G PROTEIN COUPLED RECEPTOR (GPCR)

G-protein-coupled receptors (GPCRs)

• constitute a large and diverse family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals

• Among the largest and most diverse protein families in mammalian genomes.

• On the basis of homology with rhodopsin, they are predicted to contain seven membrane-spanning helices, an extracellular N-terminus and an intracellular C-terminus.

• This gives rise to their other names,: the 7-TM receptors or the heptahelical receptors.

• GPCRs transduce extracellular stimuli to give intracellular signals through interaction of their intracellular domains with heterotrimeric G proteins.

• This class of membrane proteins can respond to a wide range of agonists, including photon, amines, hormones, neurotransmitters and proteins.

• Some agonists bind to the extracellular loops of the receptor, others may penetrate into the transmembrane region.

GPCRs..contd• Their presence in the genomes of bacteria, yeast,

plants, nematodes and other invertebrate groups argues in favor of a relatively early evolutionary origin of this group of molecules.

• The diversity of GPCRs is dictated both by the multiplicity of stimuli (include light, neurotransmitters, odorants, biogenic amines, lipids, proteins, amino acids, hormones, nucleotides,

chemokines) to which they respond and by the variety of intracellular signalling pathways they activate.

• There are at least 18 different human G proteins to which GPCRs can be coupled.

• These G proteins form heterotrimeric complexes with Gß subunits, of which there are at least 5 types, and G subunits, of which there are at least 11 types

Signal molecules • A wide variety of ligands use GPCRs to stimulate cytoplasmic and nuclear

targets through heterotrimeric G-protein-dependent and -independent pathways.

• Biogenic amines: noradrenaline, dopamine, 5-HT, histamine, acetylcholine, epinephrine, norepinephrine

• Amino acids and ions: glutamate, Ca2+, GABALipids: LPA (lysophosphatidic acid), PAF (platelet-activating factor), prostaglandins, leukotrienes, anandamine, S1P (sphingosine-1-phosphate)

• Peptides and proteins: Tripeptide N-formyl-Met-Leu-Phe, GnRH (gonadotropin-releasing hormone), angiotensin, bradykinin, thrombin, bombesin, glucagon, calcitonin, vasoactive intestinal peptides, PTH (parathyroid hormone), FSH (follicle-stimulating hormone), LH (leuteinizing hormone), TSH (thyroid-stimulating hormone), endorphins

• Nucleotides: adenosine nucleotides, adenine nucleotides, urdine nucleotides Others: light, odorants, pheromones, opiates, cannabinoids

G-Protein-Coupled Receptor (GPCR): Structure and Function

The largest family of integral membrane protein involved in many biological process and pathologies; 50% of all modern drugs and 25% of the top 200 best selling drugs are estimated to target GPCRs; Transduce the signals mediated by diverse signaling molecules, such as ions, peptides, lipids and photons, to induce different intracelluar function; Bind their ligand and to activate different G proteins;

G-Protein-Coupled Receptor (GPCR)

GPCR-Gα Fusion Protein

Schematic of a G-protein-coupled receptor (GPCR)-Gα fusion protein

STRUCTURE• The receptor's amino (N) terminal is extracellular (above the plane

of the membrane), and its carboxyl (C) terminal intracellular. • The terminals are connected by a polypeptide chain that traverses

the plane of the membrane seven times.• The hydrophobic transmembrane segments (light color) are

designated by roman numerals (I–VII).• The agonist (Ag) approaches the receptor from the extracellular

fluid and binds to a site surrounded by the transmembrane regions of the receptor protein.

• G proteins (G) interact with cytoplasmic regions of the receptor, especially with portions of the third cytoplasmic loop between transmembrane regions V and VI.

• The receptor's cytoplasmic terminal tail contains numerous serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated.

• This phosphorylation may be associated with diminished receptor-G protein interaction.

Transmembrane topology of a typical serpentine receptor

. The receptor's amino (N) terminal is extracellular (above the plane of the membrane), and its carboxyl (C) terminal intracellular. The terminals are connected by a polypeptide chain that traverses the plane of the membrane seven times. The hydrophobic transmembrane segments (light color) are designated by roman numerals (I–VII). The agonist (Ag) approaches the receptor from the extracellular fluid and binds to a site surrounded by the transmembrane regions of the receptor protein. G proteins (G) interact with cytoplasmic regions of the receptor, especially with portions of the third cytoplasmic loop between transmembrane regions V and VI. The receptor's cytoplasmic terminal tail contains numerous serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated. This phosphorylation may be associated with diminished receptor-G protein interaction.

