1. G-protein coupled
receptors and drugs
modulating them

2. • 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

3. 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.

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
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.

8. 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

9. Signals to which cell 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
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, 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
 A 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”.

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
second messenger
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,Adenylyl cyclase
 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
4 cAMP
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
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

19. 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

20. Second messengers

23. Targets of G proteins
 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 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

26. 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

27. Phosphodiesterases
 Hydrolyze 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

28. ZAqw

29. Energy metabolism

30. cAMP and Immunomodulation
Am J 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
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

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 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

40. 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

41. 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

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

43. RECENT
DEVELOPMENTS

44. 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

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
 Cellular 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

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
 200 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

50. British Journal of Pharmacology (2008) 153 S339–S346

51. 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

52. mAbs 2:6, 594-606; November/December 2010; © 2010 Landes Bioscience

53. Tools for GPCR drug 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 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

57. Let the
future begin