Membrane FunctionMembrane Function

Signal Transduction

I. Introduction to I. Introduction to Receptors & Signal Receptors & Signal

TransductionTransduction

The PlayersThe Players

Signaling molecules Receptors G-proteins Second messenger systems Effector proteins

Signaling MoleculesSignaling Molecules

Neurotransmitters Hormones Growth factors Drugs Other nomenclature

Ligand Agonist / Antagonist

ReceptorsReceptors

Receptors are proteins associated with cell membranes

Receptors “recognize” signaling molecules by binding to them.

Binding of receptors by signaling molecules ---> Cell behavior change

Figure 1: Overview of Figure 1: Overview of SignalingSignaling

TyrosineKinase

mRNASynthesis

Protein Synthesis

SecondMessangers

Protein Kinases

IonChannels

Hormones:SteroidsThyroid

GrowthFactors

TransmittersTransmittersHormones

Neurotansmitters: Neurotansmitters: Biogenic Amines.Biogenic Amines.

Catecholamines Epinephrine Norepinephrine Dopamine

Esters: Acetylcholine Indolamines

Histamine 5-HT

Neurotransmitters: Neurotransmitters: PeptidesPeptides

Substance P Neuropeptide Y (NPY) Enkephalins Somatostatin VIP

Neurotransmitters: Amino Neurotransmitters: Amino AcidsAcids

Excitatory Glutamate Aspartate

Inhibitory -aminobutyric acid (GABA) Glycine

Neurotranmitters: OtherNeurotranmitters: Other

Nitric Oxide Arachadonic acid Carbon Monoxide PAF Zinc

The G-ProteinsThe G-Proteins

Involved in most signaling processes

Link receptor proteins to effector proteins.

Trimeric proteins composed of , , and -subunits.

Figure 2: G-Protein CyclingFigure 2: G-Protein Cycling

Adenylate CyclasePhospholipase C

Ion ChannelsPhospholipase A2

Phosphodiesterase

A

A

A

A

R

R

R

R

GTP(GTPase)

-Pi

GTP

GTP

GDP

GDP

Functional G-Protein UnitsFunctional G-Protein Units

GTP-activated -subunit produce second messenger and/or opens ion channels.

-complexes Initially thought to be inert. Probably not inert Exact role currently ill-defined.

Second messengers Second messengers produced by G-protein produced by G-protein

activation.activation. Adenylate Cyclase cAMP

Phospholipase C (PLC) Inositol triphosphate (IP3) Diacylglycerol (DAG)

Ion Channel Activity

Families of G-proteinsFamilies of G-proteins

Unique structure of their -subunits. subunits appear to be similar

across families. Main families:

Gs

Gi

Gq

II. cAMP Second II. cAMP Second Messenger SystemMessenger System

Figure 3: Adenylate Figure 3: Adenylate CyclaseCyclase

AdenylateCyclaseR1 R2

As

Gs Gi

Ai

GTPGDP

GTPGDP

PDEAMP cAMPATP-Mg++

Reg RegC

C

C

C

Protein

Protein-PProtein Kinase A

(PKA)PKA

Summary of Adenylate Summary of Adenylate Cyclase ActivationCyclase Activation

Receptors which associate with Gs -type G-protein Stimulates adenylate cyclase. Increases cAMP

Receptors which associate with Gi -type G-protein Inhibit adenylate cyclase. Decreases cAMP

Summary of cAMP actionSummary of cAMP action

cAMP exerts its effect by activating protein kinase A (PKA)

PKA phosphorylates proteins Enzymes, pumps, and channels Phosphorylation can either increase

or decrease activity depending on the protein.

Adenylate CyclaseAdenylate Cyclase

Family of membrane spanning enzymes.

Types I through IV have been well characterized. Additional types probably exist.

Types differ with respect to activity modulation by other second messenger systems

Adenylate Cyclase Activity Adenylate Cyclase Activity and Other Messenger and Other Messenger

SystemsSystems Kinases (PKA, PKC, other) can phosphorylate adenylate cyclase in some cells.

Binding of adenylate cyclase by: -subunits of other G-proteins Ca++/calmodulin complexes

Allows other second messenger systems to interact with cAMP system

III. The Phospholipase C III. The Phospholipase C Second Messenger Second Messenger

System:System:IPIP33 and DAG and DAG

Figure 4: Phospholipase C Figure 4: Phospholipase C SystemSystem

R

Ca++

PKC

Ca++Endoplasmic Reticulum

Gq

PLC

ProteinProtein-P

A

DAG

IP3

PIP2

Summary of the Summary of the Phospholipase C Phospholipase C

MessengersMessengers Agonist binds receptor Occupied Receptor ---> activation of

PLC (Gq -mediated) PLC Produces second messengers:

IP3 and DAG PLC activation associated with

Ca++-channel activation

Action of IPAction of IP33

IP3 binds to IP3-receptors on the endoplasmic reticulum

Releases intracellular Ca++ stores.