G-protein cycling. Rate-limiting receptor-promoted GDP dissociation;

STRUCTURAL

• Three Families: A,B,C• Based on sequence

homology• Sequences within each family

generally share over 25% sequence identity in the transmembrane core region, and a distinctive set of highly conserved residues and motifs.

• Among the three families, little similarity is evident beyond the predicted 7TM architecture

FUNCTIONAL

• Six Classes : A,B,C,D,E,F• Based on chemical

intercation• .According to the binding of

GPCRs with different ligand types

• The correlation between sub-family classification and the specific binding of GPCRs to their ligands can be computationally explored for Level 2 subfamily classification

GPCR CLASSIFICATION

GPCR CLASSIFICATION• Class A: Rhodopsin like- Subfamilies• Class B: Secretin like• Class C: Metabotropic glutamate / pheromone• Class D: Fungal pheromone• Class E: cAMP receptors

Functional classification

GPCR Classes* Class A Rhodopsin like o Amine o Peptide o Hormone protein o (Rhod)opsin o Olfactory o Prostanoid o Nucleotide-like o Cannabinoid o Platelet activating factor o Gonadotropin-releasing hormone o Thyrotropin-releasing hormone & Secretagogue o Melatonin o Viral o Lysosphingolipid & LPA (EDG) o Leukotriene B4 receptor o Class A Orphan/other

* Class B Secretin like o Calcitonin o Corticotropin releasing factor o Gastric inhibitory peptide o Glucagon o Growth hormone-releasing hormone o Parathyroid hormone o PACAP o Secretin o Vasoactive intestinal polypeptide o Diuretic hormone o EMR1 o Latrophilin o Brain-specific angiogenesis inhibitor (BAI)

* Class C Metabotropic glutamate / pheromone o Metabotropic glutamate o Calcium-sensing like o Putative pheromone receptors o GABA-B o Orphan GPRC5 o Orphan GPCR6 o Bride of sevenless proteins (BOSS) o Taste receptors (T1R)

* Class D Fungal pheromone o Fungal pheromone A-Factor like (STE2,STE3) o Fungal pheromone B like (BAR,BBR,RCB,PRA) o Fungal pheromone M- and P-Factor

* Class E cAMP receptors

* Frizzled/Smoothened family o frizzled o Smoothened

Putative families:

* Ocular albinism proteins * Insect odorant receptors * Plant Mlo receptors * Nematode chemoreceptors * Vomeronasal receptors (V1R & V3R) * Taste receptors T2R

Orphans:

* Putative / unclassified GPCRs

non-GPCR families:

* Class Z Archaeal/bacterial/fungal opsins

Other families

• Frizzled/Smoothened familyPutative families: * Ocular albinism proteins * Insect odorant receptors

* Plant Mlo receptors * Nematode chemoreceptors * Vomeronasal receptors (V1R & V3R) * Taste receptors T2R • Orphans:• * Putative / unclassified GPCRs

• non-GPCR families:• * Class Z: Archaeal/bacterial/fungal opsins

GPCR Ligands

Rhodopsin family: amine receptors Acetylcholine (muscarinic) Adrenaline Dopamine Histamine Serotonin Octopamine Trace amine

Rhodopsin family: other receptors Rhodopsin Olfactory Prostaglandin Prostacyclin Thromboxane Adenosine Purine / pyrimidine Cannabinoid Platelet activating factor Gonadotropin-releasing hormone Thyrotropin-releasing hormone

• Rhodopsin family: peptide receptors• Angiotensin• Apelin• Bombesin• Bradykinin• C5a anaphylatoxin• CC Chemokine• CXC Chemokine• CX3C Chemokine• C Chemokine• Cholecystokinin• Endothelin• fMet-Leu-Phe• Galanin• Ghrelin

• KiSS1-derived peptide• Melanocortin• Motilin• Neuromedin U• Neuropeptide FF• Neuropeptide S• Neuropeptide Y• Neuropeptide W / neuropeptide B• Neurotensin• Orexigenic neuropeptide QRFP• Opioid• Orexin• Oxytocin• Prokineticin• Somatostatin• Tachykinin• Urotensin II• Vasopressin• Protease-activated (thrombin)

