1.
G-Protein-Coupled Receptors
Dr. Prashant Shukla
Junior Resident
Dept of Pharmacology

2.
2
Contents
 Introduction
 Historical Background
 GPCR - Basics
 GPCR as Targets for Drug Designing
 GPCR associated Diseases
 Concept of Orphan GPCR
 Future Prospects & Conclusions

3.
Introduction
3
Receptors are the sensing elements in
the system of chemical
communications that coordinates the
function of all the different cells in the
body.
The chemical messengers can be
hormones, transmitters and other
mediators.

4.
4
Types of receptors

5.
 The G protein-coupled receptor (GPCR)
superfamily comprises the largest and
most diverse group of proteins in
mammals.
 Synonym: “seven-transmembrane” (7-
TM), “serpentine”
receptors, heptahelical
receptors, serpentine receptor, and G
protein–linked receptors (GPLR).
5
GPCRs

6.
GPCRs
 It is involved in information transfer
(signal transduction) from outside the cell
to the cellular interior.
 GPCRs are responsible for every aspect
of human biology from vision, taste,
sense of smell, sympathetic and
parasympathetic nervous functions,
metabolism, and immune regulation to
reproduction.
 ~45% of all pharmaceutical drugs are
6

7.
Receptors associated with
GPCRs
1. GABAB Receptors
 GABABR1 and GABABR2
2. Taste Receptors
 T1R3 and T1R2
3. Adrenergic Receptors
 Three subfamilies (α 1, α 2 and β )
 Family A (rhodopsin-like) GPCRs
4. Opioid Receptors
 Three cloned subtypes: δ, қ and μ
7

8.
Receptors associated with
GPCRs
5. Somatostatin Receptors
 Five subtypes (SSTR1-5)
6. Purinergic Receptors
 Neurotransmitters in the CNS, CVS, immune
system, and other tissues i.e. adenosine and
ATP
7. Olfactory Receptors
 Represent the largest family of GPCRs, with
>300 members
8. Vasopressin, Oxytocin and Other
Receptors 8

9.
The importance of GPCRs
1. Number (C.elegans 1100; H. sapiens, ~1000;
D. melanogaster, 160; reflects number of
olfactory receptor genes in worm [~1000] and
mammal [several hundreds]), a few % of
genome; 300-400 non-olfactory GPCRs)
2. Diversity (mostly small molecule ligands)
3. Evolutionarily conserved yeast to man (yeast
Ga 45% identical to mammalian Gia)
4. Pharmaceutical importance: ~500 known
molecular targets of drugs, 60% of these are
cell surface receptors, 75% of these are
GPCRs (GPCRs = ~45% of all known drug
targets) 9

10.
Historical background
10

11.
 Robert Lefkowitz and Brian Kobilka:
the 2012 Nobel Prize in Chemistry for
groundbreaking discoveries that reveal
the inner workings of an important family
of receptors: G-protein–coupled
receptors.
11
Historical background

12.
12
Common Experimental Tools used to
Study GPCRs

13.
GPCR- basics
13
 Structure
 Classification
 Signal molecules/ Ligands
 Physiological role
 G proteins
 Mechanism of action

14.
GPCR- Basic structure
CYTOSOL
EXTRACELLULA
R
14
Extracellular loops
Intracellular loops
Plasma
membrane

15.
15
GPCR- Basic structure

16.
GPCR: Classification
Based on Sequence homology and
functional similarity
◦ Class A (or 1) (Rhodopsin-like)
◦ Class B (or 2) (Secretin receptor family)
◦ Class C (or 3) (Metabotropic glutamate/
pheromone)
◦ Class D (or 4) (Fungal mating pheromone
receptors)
◦ Class E (or 5) (Cyclic AMP receptors)
◦ Class F (or 6) (Frizzled/Smoothened)
16

17.
Based on phylogenetic origin:
The GRAFS classification system has
been proposed
1. Glutamate
2. Rhodopsin
3. Adhesion
4. Frizzled/Taste
5. Secretin
17
GPCR: Classification

18.
18
GPCR
Tree

19.
Signal molecules/ Ligands of
GPCRs
GPCRs interact with a number of ligands
ranging from photons, ions, amino
acids, odorants, pheromones,
eicosanoids, neurotransmitters,
peptides, proteins, and hormones.
Nevertheless, for the majority of GPCRs,
the identity of their natural ligands is still
unknown, hence remain orphan
receptors.
19

