g protein coupled receptor and pharmacotherapeutics

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G-protein coupled receptors

G-protein coupled receptors and drugs modulating themDR. NISHIKANT SHARMA

DR. PRIYANKA KUMAWAT

General description of Receptors and signaling G- Protein coupled receptor and its mechanismClasses of GPCRSecond messenger and its applied pharmacologyRecent developmentTools for drug discoveryConclusion

Brief outlineHistory1967. Ragnar Granit, Haldan Keffer Hartline and George Wald-Physiological and chemical processes underlying photoreception. 1971. Earl W. Sutherland, Jr.-cyclic AMP (cAMP).1988. Sir James W. Black-Discovery of propranolol, which blocks the -adrenergic receptor, and the H2 histamine receptor blocker cimetidine. 1994. Martin Rodbell and Alfred G. Gilman-Heterotrimeric G-proteins.2004. Linda B. Buck and Richard Axel- Odourant receptors. 2012. Brain kobilka and Robert Lefkowitz-Studies of G-protein coupled receptors.

RECEPTORSINTRACELLULAR RECEPTORS- Cytoplasmic Nuclear receptorsCELL SURFACE RECEPTORSION CHANNEL RECEPTOR

Ligand gated ion channelsControlled by neurotransmittersPresent in neuronsEg: Ach cation channelG-PROTEIN LINKED RECEP

Act via second messengers-cAMP, IP3/DAG,cGMPENZYME LINKED RECEP

Eg: Protein kinaseTyrosine kinaseTyrosine phosphotaseSerine/threonine kinaseGuanylyl cyclaseHistidine kinase

Concept of Cell Signaling

Process in which cells sense the extracellular stimuli through membranous or intracellular receptors, transduce the signals via intracellular molecules -Regulate the biological function of the cells.

Features of signal transductionSpecificity- Signal molecules fits binding site on its complementary receptor, other signals do not. Affinity- High affinity of receptors for signal moleculesAmplification-Signal receptor activate many molecules of second enzyme, which activates many molecules of the third enzyme and so onDesensitization Feedback circuit that shuts off the receptor or remove it from the cellIntegration Two signals with opposite action on second messenger, the regulatory outcome results from integrated output from both the receptors

Specificity-G-protein With a common pool of promiscuous G-proteins linking the various receptors and effector systems in a cell, it might seem that all specificity would be lost, but this is clearly not the case.it show selectivity with respect to both the receptors and the effectors with which they couple, having specific recognition domains in their structure complementary to specific G-protein-binding domains in the receptor and effector molecules8Signals to which cell respondAntigenCell surface glycoproteins/ oligosaccharidesExtracellular matrix componentGrowth factorsHormonesNeurotransmitters

LightMechanical touchNutrientsOdorantsPheromonesTastants

Primary Messengers Secondary TertiaryTransmit the signal from receptor to the enzyme and activate it to produce secondary messenger

Eg:G,G Transmit signals in form of either direct cellular response eg:cAMP, cGMPOr activate further enzymes to produce response eg:IP3,DAG Release after action of second messenger on an organelle (ER) and act directly or in conjunction to give cellular responses

Eg:Ca+2G-protein coupled receptor structureSeven transmembrane (7TM) helices coupled to effecter system (enzyme/ channel) through GTP/GDP binding protein called G-proteins An extracellular domain which binds to the ligand (drug/ neurotransmitter) An intracellular domain which couples to G-protein

GPCRs are one of the largest superfamily of membrane receptors which present in almost every organ of the body.and they play an extremely important role in transducing extra cellular signals and regulates many basic physicochemical processess in the body eg:smell,taste,vision,secretion,neurotransmission,metabolism,cellular differentiation,growth,inflammation and immune response and for these reasons GPCRs have been the most important and common target for pharmacological intervention.At present 40% of the drugs available in the market are targetting GPCRs

11G- proteinA family of membrane proteins anchored to the membrane. Recognize activated GPCRs and pass the message to the effector system.Named as G-protein because of their interaction with guanine nucleotides (GTP/GDP)Consist of three subunits: , and . Guanine nucleotides bind to the subunit, has GTPase enzymic activity Functions as a molecular switches. when bind with GTP they are on & when with GDP they are off.

which convert GTP to GDP. The and subunits remain together as a complex.

12Types of G-proteinLarge" G proteins (Heterotrimeric)Activated by GPCRsMade up of alpha (), beta (), and gamma ()subunits.

Small" G proteins- Belong to theRassuperfamily ofsmall GTPases. Homologous to the alpha () subunitAlso bind GTP and GDP and are involved insignal transduction.

G-protein subunits with second messenger GsGiGqcAMP stimulation receptorHistamineSerotoninDopamine

cAMP inhibition2 receptorM2 receptorOpioid receptorD2 receptor5HT1 receptorPLC(IP3 & DAG)1 M1AT15HT2Vasopressin Activate potassium channels Inhibit voltage-gated calcium channels Activate mitogen-activated protein kinase cascade.

