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G-protein coupled receptors and drugs
modulating them
DR. NISHIKANT SHARMA
DR. PRIYANKA KUMAWAT
• General description of 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
History
1967. 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 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.
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 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 transduction
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
Signals to which cell respond
Antigen
Cell surface glycoproteins/ oligosaccharides
Extracellular matrix component
Growth factors
Hormones
Neurotransmitters
Light
Mechanical touch
Nutrients
Odorants
Pheromones
Tastants
Primary
Messengers
Secondary
Tertiary
Transmit the signal from 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, 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+2
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
G- proteinA family 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”.
Types of G-protein
1. “Large" G proteins (Heterotrimeric)Activated by GPCRsMade 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) subunitAlso bind GTP and GDP and are involved in signal
transduction.
G-protein subunits with second messenger
β γ α
Gs Gi Gq
cAMP stimulationβ receptorHistamineSerotoninDopamine
cAMP inhibitionα2 receptorM2 receptorOpioid receptorD2 receptor5HT1 receptor
PLC(IP3 & DAG)α1
M1
AT1
5HT2
Vasopressin
•Activate potassium channels• Inhibit voltage-gated calcium channels• Activate mitogen-activated protein kinase cascade.
Golf-Odorant receptor,Adenylyl cyclase
Gt- cGMP phosphodiesterase , cGMP
Gα12/13 -Rho family GTPase signaling and control
cell cytoskeleton remodeling and regulating cell migration.
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
GTP
GDP
GDPGTP
4 ATP
4 cAMP
Cell response
AT
Protein kinase
ADP
PInactive protein
Active protein
hormone
Adenylate cyclase Signaling System
AC
RS
Inhibitor
Ri
Phospholipase-c signaling systemPIP2
IP3 DAG
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
GPCR classes Class A- 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
Second messengers
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 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
Cyclic AMP dependent protein 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
Cyclic Amp–Regulated Guanine Nucleotide Exchange 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
PhosphodiesterasesHydrolyze the cyclic 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
ZAqw
Energy metabolism
cAMP and Immunomodulation
Am J Respir Cell Mol Biol Vol 39. pp 127–132, 2008
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
Diacylglycerol and protein kinase C
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
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
Ca2+
In skeletal and 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
Ion channels as targets 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
The Rho/Rho kinase system
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
DesensitizationReceptor 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
RECENT DEVELOPMENTS
GPCR dimerisation The conventional 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
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.
Agonist specificityCellular effects 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
GPCR and arrestinsFollowing 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
GPCR and arrestinsShown 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
Orphan GPCRs200 or 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
British Journal of Pharmacology (2008) 153 S339–S346
GPCR mutations, disease and 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
mAbs 2:6, 594-606; November/December 2010; © 2010 Landes Bioscience
Tools for GPCR drug discovery
Receptor binding assay
G-protein dependent functional assaysGTPγS binding assaycAMP assayIP3/IP1 and Ca2+ assaysReporter assay
Generic G-protein independent functional assaysReceptor internalization assayβ-arrestin recruitment assayLabel-free whole cell assaysReceptor dimerization assay
Acta Pharmacol Sin. 2012 March; 33(3): 372–384
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
REFERENCES Goodman and Gilman’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
Let the future begin THANK YOU