G-protein coupled receptors (GPCRs) are the largest family of membrane receptors and the target of about 30-40% of drugs. They signal through heterotrimeric G proteins which activate downstream effectors like adenylyl cyclase, phospholipase C, and ion channels. This leads to the production of second messengers cyclic AMP, inositol trisphosphate, and diacylglycerol which activate kinases and regulate important cellular processes. Modulation of GPCRs and their associated G proteins and effectors provides opportunities for drug development.

1. G-protein coupled
receptors and drugs
modulating them

2. 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.

3. 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”.

4. 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.

5. G-protein subunits with
second messenger
β γ
α
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.

6.  Golf-Odorant receptor, Adenylyl cyclase
 Gt- cGMP phosphodiesterase  , cGMP 
 Gα12/13 -Rho family GTPase signaling and control
cell cytoskeleton remodeling and regulating cell
migration.

7. 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

8. G-protein Coupled Receptors (GPCR) Families
Largest class of membrane proteins,
more than 700 GPCRs identified
Target of about 30-40% of all drugs on
the market (but target only a small
fraction of the receptors
Sometimes called 7 transmembrane
receptors
Conserved structure:

9. G-protein coupled receptor

10. Family Class A Class B
(Secretin)
Class C
(Glutamate)
Adhesion Frizzled
Receptors w/ligands 197 15 12 0 11
Orphan 87 8 26 0
Olfaction 390
Vision 10
Taste 30 3
Pheromone 5
Total 719 15 23 26 11
GPCR Families
http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=694
GPCRs are classified into 5 families, which is defined by IUPHAR/BPS

11. 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

12. Rhodopsin family (Class A) which includes receptors for a wide variety of small molecules,
neurotransmitters, peptides and hormones, together with olfactory receptors, visual pigments, taste type 2
receptors and five pheromone receptors (V1 receptors).
Secretin family (Class B), encoded by 15 genes in humans. The ligands for receptors in this family are
polypeptide hormones; nine of the mammalian receptors respond to ligands that are related (glucagon,
glucagon-like peptides (GLP-1, GLP-2), glucose-dependent insulinotropic polypeptide (GIP), secretin,
vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP) and
growth-hormone-releasing hormone (GHRH).
Glutamate family (Class C), which includes metabotropic glutamate receptors, a calcium-sensing
receptor and GABAB receptors, as well as three taste type 1 receptors and a family of pheromone
receptors (V2 receptors) that are abundant in rodents but absent in man.
Adhesion Family GPCRs are related to class B receptors, from which they differ by possessing large
extracellular N-termini that are autoproteolytically cleaved.
Frizzled Family consists of 10 Frizzled proteins (FZD(1-10)) and Smoothened (SMO). The FZDs are
activated by secreted lipoglycoproteins of the WNT family, whereas SMO is indirectly activated by the
Hedgehog (HH) family of proteins acting on the transmembrane protein Patched (PTCH).
GPCR Families

13. GPCR Structure
Ligand Binding Domain
G-protein interaction domain
Phosphorylation sites
(desensitization,
internalization, Arrestin
binding

14. Tautermann C 2014 Bioorg Med Chem Ltrs 24:4073-79
Meng, E.C. and Bourne, H.R. 2001. Trends in Pharmacol Sci, 22: 587-593.
GPCR Structure
Generally:
Ligand binding occurs in TM2-TM5 domains
Affects the positioning of TM6 and TM7 changing the interaction with the G-Protein

15. Heterotrimeric G-proteins
Heterotrimeric G-proteins
a-subunit with GTPase activity.
b-subunit
g-subunit
16 Gα subunit genes,
6 Gβ subunits encoded by 5 genes
12 reported Gγ subunits in human

16. G-protein cycle and GTP hydrolysis
G-protein cycle - simplified
1. In the resting state (no
ligand), receptor can
associate with G-protein.
2. Ligand binding promotes
exchange of GDP for GTP
3. This promotes dissociation of
the G-protein a and bg
subunits and initiation of
signaling
4. Ga subunit hydrolyzes GTP,
promoting reformation of the
heterotrimeric G-protein

17. G-protein Subtypes Expression Signaling
Gs-family
as
aolf
1
1
(stimulation of adenylyl cyclase)
Ubiquitous
Olfactory epithelium, some
neurons
AC (subtypes 1-9)
AC (subtypes 1-6)
Gi-family
ao (A,B)
ai (1-3)
az
2
3
1
(inhibition of adenylyl cyclase)
Neurons, neuorendocrince cells,
astroglia, cardiac muscle
Ubiquitous
Platelets
PLCb
AC (subtypes 1-10)
AC
Gq-family
aq,
(q,11,14,15,16)
5 Ubiquitous PLCb
Unrelated
at
agust
a12/13
1
2
2
Retina
Taste buds
Ubiquitous
gPDE
PLCb
GTPases
G-protein a-subunits

18. Targets of G proteins
 Adenylyl cyclase
 IP3/DAG Phospolipase C
system
 Ion channels esp. potassium
and calcium
 Rho a/ Rho kinase system

19. GTP
GDP
a GDP
GTP
a
4 ATP
4 cAMP
Cell response
AT
Protein
kinase
ADP
P
Inactive
protein
Active
protein
hormone
Adenylate cyclase
Signaling System
AC
RS
Inhibitor
Ri
g
b

20. Gs/Gi mediated signaling pathways: Adenylyl Cyclase
Gs/Gi-coupled receptor signaling
1. Receptor binds ligand leading to
activation of Gas or Gai
2. Gas activates adenylyl cyclase (AC),
whereas Gai inhibits AC
3. AC hydrolysis ATP to produce cAMP
a second messenger.
4. cAMP can activate cAMP-dependent
protein kinase/protein kinase A.
5. Phosphodiesterases (PDE) mediate
the breakdown of cAMP.