-mediated effects occur at higher levels of receptor occupancy than -mediated effects otherwise the action of it is same to alpha14Golf-Odorant receptor,Adenylyl cyclase

Gt- cGMP phosphodiesterase , cGMP

G12/13-Rho family GTPase signaling and control cell cytoskeleton remodeling and regulating cell migration.

guanine nucleotide exchange factor (GEF) 15Mechanism of GPCRThe ligand comes to the extracellular binding site of receptor make some conformational changes in receptor which attract G-proteinCoupling of the subunit to an agonist-occupied receptor causes the bound GDP to exchange with intracellular GTP -GTP complex then dissociates from the receptor and from the complexThis -GTP complex interacts with a target protein (target- adenylyl cyclase/ ion channel/PLC)The complex may also activate a target protein (target) These effectors then form the second messengers to initiate the cell responses e.g.; cAMP 2nd messenger for Adenylyl cyclase, IP3/DAG for PLC and cGMP for guanylyl cyclaseTarget 2? The GTPase activity of the subunit is increased when the target protein is bound, leading to hydrolysis of the bound GTP to GDP, whereupon the subunit reunites with .

16GTPGDP GDPGTP 4 ATP4 cAMPCell responseATProtein kinaseADPPInactive proteinActive proteinhormoneAdenylate cyclase Signaling SystemACRSInhibitorRiPhospholipase-c signaling systemPIP2IP3DAGRelease of Ca+2from ERintracellular Ca+2 Along with Ca+2 Activate Protein Kinase-CCellular functions- Proliferation, differentiation, apoptosis, cytoskeletal Remodeling, vesicular trafficking, ion channels conductance,neurotransmissionPLCAn intracellular enzyme play an imp. Role in signal transduction participate in phosphatidylinositol 4,5-bisphosphonate(PIP2) and lipid signaling in a calcium dependent manner.18GPCR classesClass A- Rhodopsin like-receptors e.g.: Retinal, odorants, catecholamine(2),adenosine(A2), opiates, enkephalins, anandamide, thrombin.Class B- Secretin like- Secretin, Glucagon, PTH, Calcitonin, VIPClass C- Metabotropic glutamate- GlutamateClass D- Pheromone- Used for chemical communication Class E- cAMP receptor(Dietyostelium)Class F- Frizzled/smoothened family-Wnt binding, a key regulator of animal development (embryonic life)Ocular albinism proteinsPutative families- Vomeronasal receptors (V1R & V2R),Taste receptors(T2R)Orphan GPCR- putative unclassified

Second messengers

21

Targets of G proteinsAdenylyl cyclase

IP3/DAG Phospolipase C system

Ion channels esp. potassium and calcium

Rho a/ Rho kinase system

The Adenylyl cyclase/cAMP systemcAMP is a nucleotide Synthesized within the cell from ATP by membrane-bound, adenylyl cyclaseProduced continuously Inactivated by hydrolysis to 5-AMP, by the PhosphodiesterasesCommon mechanism, namely the activation of protein kinasesInvolved in Energy metabolismCell division and cell differentiationIon transport, ion channelsContractile proteins in smooth muscleCyclic AMP dependent protein kinase Best understood target of cyclic AMPCan phosphorylate a diverse array of physiological targets Metabolic enzymesTransport proteinsNumerous regulatory proteins including other protein kinasesIon channelsTranscription factorsFor example cAMP response elementbinding protein(CREB) leads to Tyrosine hydroxylase, iNOS, AchR, Angiotensinogen, Insulin, the glucocorticoid receptor, and CFTRCyclic AmpRegulated Guanine Nucleotide Exchange Factors (Gefs)

Monomeric GTPases and key regulators of cell functionIntegrate extracellular signals from membrane receptors with cytoskeletal changesEPAC pathway provides an additional effector system for cAMP signaling and drug action that can act independently or cooperatively with PKA Activation of diverse signaling pathways, regulate PhagocytosisProgression through the cell cycleCell adhesionGene expressionApoptosisPhosphodiesterasesHydrolyze the cyclic 3',5'-phosphodiester bond in cAMP and cGMP>50 different PDE proteins divided into 11 subfamilies Drug targets for AsthmaCardiovascular diseases such as heart failureAtherosclerotic coronary and peripheral arterial diseaseNeurological disorders

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Energy metabolism

cAMP and Immunomodulation

Am J Respir Cell Mol Biol Vol 39. pp 127132, 2008

The Phospholipase C/ inositol phosphate system1950s by Hokin and HokinPIP2 is the substrate for a membrane-bound enzyme, phospholipase C (PLC), Which splits it into DAG and inositol (1,4,5) trisphosphate (IP3) Both function as second messengers

After cleavage of PIP2,

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