20.
Signal molecules
20
Biogenic amines: Adrenaline,
noradrenaline, dopamine, 5-HT,
histamine, acetylcholine
Amino acids and ions: Glutamate,
Ca2+, GABA
Lipids : PAF, prostaglandins,
leukotrienes, anandamine

21.
21
Peptides / proteins :
GnRH, angiotensin, bradykinin, thrombin,
bombesin, glucagon, calcitonin,
vasoactive intestinal peptides, PTH,
FSH, LH, TSH
Nucleotides : Adenosine nucleotides,
adenine nucleotides, uridine nucleotides
Others : Light, odorants, pheromones,
opiates
Signal molecules…

22.
Physiological roles
22
1. Visual sense: Rhodopsin
2. Sense of smell: Olfactory receptor
3. Behavioral and mood regulation:
Serotonin, dopamine, GABA and glutamate
4. Immune system activity and
inflammation: Chemokine receptors,
histamine receptors
5. ANS transmission: β adrenergic receptors
6. Apoptosis

23.
Structure of G Protein
G proteins, also known as guanine
nucleotide-binding proteins, involved in
transmitting signals and function as
molecular switches.
Their activity is regulated by factors that
control their ability to bind to and hydrolyze
GTP to GDP. When they bind GTP, they are
'on', and, when they bind GDP, they are 'off '.
23

24.
 G protein complexes are made up of
 20 alpha (α)
 6 beta (β)
 12 gamma (γ) subunits.
 Beta and gamma subunits
can form a stable dimeric
complex referred to as the
beta-gamma complex.
α subunit
β subunit
γ subunit
24

25.
Types of G Proteins
25

26.
G protein cycle
26
Basal state
Activated state

27.
GPC Receptors
G
Protein
Receptors Signaling Pathway
GS
Beta adrenergic receptors,
glucagon, histamine, serotonin
Increase CAMP
Excitatory effects
Gi
Alpha2 adrenergic receptors,
mAchR, opioid, serotonin
Decrease CAMP
Cardiac K+ channel
open- decrease heart rate
Gq
mAchR, H1, α1, Vasopressin
type 1, 5HT1C
PLC- IP3 , DAG
Increase Cytoplasmic Ca
Gt
Rhodopsin and colour opsins
in retinal rod and cone cells
Increase cGMP
phosphodiesterase.
Decrease cGMP
27

28.
G Protein Mediated
Pathways
Secondary messenger Systems
Involved In Signal Transduction:
Adenylate cyclase cAMP mediated
pathway
 Phospholipase mediated pathway
GPCR s can also directly activate the
ion channels
28

29.
cAMP Mediated Pathway
The cAMP-dependent pathway, also known
as the adenylyl cyclase pathway, is a G
protein-coupled receptor triggered signaling
cascade used in cell communication.
 Gs cAMP Dependent Pathway
 Gi cAMP Dependent Pathway
29

30.
GTP
GDP
 GDP
GTP

ATP
cAMP
Cell response
AT
Protein
kinase
ADP
P
Inactive
protein
Active
protein
hormone
Adenylate cyclase
Signaling
System
AC
RS
Inhibitor
Ri


CYTOSOL
EXTRACELLULA
R
30
Gs cAMP Dependent Pathway

31.
Gi cAMP Dependent Pathway
31

32.
32
CYTOSOL
EXTRACELLULA
R
Gq Protein Coupled Receptor

33.
33
Gt PCR: involved in photo transduction.
Gt Protein Coupled Receptor

34.
Signal Amplification through G
proteins
34

35.
Regulation of GPCRs
Turning GPCRs Off
 A cell must also be able to stop
responding to protect overstimulation
 High activation of a receptor leads to a
reduced ability to be stimulated in the
future (desensitization)
 Can also significantly limit therapeutic
usefulness of many receptor agonists.
35

36.
36
Desensitization mechanisms include
1. “down-regulation” or reduction of
receptor number
2. “sequestration” or apparent shielding
of the receptors from interacting
ligands
3. “uncoupling” from G-proteins.
Regulation of GPCRs

37.
 Homologous desensitization: The
activation dependent regulation of
receptors.
 Heterologous desensitization: Receptor
activation-independent regulation of
receptors.
37
Regulation of GPCRs

38.
38

39.
Homologous desensitization
 The activated state of GPCRs serves not
only as an activator of G proteins, but
also as the substrate for GPCR kinases
(GRKs).
39