GPCR Ligands

Melatonin Lysosphingolipid and LPA (EDG) Leukotriene B4 receptor SREB Mas proto-oncogene & Mas-related (MRGs) RDC1 EBV-induced Relaxin LGR like Free fatty acid G protein-coupled bile acid Nicotinic acid GPR GPR45 like Cysteinyl leukotriene Putative / unclassified Class A GPCRs

Metabotropic glutamate family Glutamate (metabotropic) Extracellular calcium-sensing GABA-B Pheromone (V2R) Taste receptors (T1R) Orphan GPRC5 Orphan GPCR6 Bride of sevenless proteins (BOSS) Putative / unclassified Class C GPCRs

Other families Frizzled / Smoothened family Ocular albinism proteins Vomeronasal receptors (V1R) Taste receptors (T2R) Insect odorant receptors Nematode chemoreceptors Plant Mlo receptors Fungal pheromone cAMP (Dictyostelium) Bacterial rhodopsin

• Secretin family• Calcitonin• Corticotropin releasing factor• Gastric inhibitory peptide• Glucagon• Growth hormone-releasing hormone• Parathyroid hormone• PACAP• Secretin• Vasoactive intestinal polypeptide• EMR1• Latrophilin• Brain-specific angiogenesis inhibitor (BAI)• Methuselah-like proteins (MTH)• Cadherin EGF LAG (CELSR)• Putative / unclassified Class B GPCRs

•

GPCR: Family A

• The largest group • Includes the receptors for light (rhodopsin)

and adrenaline (adrenergic receptors) and most other 7TM receptor types, including the olfactory subgroup.

• The olfactory receptors constitute most of these sequences, but nearly 200 non-olfactory 7TM receptors that recognize over 80 distinct ligands have also been functionally characterized.

GPCR: Family B• Contains only 25 members, including the

receptors for the gastrointestinal peptide hormone family (secretin, glucagon, vasoactive intestinal peptide (VIP) and growth-hormone-releasing hormone), corticotropin-releasing hormone, calcitonin and parathyroid hormone

• All family B receptors seen to couple mainly to activation of the effector adenylyl cyclase through the G protein Gs

GPCR : Family C• Relatively small • Family C contains the metabotropic glutamate

receptor family, the GABA receptor, and the calcium-sensing receptor, as well as some taste receptors.

• All family C members have a very large extracellular amino terminus that seems to be crucial for ligand binding and activation.

Three Families of GPCR

Ligands: neurotranmitters (dopamine and serotonin)

Ligands: hormones (glucagon, secretin, PTH) Example: mGluR, CaR

• To date, the only member of the receptor superfamily for which a crystal structure has been solved is Rhodopsin.

• This structure, which corresponds to the inactive receptor, confirms the presence of an anticlockwise bundle of 7 TM alpha helices (viewed from the intradiscal or extracellular side) that are connected by loops of varying lengths.

General structure

• N-terminal segment

• Seven TMs, Three exoloops, Four cytoloops,

• C-terminal segment.

Family 1 GPCRs: Rhodopsin-like Receptors

Represent the predominant class of GPCRs; Several highly conserved amino acid (red circles); Disulfide bridge between first and second extracellular loops (ECLs), palmitoylated cys in C-tail; The binding of small molecule ligands occures within the TM region; Example: Rhodopsin, Dopamine receptor, Chemokine receptors;

• The rhodopsin crystal structure provides a structural basis for understanding the function of other G protein–coupled receptors (GPCRs).

• The major structural motifs observed for rhodopsin are expected to carry over to other GPCRs, and the mechanism of transformation of the receptor from inactive to active forms is thus likely conserved.

• Moreover, the high expression level of rhodopsin in the retina, its specific localization in the internal disks of the photoreceptor structures [termed rod outer segments (ROS)], and the lack of other highly abundant membrane proteins allow rhodopsin to be examined in the native disk membranes by a number of methods.

• Evidence of the propensity of rhodopsin and, most likely, other GPCRs to dimerize, a property that may be pertinent to their function.

