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G protein coupled receptor and pharmacotherapeutics
- 1. G-protein coupled receptors anddrugs modulating them
- 2. • General descriptionof Receptors and signaling • G- Protein coupled receptor and its mechanism • Classes of GPCR • Second messenger and its applied pharmacology • Recent development • Tools for drug discovery • Conclusion Brief outline
- 3. History 1967. RagnarGranit, 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 β- 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.
- 4. RECEPTORS INTRACELLULAR RECEPTORS-Cytoplasmic Nuclear receptors CELL SURFACE RECEPTORS ION CHANNEL RECEPTOR • Ligand gated ion channels • Controlled by neurotransmitters • Present in neurons • Eg: Ach cation channel G-PROTEIN LINKED RECEP • Act via second messengers-cAMP, IP3/DAG,cGMP ENZYME LINKED RECEP • Eg: Protein kinase Tyrosine kinase Tyrosine phosphotase Serine/threonine kinase Guanylyl cyclase Histidine kinase
- 7. Concept of Cell Signaling Processin which cells sense the extracellular stimuli through membranous or intracellular receptors, transduce the signals via intracellular molecules -Regulate the biological function of the cells.
- 8. Features of signaltransduction Specificity- Signal molecules fits binding site on its complementary receptor, other signals do not. Affinity- High affinity of receptors for signal molecules Amplification-Signal receptor activate many molecules of second enzyme, which activates many molecules of the third enzyme and so on Desensitization –Feedback circuit that shuts off the receptor or remove it from the cell Integration – Two signals with opposite action on second messenger, the regulatory outcome results from integrated output from both the receptors
- 9. Signals to whichcell respond Antigen Cell surface glycoproteins/ oligosaccharides Extracellular matrix component Growth factors Hormones Neurotransmitters Light Mechanical touch Nutrients Odorants Pheromones Tastants
- 10. Primary Messengers Secondary Tertiary Transmit the signalfrom receptor to the enzyme and activate it to produce secondary messenger Eg:Gα,Gβγ Transmit signals in form of either direct cellular response eg:cAMP, cGMP Or 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+2
- 11. G-protein coupled receptor structure Seven transmembrane (7TM) α 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
- 12. G- protein Afamily of membrane proteins anchored to the membrane. Recognize activated GPCR’s 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: α, β and γ. Guanine nucleotides bind to the α subunit, has GTPase enzymic activity Functions as a molecular switches. when bind with GTP they are “on” & when with GDP they are “off”.
- 13. Types of G-protein 1.“Large" G proteins (Heterotrimeric) Activated by GPCRs Made up of alpha (α), beta (β), and gamma (γ) subunits. 2. ”Small" G proteins- Belong to the Ras superfamily of small GTPases. Homologous to the alpha (α) subunit Also bind GTP and GDP and are involved in signal transduction.
- 14. G-protein subunits with secondmessenger β γα Gs Gi Gq cAMP stimulation β receptor Histamine Serotonin Dopamine cAMP inhibition α2 receptor M2 receptor Opioid receptor D2 receptor 5HT1 receptor PLC (IP3 & DAG) α1 M1 AT1 5HT2 Vasopressin •Activate potassium channels • Inhibit voltage- gated calcium channels • Activate mitogen- activated protein kinase cascade.
- 15. Golf-Odorant receptor,Adenylylcyclase Gt- cGMP phosphodiesterase , cGMP Gα12/13 -Rho family GTPase signaling and control cell cytoskeleton remodeling and regulating cell migration.