40.
40

41.
41
Homologous desensitization

42.
 Based on feedback regulation of
receptors by the second-messenger-
regulated kinases.
 Eg. Upon stimulation, β- receptors leads
to ↑ cAMP, which activates PKA. PKA
can then phosphorylate the β- receptors
themselves, even those particular
receptor proteins that were not activated
by the current stimulation. These PKA-
phosphorylated receptors are less able to
mount a response. 42
Heterologous desensitization

43.
Receptor Drugs and some key indications
AT1 angiotensin
II receptor
Antagonists e.g. losartan in treatment of HT or
CHF
α1A-c receptor
Antagonists e.g. tamsulosin to treat disorders
asso. with enlarged prostate
β1- receptor
Antagonists e.g. propranolol, atenolol, metoprolol,
carvedilol to treat essential HT or CHF
β2- receptor
Agonists e.g. terbutaline, salbutamol, formoterol for
treatment of COPD or Bronchial asthma
D2 receptor
Antagonists e.g. Haloperidol & clozapine to treat
schizophrenia
Agonists e.g. levodopa for Parkinsonism
43
GPCR as drug targets

44.
44
Receptor Drugs and some key indications
D3 receptor Antagonists e.g. haloperidol in schizophrenia
5-HT2A receptor
Antagonists e.g. clozapine for schizophrenia.
Indirect agonists e.g. fluvoxamine for
depression
5-HT2C
receptor
Antagonists e.g. clozapine for schizophrenia
CCR5
Associated with progression of AIDS e.g.
Aplaviroc and maraviroc
M3
Antagonists e.g. Atropine to dilate pupil;
Scopolamine for motion sickness
Neuropeptide S
receptor
Asthma susceptibility e.g. Neuromedin and
neurotensin
Associated with bleeding diathesis e.g.
GPCR as drug targets…

45.
45
GPCR as drug targets…

46.
Diseases associated with G-
proteins
Abnormal G protein signalling can result
by
1. Bacterial toxins (Cholera and pertussis)
2. Gene mutations
 Loss of function mutations
 Gain of function mutations
3. Altered GPCR folding
46

47.
Mutations in GPCR
Mutations in genes encoding are an
important
cause of human disease
Help to define critical structure-function
relationships
Two types –
 Loss-of-function : Block signalling in
response to the corresponding agonist(s)
 Hormone resistance, mimicking hormone deficiency
 Gain-of-function : Lead to constitutive,
agonist-independent activation of signaling
 Mimic states of hormone excess 47

48.
Cone opsins Colour blindness X-linked, AR
Rhodopsin Retinitis pigmentosa AD; AR
V2
vasopressin
Diabetes insipidus X-Linked
ACTH Familial ACTH resistance AR
LH ♂ pseudohermaphrodite AR
TSH Cong. hypothyroidism AR
TRH Central hypothyroidism AR
Diseases caused by Loss of function
Mutation
48

49.
49
FSH Hypergonadotropic
Ovarian failure
AR
Ca2+
sensing
Hypocalciuric
hypercalcaemia
AD
Ca2+ sensing Neonatal hyperthyroidism AR
GHRH G H deficiency AR
GnRH Central hypogonadism AR
Endothelin-B Hirschsprung disease Complex
Melanocortin
4
Extreme obesity Co-dominant
PTH/PTHrP Chondrodysplasia AR
Diseases caused by Loss of function
Mutation

50.
50
Rhodopsin Congenital night blindness AD
LH Familial ♂ precocious
puberty
AD
LH Sporadic Leydig cells
tumours
Somatic
TSH Familial non-autoimmune
hyperthyroidism
AD
TSH Sporadic hyperfunctional
thyroid adenomas
Somatic
Ca2+ sensing Familial hypocalcaemia AD
PTH/PTHrP Jansen metaphyseal
chondrodysplasia
AD
Diseases caused by Gain of function
Mutation

51.
Mis-folded GPCRs
Point mutations resulting in protein sequence
variations may result in production of mis-folded
and disease-causing proteins
 Retain proper function but end up in parts of
cell where function is inappropriate, or even
deleterious, to cell function.
51

52.
Disease/
Abnormality
GPCR
Pharmacoperones
Retinitis pigmentosa Rhodopsi
n
9-cis-retinal, 11-cis-retinal, 11-cis-
7-ring retinal
Nephrogenic
diabetes insipidus
V2R
SR121463 (satavaptan),
SR49059 (relcovaptan), VPA-985,
YM087, OPC41061 (tolvaptan),
OPC31260
Hypogonadotropic
hypogonadism
GnRHR
Indoles, quinolones,
erythromycin-derived macrolides
Familial hypocalciuric
hypercalcemia
Ca2+
sensing
NPS R-568
Diseases due to GPCR misfolding
52