Rod cell specific visual pigment protein found in the vertebrate retina Responsible for achromatic vision under dim light conditions 348 amino acids, 7-TM, 11-cis-retinaldehyde (chromophore, derived from Vit A) Posttransliational modifications: acetylation (M1), N-Glycosylation (N2, N15), S-palmitoylation (C322, C323) one ROS stack contains 1000-2000 discs• 6.4 million retinal cells (70% rods

[B&W], <2% cones [color])• ~50% of membrane area is rhodopsin

(balance is phospholipids and cholesterol)

1

3

2

4

5 6

7

N

C

Rhodopsin

G Protein–Coupled Receptor RhodopsinKrzysztof Palczewski

Annu. Rev. Biochem.2006. 75:743–767

Visual system signaling cascade

Lower [cGMP] leads to Na+ channel inactivation,which in turn leads to hyperpolarization

GTP:G separates from G

signaling state, binds GDP:G

Visual system signaling cascade

Visual Signal Transduction

Family 2 GPCRs: Secretin-like Receptors

15 peptide-binding receptors in human; Relatively long N-terminus (~100-160 residues) and a juxtamembrane doamin of 7 membrane-spanning α-helices (J-domain); Tertiary structure is stabilized by 3 disulfide bonds within 6 highly conserved Cys Share little sequence homology with family 1 and 3 GPCRs; Example: Corticotropin-releasing factor receptor, Glucagon receptor, PTH receptor;

Family 2 GPCRs: Secretin-like Receptors

Corticotropin-Releasing Factor Receptor (CRF)

PNAS, 2004;101: 12836-12841.

Related to human stress response; NMR structure of ECD1-CRF-R2β; Clustered in the cleft region between the tip of the first β-sheet and the edge of the “palm” of the second β-sheet; The central core contains a salt bridge sandwiched between aromatic side chains;

Family 3 GPCRs: mGluR-like Receptors

Very long N-terminus; Ligand-binding domain is located in N-terminus and is thought to resemble a Venus fly trap (VFT) that can open and close with the agonist bound inside; Cys residues are conserved, the tertiary structures are likely highly conserved; Share similar ligands and downstream signaling pathway; Constitutive homo- or heterodimers: mGluR (homo), GAGAβ(hetero); Example: metabotropic glutamate receptor (mGluR), CaR, GABAβ

Structure of mGluR and CaR

Glutamate is a neurotransmitter in CNS and functions in long-term potentiation, learning and memory; Metabotropic glutamate receptor (mGluR) mediate excitatory transmission on the cellular surface through initial binding of glutamate

Calcium sensing receptor (CaR) shares 27% sequence identity with (mGluR); Response to [Ca2+]o, L-amino acids,

polyamines, ionic strength and pH; Parathyroid CaR plays a central role in systemic PTH level and Ca2+ homeostasis;

Nature 2006, 407: 971-977; Current Opinion in Neurobiology 2003, 13: 271-278

G-PROTEIN-COUPLED RECEPTORS

• The seven α-helical membrane-spanning domains probably form a circle around a central pocket that carries the attachment sites for the mediator substance.

• Binding of the mediator molecule or of a structurally related agonist molecule induces a change in the conformation of the receptor protein, enabling the latter to interact with a G-protein (= guanyl nucleotide-binding protein).

• G-proteins lie at the inner leaf of the plasmalemma and consist of three subunits designated α, β, and γ.

• There are various G-proteins that differ mainly with regard to their α-unit. Association with the receptor activates the G-protein, leading in turn to activation of another protein (enzyme, ion channel).

• A large number of mediator substances act via G-protein-coupled receptors

G PROTEIN

• GPCRs are known for their involvement in physiological functions including neurotransmission, vision, olfaction, hormone action, platelet aggregation, and leukocyte chemotaxis.

• Induction of these diverse biological functions results from the activation of a collection of heterotrimeric G-proteins, which consist of alpha subunits and closely associated beta-gamma subunits.

• The alpha subunit is responsible for GTP and GDP binding and for GTP hydrolysis, whereas the beta and gamma subunits are associated in a tightly linked beta-gamma complex.

• The complexity of the signaling pathways initiated by GPCRs is illustrated by the presence of numerous G-proteins, including 18 alpha subunits, which can be classified into four groups, 12 beta subunits, and 5 gamma subunits.

G proteins

• Generally referred to by their alpha subunits.• So, the Gs heterotromeric complex contains

Galphas; • Gq contains Galphas; and so on. • Four distinct gamma subunit subfamilies are

recognized. :

The four major families of G-protein