- 16. Mechanism of GPCR The ligand comes to the extracellular binding site of receptor make some conformational changes in receptor which attract G-protein Coupling of the α subunit to an agonist-occupied receptor causes the bound GDP to exchange with intracellular GTP α-GTP complex then dissociates from the receptor and from the βγ complex This α-GTP complex interacts with a target protein (target- adenylyl cyclase/ ion channel/PLC) The βγ 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 cyclase
- 17. GTP GDP GDP GTP 4 ATP 4cAMP Cell response AT Protein kinase ADP P Inactive protein Active protein hormone Adenylate cyclase Signaling System AC RS Inhibitor Ri
- 18. Phospholipase-c signaling system PIP2 IP3DAG Release of Ca+2 from ER intracellular Ca+2 Along with Ca+2 Activate Protein Kinase-C Cellular functions- Proliferation, differentiation, apoptosis, cytoskeletal Remodeling, vesicular trafficking, ion channels conductance, neurotransmission PLC
- 19. GPCR classes ClassA- Rhodopsin like-receptors e.g.: Retinal, odorants, catecholamine(β2),adenosine(A2), opiates, enkephalins, anandamide, thrombin. Class B- Secretin like- Secretin, Glucagon, PTH, Calcitonin, VIP Class C- Metabotropic glutamate- Glutamate Class 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 proteins Putative families- Vomeronasal receptors (V1R & V2R),Taste receptors(T2R) Orphan GPCR- putative unclassified
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- 23. Targets of Gproteins Adenylyl cyclase IP3/DAG Phospolipase C system Ion channels esp. potassium and calcium Rho a/ Rho kinase system
- 24. The Adenylyl cyclase/cAMP system cAMP is a nucleotide Synthesized within the cell from ATP by membrane-bound, adenylyl cyclase Produced continuously Inactivated by hydrolysis to 5´-AMP, by the Phosphodiesterases Common mechanism, namely the activation of protein kinases Involved in Energy metabolism Cell division and cell differentiation Ion transport, ion channels Contractile proteins in smooth muscle
- 25. Cyclic AMP dependentprotein kinase Best understood target of cyclic AMP Can phosphorylate a diverse array of physiological targets Metabolic enzymes Transport proteins Numerous regulatory proteins including other protein kinases Ion channels Transcription factors For example cAMP response element–binding protein(CREB) leads to Tyrosine hydroxylase, iNOS, AchR, Angiotensinogen, Insulin, the glucocorticoid receptor, and CFTR
- 26. Cyclic Amp–Regulated Guanine NucleotideExchange Factors (Gefs) Monomeric GTPases and key regulators of cell function Integrate extracellular signals from membrane receptors with cytoskeletal changes EPAC 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 Phagocytosis Progression through the cell cycle Cell adhesion Gene expression Apoptosis
- 27. Phosphodiesterases Hydrolyze thecyclic 3',5'-phosphodiester bond in cAMP and cGMP >50 different PDE proteins divided into 11 subfamilies Drug targets for Asthma Cardiovascular diseases such as heart failure Atherosclerotic coronary and peripheral arterial disease Neurological disorders
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- 30. cAMP and Immunomodulation AmJ Respir Cell Mol Biol Vol 39. pp 127–132, 2008
- 34. The Phospholipase C/inositol phosphate system 1950s by Hokin and Hokin PIP2 is the substrate for a membrane-bound enzyme, phospholipase Cβ (PLCβ), Which splits it into DAG and inositol (1,4,5) trisphosphate (IP3) Both function as second messengers After cleavage of PIP2, the status quo is restored Lithium blocks this recycling pathway IP3 receptor- a ligand-gated calcium channel present on the membrane of the endoplasmic reticulum
- 36. Diacylglycerol and protein kinaseC DAG, unlike the inositol phosphates, is highly lipophilic and remains within the membrane Binds to a specific site on the PKC molecule, which migrates from the cytosol to the cell membrane in the presence of DAG, thereby becoming activated 10 different mammalian PKC subtypes Kinases in general play a central role in signal transduction, and control many different aspects of cell function
- 38. Ca2+ IP3 receptor– a ligand-gated Ca2+ channel found in high concentrations in the membrane of the ER 10-9 m range enhance Ca2+ release, but concentrations near 10-9 m inhibit release Phosphorylation of the IP3 receptor by PKA enhances Ca2+ release, Phosphorylation of an accessory protein, IRAG, by PKG inhibits Ca2+ release In smooth muscle, this effect of PKG represents part of the mechanism by which cyclic GMP relaxes vessel tone
- 39. Ca2+ In skeletaland cardiac muscle - Ca2+ release from intracellular stores occurs through a process -Ca2+-induced Ca2+ release Primarily mediated by the ryanodine receptor (RyR) Ca2+ entry into a skeletal or cardiac myocyte through L-type Ca2+ channels causes conformational changes in the ryanodine receptor Induce release of large quantities of Ca2+ into the sarcoplasm. Drugs that activate the RyR include caffeine; drugs that inhibit the RyR include Dantrolene
- 40. Ion channels astargets for G- proteins Directly by mechanisms that do not involve second messengers In cardiac muscle, for example, mAChRs are known to enhance K+ permeability Opiate analgesics reduce excitability by opening potassium channels Actions are produced by direct interaction between the βγ subunit of G0 and the channel, without the involvement of second messengers
- 41. The Rho/Rho kinasesystem Activated by certain GPCRs (and also by non-GPCR mechanisms), which couple to G-proteins of the G12/13 type Rho-GDP, the resting form, is inactive When GDP-GTP exchange occurs, Rho is activated In turn activates Rho kinase Smooth muscle contraction and proliferation, angiogenesis and synaptic remodeling Important in the pathogenesis of pulmonary hypertension
- 42. Desensitization Receptor phosphorylation Phosphorylation by PKA and PKC Not very selective, receptors other than that for the desensitizing agonist will also be affected Heterologous desensitization Phosphorylation by GRKs Receptor-specific to a greater or lesser degree Affects mainly receptors in their activated (i.e. agonist-bound) state Homologous desensitization
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- 44. GPCR dimerisation Theconventional view first overturned by work on the GABAB receptor Most, if not all, GPCRs exist as oligomers Within the opioid receptor family, stable and functional dimers of κ and δ receptors have been found whose pharmacological properties differ from those of either parent Functional dimeric complexes between angiotensin (AT1) and bradykinin (B2) receptors occur in human platelets Show greater sensitivity to angiotensin than 'pure' AT1 receptors Pre-eclampsia number of these dimers increases due to increased expression of B2 receptors Resulting-paradoxically- in increased sensitivity to the vasoconstrictor action of angiotensin
- 45. Constitutively active receptors Spontaneously active in the absence of any agonist β-adrenoceptor, histamine H3 Inverse agonists, which suppress this basal activity, may exert effects distinct from those of neutral antagonists, which block agonist effects without affecting basal activity.
