expressed sequence tags (ESTs), small sequenced fragments of expressed genes,

have been identified and placed within computer dataDases, representing about

75% of the total human expressed sequences. Currently, approximately 1,500

sequences per day are added to the existing inforration base. Advances in

cloning, mapping, sequencing technologies and new instrumentation are jointly

contributing to an explosion in the volume of humari sequence data which is

expected to lead to structure of all of the roughly 100,C100 different human genes

by about 2003. To interpret the vast database of genonic data, the nascent field

of bioinformatics is providing computational algorithms and methods for searching

EST databases to allow classification and construction c f receptor protein function

from numerous new sequences. As a consequence, i t can be anticipated that the

impact of this effort will be an exponential increase in tt-e number of novel orphan

receptors requiring further study to define their potentsat role as targets for drug

discovery. At the conclusion of this project, the identification and sequence of all

receptor genes in the human genome will be available for study. Based upon

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estimates that 1-2% of the genome is likely to consist of receptors, this would

suggest that the total number of receptors to be identified will be approximately

1000-2000.

One focus of these efforts is to attempt to identify single disease-causing

genes which are responsible for an estimated 3000 common or inherited diseases.

Numerous examples exist for disease states that are associated with either

deficient receptor responses or enhanced responses to neurotransmitter or

hormonal signaling, indicating the biomedical importance for identifying the

molecular etiology of a disease skate and gaining access to disease-relevant

receptor targets, The recent cloning of a Karposi's sarcoma-associated herpevirus

(KSHV, or human herpesvirus 8) genome fragment revealed an open reading frame

encoding a putative GPCR that was homologous to human CXCRI and CXCR2

(interleukin-8) receptors (270). Expression studies showed that it was indeed a

bona fide signaling receptor that exhibited binding characteristics of a chemokine

receptor, with affinity for a range of chemokines in the CXC and CC families. The

receptor demonstrated constitutive (agonist-independent) activity in COS cells and

stimulated cellular proliferation, making i t a candidate viral oncogene.

One approach will be to isolate the receptor gene with direct relevance to the

disease process and subsequently employ the gene product as a highly specific

target for the design of new drugs. As an example. important development in this

area has been the understanding of the role of orphan GPCR chemokine receptors

in virus-host cell interactions. The recent identification of the human chemokine

stromal cell-derived factor 1 (SDF-1) as the natural hgand for the orphan

LESTR/fusin chemokine receptor (now termed CXCR4, Table 1) and the

characterization of CXCR4 function as a coreceptor for lymphocyte-tropic HIV

strains has made this little studied chernokine receptor critical to understanding the

dynamics of HIV pathogenesis and transmission (271,272). Concurrent with identification of the orphan receptor was the finding that SDF-1 was a powerful

inhibitor of infection of cells expressing CXCR4 and CD4 by T-cell adapted strains

of HIV-1. Additionally, several laboratories have shown that the chemokine

receptors, CCR3 and CCR5 (CC chemokine receptor subtypes, Table II), also serve

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758 HERZ, THOMSEN. A N D YARBROUGH

as the coreceptors for specific HIV-1 strains. Naturz I ligands for the CCRS and

CCRS chemokine receptors have been shown to inhit'it HIV-1 entry into microglia

for isolates that use these co-receptors (273). A incitant allele of CCR5 that

produces a truncated receptor which fails to mediate ctiemokine signaling appears

to confer natural resistance to HIV-1 infection in pEople who are homozygous

mutants. The identification of these CC-chemokine GPCR co-receptors and the

function of their endogenous ligands suggests new thei.apeutic strategies to inhibit

HIV-1 replication in the CNS.

Technological extensions based upon the hiAman genome sequencing

platform that enhance receptor-based drug discover:/ include development and

construction of biochips containing DNA microarrays ,f gene sequences. These

biochips are anticipated to enable high-throughput screening using fluorescent

hybridization of total cDNA libraries from particular cell types or rare tissues and will

permit quantitative examination of large numbers of specific receptor sequences

expressed in normal and disease samples. An additional application of this

technology should make it possible to assess the relative expression levels of a

large group of highly related receptors from specific cel s or a small tissue sample.

Potential applications of this new technology for gene cjciantitation and expression

may include target identification for drug discovery, compound screening and drug

development.

CONCLUSION

In summary, the application of molecular genetic: to large scale sequencing,

analysis of raw DNA sequence information, and new experimental approaches for

rapid functional analysis of receptor molecules fron- these sequences can be

expected to lead to the identification of an abundance of novel receptors that will

become molecular targets for the development of highly selective drugs. Indeed,

gene-based discoveries are already providing an en:irely new approach to the

development of a novel drug pharmaceuticals. The utilization 3f a family of cloned,

expressed human receptors subtypes in high-throughput primary screening

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programs has demonstrated the value of this approach for the identification of receptor-subtype specific drugs. This is an exciting development in the drug

discovery field since drugs with therapeutic activity that emerge from such

screening programs will possess the advantage of molecular specificity inherent jr i

using receptor subtype specific-targets as the basis for discovery efforts.

The current challenge will be to develop successful experimental strategies

and new technologies for the identification of ligands that can activate orphan

receptors or inhibit these receptors should they exhibit constitutive activity.

Although progress has been made, the continued application of molecular

biological approaches to receptor pharmacology coupled with advances in

biophysical methods should serve as tools that will enable a precise delineation of

receptor binding sites and drug-receptor interactions at the molecular level. A

more complete understanding the molecular events which transduce ligand binding

into receptor activation will ultimately aid in the in the discovery of more selective

agents for specific conformational states of existing receptor subtypes. As we move

towards the goal of sequencing the entire human genome within the next several

years, opportunities for the development of iinproved pharmaceuticals targeted to

specific receptor subtypes for existing diseases, and new drugs for diseases not

previously considered amenable to pharmacological therapy, should continue to

expand.

ACKNOWLEDGEMENTS

We thank Richard Mitchell for his excellent assistance in preparing the section on intracellular hormone receptors and Drs. Eric Prossnitz and Mona Mehdy for critical reading of the manuscript.

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