53.
POLYMORPHISMS OF GPCR
 Variations in GPCR gene sequence
can have important consequences
beyond causing Mendelian diseases
 As more polymorphisms are
discovered more examples of
variations in GPCR gene sequence will
be found
53

54.
POLYMORPHISMS.... Challenges Ahead
Whether such differences are
important in individual variation
in drug response
(pharmacogenomics)
Whether they could confer
susceptibility to disease.
54

55.
Allosteric Modulators of G-
protein
•Bind receptor domains topographically distinct
from orthosteric site, altering biological activity
of orthosteric ligand by changing its binding
affinity, functional efficacy or both.
• Potential for engendering greater GPCR
subtype-selectivity
• Challenge for detecting /validating allosteric
behaviors
• Contribute to physical or pathophysiological
processes.
55

56.
ORPHAN GPCRs
 Lack their pharmacological identities
 Pre-genome era: Most GPCRs were found
by sequence similarity using nucleic acid-
based homology screening approaches
 After genome sequencing: 150 Orphan
GPCRs using bio-informatic analysis
 First Orphan GPCR was G21, later found to
be 5HT1A receptor in 1988
Focus of intense research effort, both in academia and in industry
56

57.
57
GPCR types No. of members Orphan receptors
Glutamate-
class GPCRs
22 Two third (15)
Rhodopsin-
class GPCRs
701 63
Adhesion-
class GPCRs
33 Majority
Frizzled/ taste
GPCRs
36 (11 frizzled and
25 taste)
None among
frizzled ; Most
taste
Secretin-class
GPCRs
15 None

58.
The de-Orphanization of GPCRs
 Evolutionarily conserved and thus are
expected to be active
 Reverse Pharmacological Approaches based
on receptor reactivity & receptor binding are
applied
 Isolating natural ligand provides a first hint of
function, structural cues for lead design
 Once de-orphanised, GPCRs can be used
for designing new drugs.
58

59.
Tools for de-orphanization
High -throughput screening
 GPCR over expressing cells
 Ligand libraries: chemicals, serum, peptides
 Finding a robust marker: Measure receptor
binding or Receptor reactivity
 Finding an endogenous ligand
59

60.
Search available orphans that have an effect
on a specific therapeutic area
Analyze Orphans using:
1. Laser capture micro-dissection to determine the localization
2. Microarray to compare the level of transcript expression
Screening against Compound Libraries
Identify compound hits and optimize for pre-clinical
and, if successful, clinical trials
60

61.
Issues of Orphan GPCR
research
 Deorphanization is a risky, lengthy and
demanding endeavour
 GPCRs exist not only as monomers but as
dimers or higher oligomers
 Concentration of transmitters in their
natural environment.
61

62.
GPCR Screening
 Cell-based screens performed with calcium-
sensitive or membrane-potential-sensitive
dyes
 Gs- and Gi-coupled GPCRs are assayed via
cAMP determinations using either a cell-
based real time cAMP assay or other
validated cAMP assay platform
 All screens include positive controls and a
comprehensive report.
62

63.
Recent developments
 Ligand-induced selective signaling (LiSS):
It states that different ligands selectively recruit
different intracellular signaling proteins to
produce different phenotypic effects in cells .
 Terry Kenakin proposed this concept and is
rapidly becoming a generic theme for
GPCRs.
 This phenomenon is referred to by different
groups using a variety of terms such as:
“functional selectivity,” “biased agonism,”
“ligand-selective agonism,” “agonist-directed
trafficking of signaling,” or “agonist-receptor 63

64.
It has important implications in specific drug
development and in minimizing side effects.
E.g. the effects of the two naturally occurring
GnRHs, GnRH I and GnRH II, operating
through the single GnRH type I receptor. GnRH
I is much more potent in generating inositol
phosphate than in its antiproliferative effects on
certain cells, whereas GnRH II does not show
much difference between these two effects. An
extreme example is a GnRH antagonist, which
has no intrinsic stimulation of inositol
phosphate generation but has potent
antiproliferative activity. It has been shown that
the Tyr8 of GnRH II is the main determinant of
selective antiproliferative effects and identified
residues in the TM domains and ECLs of the
64