21. 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

22. Class I
Class II
Class III
Class IV
Brain
Olfactory, pancreas
Brain, pancreas
Brain, lung
Heart, striatum
Ubiquitous
Ubiquitous
Ubiquitous
Heart, kidney
Gs Gi
Adenylyl Cyclase
Willoughby D. and Cooper DM. Amer J Physiol. 2007 87: 965.
Adenylyl Cyclase (AC) has multiple isoform divided into 4 family types that show differential
tissue distribution and regulation by Gas and Gai
C1 and C2 are the catalytic domains and dimerize to form an active enzyme
TM1 and TM2 are transmembrane domains

23. 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

24. 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

25. 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

26. Phosphodiesterases
Phosphodiesterases are enzymes that degrade cyclic monoamines – cAMP and cGMP
There are three types:
cAMP-specific: PDE 4, 7, 8
cGMP-specific: PDE 5, 6, 9
Dual substrate-specific: PDE 1, 2, 3, 10, 11
Maurice DH et al 2014 Nat Rev Drug Disc 13 290.

27. 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

28. Gq mediated signaling pathways: Phospholipase C
Gq-coupled receptor signaling
1. Receptor binds ligand leading to activation of Gaq
2. Gaq activates Phospholipase C (PLC) leading to cleavage of
phosphotidylinositol 4,5 bisphosphate (PIP2) in the
membrane.
3. Cleavage of PIP2 produces inositol 1,4,5, trisphosphate (IP3)
and diacylglycerol (DAG).
4. IP3 mediates release of Ca2+ from intracellular stores (ER)
through activation of the IP3 receptor
5. DAG required for activation of protein kinase C (PKC)
6. Inositol phosphates are degraded by inositol phosphate
phosphatases.

29. 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

31. 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

32. 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

33. 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

34. 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

35. Gq mediated signaling pathways: Protein Kinase C
Steinberg S., 2012 Amer J Physiol. 27: 130.
PKC family members
1. Conventional PKCs: a, bI, bII, g
DAG and Ca2+ required for activation
2. Novel PKCs: d, e, h, q
Ca2+ dependent, DAG independent
3. Atypical PKCs z, l
Ca2+ and DAG independent, activated
by lipids like phosphatidyl inositols
phosphatidic acid, arachadonic acid
PKC implicated in multiple process in cardiac myocytes
1. Inotropy – through phosphorylation of sarcomeric proteins
2. Cell death/Survival – multiple targets
3. Hypertrophy
PKC mediated cardiac hypertrophic signaling
1. Gq-coupled receptor activate multiple isoforms of PKC in adult
cardiac myocytes
2. PKCa, bII, d, e all have been implicated in hypertrophic signaling
3. nPKCs, d and e, proposed to activate PKCm, which has been
reclassified as protein kinase D (PKD)
4. PKD induces phosphorylation of Histone Deacetylases – relief of
transcriptional repression – increased transcription.

36. 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

37. G12/13 mediated signaling pathways
G12/13-coupled receptor signaling
1. Receptor binds ligand leading to
activation of Ga12/13
2. Ga12/13 link to RhoGEF, and Rho family
of GTPases (members of the Ras
superfamily)
3. Involved in cytoskeletal regulation, and
actin dynamics
Link to Gq-coupled receptor signaling
1. Gaq is linked to growth responses
(mitogenic in tumors, hypertrophic
growth in heart) that are not mediated
by PLCb, but rather activation of Rho.
2. This is achieved by signaling through a
Rho-GEF (guanine exchange factor) –
which bind to “Dbl-homologous" (DH)
domains
3. In this regard, both Gq and G12/13-
coupled receptors can activate Rho-
mediated signaling pathways.

38. GPCR Dimerization
Possible states
Pharmacology
Adenosine A1-P2Y1
a2AR-b1AR
AT1A-B2
D1-a1AAR
D2-SSTR5
D2-D3
M2-M3
dOR-mOR
dOR-kOR
Internalization
a1AAR-a1BAR
a1BAR-a1DAR
a2AR-b1AR
A2a-D2
AT1-B2
b1AR-b2AR
b2AR-b3AR
b2AR-kOR
b2AR-mOR
CCR2-d, m, kOR
CCKA-CCKB
dOR-kOR
ETA-ETB
SST2a-SST3
SST2a-mOR
TRH1-TRH2
Trafficking
AT1A-B2
CaR-mGluR1
CaR0mGluR5
Signaling
a1AAR-a1BAR
mGluR1a-A1
mGluR1a-A2
AT1A-AT2
AT1A-b2AR
AT1A-B2
b1AR-b2AR
b2AR-b3AR
CCR2-CXCR4/5
CCKA-CCKB
D1-D2
GABAbR1-GABAbR2
dOR-mOR
dOR-kOR
Functional Consequences of Receptor Dimerization
Prinster S, Hague R and Hall R 2005 Pharmacol Rev 57:289

39. 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

40. 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

41. Second messengers

44. 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.

45. 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

46. 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

47. 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

48. British Journal of Pharmacology (2008) 153 S339–

49. 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

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

51. 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

52. 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

53. 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

54. Let the
future begin