- 46. Agonist specificity Cellulareffects are qualitatively different with different ligands Existence of probably many-R* states Agonist trafficking or protean agonism If substantiated, it will add a new dimension to the way in which we think about drug efficacy and specificity
- 47. GPCR and arrestins Following continued agonist binding to GPCR Cytosolic GRKs are induced to translocate to GPCR This phosphorylation attracts -arrestins to the receptors Compete with G proteins for binding to the cytoplasmic site of the receptor Arrestins uncouple GPCRs from G proteins Causing desensitization, internalization of GPCR Universal response to agonist activation and is critical for the inactivation of GPCRs and the termination of neurotransmitter and hormone action
- 48. GPCR and arrestins Shown to have in vivo physiological roles in mediating the functions of GPCRs Implicated in development of tolerance to and dependence on drugs Safety mechanisms to prevent the over stimulation of GPCRs Could be important targets for the development of drugs to prevent tolerance development to established drugs and prolong the therapeutic activity
- 49. Orphan GPCRs 200or so known GPCRs whose endogenous ligands and functions are not known Attempts have been made to deorphanise these receptors Evidence that some recently deorphanised GPCRs, such as orexin receptor, may dimerise or associate with more classical GPCRs
- 50. British Journal ofPharmacology (2008) 153 S339–S346
- 51. GPCR mutations, diseaseand novel drug discovery Loss of function mutations in GPCRs involved in the control of endocrine systems Homozygous loss of function mutations in the type 5 chemokine receptor provides resistance to HIV infection Critical for the infectivity of this virus Gain of function mutations in GPCRs also cause disease Mutations in GPCRs could be responsible for variations in drug sensitivities among different populations
- 52. mAbs 2:6, 594-606;November/December 2010; © 2010 Landes Bioscience
- 53. Tools for GPCRdrug discovery Receptor binding assay G-protein dependent functional assays GTPγS binding assay cAMP assay IP3/IP1 and Ca2+ assays Reporter assay Generic G-protein independent functional assays Receptor internalization assay β-arrestin recruitment assay Label-free whole cell assays Receptor dimerization assay Acta Pharmacol Sin. 2012 March; 33(3): 372–384
- 55. Conclusion Nearly 40%of the drugs approved for marketing by the FDA target GPCRs 800-1,000 different GPCRs and the drugs that are marketed target less than 50 GPCRs GPCR will continue to be highly important in clinical medicine because of their large number, wide expression and role in physiologically important responses Future discoveries will reveal new GPCR drugs, in part because it is relatively easy to screen for pharmacologic agents that access these receptors and stimulate or block receptor-mediated biochemical or physiological responses
- 56. REFERENCES Goodman andGilman’s Pharmacological basis of therapeutics, 12thed Rang and Dale’s pharmacology, 7th edition Alexander SPH, Mathie A, Peters JA (2011). Guide to Receptors and Channels (GRAC), 5th edn. Br J Pharmacol 164 (Suppl. 1): S1–S324. Gurevich, E.V., et al., G protein-coupled receptor kinases: More than just kinases and not only for GPCRs,JPT Elsevier doi:10.1016j.pharmthera.2011.08.001JPT-06382; GLIDA-GPCR ligand database version 2.04 10/10/2010
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Editor's Notes
- #9 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 molecules
- #12 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
- #13 which convert GTP to GDP. The β and γ subunits remain together as a βγ complex.
- #15 βγ-mediated effects occur at higher levels of receptor occupancy than α-mediated effects otherwise the action of it is same to alpha
- #16 guanine nucleotide exchange factor (GEF)
- #17 Target 2? The GTPase activity of the α subunit is increased when the target protein is bound, leading to hydrolysis of the bound GTP to GDP, whereupon the α subunit reunites with βγ.
- #19 An intracellular enzyme play an imp. Role in signal transduction participate in phosphatidylinositol 4,5-bisphosphonate(PIP2) and lipid signaling in a calcium dependent manner.