Action of DAGAction of DAG

Remains membrane associated. Activates Protein kinase C (PKC)

which translocates from the cytosol to the membrane

Activated PKC phosphorylates other proteins and alters their function state.

PLC System and CalciumPLC System and Calcium

PLC causes the IP3-mediated Calcium

PLC also causes the influx of Ca++. Ca++ binds one of a family of Ca++

binding proteins (calmodulin). Ca++/calmodulin complex

binds to yet other proteins and changes their functional activity.

IV. Guanylate Cyclase: IV. Guanylate Cyclase: cGMP and Nitric Oxide As cGMP and Nitric Oxide As

Second MessengersSecond Messengers

Figure 5: Nitric Oxide and Figure 5: Nitric Oxide and cGMPcGMP

cGMP

NO

Ca++

GTP

GMP

IntracellularCa++ Stores Ca++

Ca++

Arginine

+Citrulline GTP

NO

PDE

Membrane BoundGuanylate Cyclase

SolubleGuanylate Cyclase

C.M.

Ion ChannelscGMP-Dependent PK

PDEase Activity

NO Synthetase

NO is Membrane Soluble.NO is Membrane Soluble.

Diffusion to nearby cells Increase cGMP levels in nearby

cells Vascular endothelial cells and

nearby smooth muscle cells.

V. SIGNALING BY V. SIGNALING BY ACETYLCHOLINEACETYLCHOLINE

Acetylcholine As a Acetylcholine As a NeurotransmitterNeurotransmitter

Both the central and peripheral nervous systems.

Binds two broad classes of receptors: Nicotinic receptors Muscarinic receptors.

Nicotinic Receptor Nicotinic Receptor FeaturesFeatures

Composed of 5 subunits: 2 , , and .

Subunits are arranged to form a central cavity that extends across the membrane.

Nicotinic receptors are also channels ACh-binding opens gates and allows

ion fluxes across the channel

Figure 6: Nicotinic Figure 6: Nicotinic ReceptorReceptorChannel

AgonistBinding

Site

Gate

Subclasses of Nicotinic Subclasses of Nicotinic ReceptorsReceptors

Skeletal muscle (N1 or Nm) Unique and subunits

Autonomic ganglia (N2 or Ng). Both N1 and N2 are gene-product

families not single receptor types.

Other Ligand-Gated Other Ligand-Gated ChannelsChannels

Structural and sequence similarity to nicotinic receptors.

Example agonists for these channels include: Serotonin (5-HT) Glutamate GABA Glycine

Muscarinic receptorsMuscarinic receptors

Muscarinic receptors are not channels.

Operate through G-proteins to alter second messenger systems.

5 muscarinic subtypes have been cloned and sequenced (M1, M2, M3, M4, M5).

Grouping Muscarinic Grouping Muscarinic ReceptorsReceptors

M1, M3, and M5 receptors: Activate Phospholipase C through Gq. PLC activation ---> increased IP3 -->

increased intracellular Ca++

Increased intracellular Ca++ --->Activation of Ca++-sensitive K+ & Cl- channels.

Grouping Muscarinic Grouping Muscarinic ReceptorsReceptors

M2 and M4 receptors Gi -coupled inhibition of adenylate

cyclase Go or Gi -coupled regulation of certain

Ca++ & K+ channels.

VI. Signaling by VI. Signaling by Epinephrine and Epinephrine and

Norepinephrine and Norepinephrine and Coupling Through Coupling Through

Adrenergic ReceptorsAdrenergic Receptors

Three Families of Three Families of Adrenergic Receptors:Adrenergic Receptors:

-receptors: Three subtypes and .

-receptors: Three subtypes AB and C

-receptors: Three subtypes A

B and C

..

All adrenergic receptors All adrenergic receptors appear to be coupled to appear to be coupled to

cellular processes cellular processes through G-proteinsthrough G-proteins

Occupation of Occupation of Adrenergic ReceptorsAdrenergic Receptors

Gs-mediated stimulation of adenylate cyclase

Increased cAMP Increased PKA activity.

Occupation of Occupation of - -Adrenergic ReceptorsAdrenergic Receptors

Mechanistic details sketchy Possibly Gq-mediated PLC

activation Increases IP3 and DAG for some

subtypes (1B)? Activates Ca++-channels for other

subtypes (1A)?

Occupation of Occupation of - -Adrenergic ReceptorsAdrenergic Receptors

Gi -mediated inhibition of adenylate cyclase.

Decreased cAMP Decreased PKA activity.