65.
 The LiSS concept has now been demonstrated for
many GPCRs and is creating a new level of
sophistication, which challenges the dogma that
ligand engagement of a GPCR consistently elicits a
specific intracellular signal. Instead, it has become
increasingly clear that the nature of the ligand and
the dynamically changing intracellular environment
alter the flavor of the signaling. Indeed, it appears
that there is a new era of drug discovery on our
doorstep, in which screening for novel ligands will
not simply involve receptor binding and/or the most
convenient high-throughput functional signal output
but instead will screen for the appropriate
intracellular signal, which reflects the desired
phenotypic response of a cell for a disease state or
pathophysiology. Equally, appropriate cells will have
to be used to ensure an appropriate intracellular
context. Although these challenges are substantial,
we believe they will bear fruit in the longer term
efforts of GPCR drug discovery in the spin-off
benefits of reduced failure in the clinic through lack
of specificity and off-target effects. 65

66.
66

67.
GPCR signaling independent of
G proteins
 There are many ways in which GPCRs
can signal independently of G proteins.
So, a case has been made for
abandoning the term “G protein-coupled
receptors” and referring to them as
“seven-transmembrane receptors.”
 The first convincing evidence for the
existence of GPCR-independent
signaling came from the works of 67

68.
 An example is angiotensin II at its
AT1 receptor activating both β-arrestin and
G proteins. When antagonists such as
angiotensin II-receptor blockers (losartan
and valsartan) engage the binding site,
neither signal is propagated. However,
another type of antagonist (SII) does not
activate the G protein pathway but
exclusively recruits β-arrestin and
activates ERK.
68
GPCR signaling independent of
G proteins…

69.
Constitutively active receptors
 G-protein-coupled receptors may also be
constitutively (i.e. spontaneously) active
in the absence of any agonist.
 This was first shown for β-adrenoceptor.
 Histamine H3 receptor also shows
constitutive activity.
 It means that inverse agonists can play a
role here.
69

70.
GPCRs and drug discovery
 Regarded as “Drug Discovery Engines”
of 21st Century
 G protein-coupled receptors (GPCRs)
represent 50-60% of the current drug
targets.
 The pace of GPCR-targeted new
molecular entities (NMEs) approved by the
USFDA in the recent years still remains to
a level near its historical average, with five
in 2010, five in 2011, seven in 2012, six in
2013, and eight in 2014. 70

71.
Novel pancreatic β-cell GPCRs
 About 20 GPCRs have been found in pancreatic β-cells, all
of which can potentially stimulate or inhibit insulin secretion.
The glucagon-like peptide 1 (GLP1) receptor is one of
these. Insulin secretion is stimulated by glucose transport
through the glucose transporter 2 into the β-cell.
 Activation of GPCRs such as GLP1 can enhance the
amount of intracellular calcium, for example through
activation of Gαq/11 and subsequent generation of IP3 and
release of Ca++ from intracellular stores, thereby
potentiating glucose stimulation of insulin secretion.
 Among the other GPCRs identified in β-cells are the newly
discovered free fatty acid receptors, GPR40, 43, and 41.
GPR40 couples to Gαq/11, so free fatty acid would enhance
the calcium response of the β-cell to glucose and increase
insulin secretion. Insulin responses to glucose are
improved in mutant mice overexpressing the GPR40
receptor and in normal rats treated with GRP40 agonists . 71

72.
GPCRs as new therapeutic targets
for type 2 diabetes
GPCRs that have received recent
attention in the field of diabetes
therapeutics include
1. Incretin receptors: GLP1R, GIPR
(GPR119)
2. Free fatty acid receptor:FFAR1
(GPR40), FFAR4 (GPR120)
3. Bile acid receptor: GPBAR1 (TGR5) 72

73.
Novel neuroendocrine GPCRs
regulating reproduction
 There have been a number of breakthroughs in
neuroendocrinology in the last year.
 After the seminal discovery that kisspeptin/GPR54
acts as a major whole-body sensor mediating
diverse effects on the GnRH neuron described
mutations in neurokinin B (NKB) and its receptor
(TACR3), which give rise to hypogonadotropic
hypogonadism and pubertal failure.
 The discovery of NKB, dynorphin A, and GnIH as
neuroendocrine regulators has provided new
opportunities for research on novel GPCRs in fine
tuning the hypothalamic-pituitary-gonadal axis and
provides new pathways in which to interrogate
feedback mechanisms and metabolic, photoperiod, 73

74.
Role of H4 receptor in asthma
H4 receptor was discovered with an orphan
GPCR gene sequence followed by
pharmacology characterization.
Animal models suggested a role for the H4
receptor in mediating asthma and chronic
pruritus associated with conditions such as
atopic dermatitis.
TheH4 antagonist JNJ 39758979 has recently
been found to have efficacy in preclinical
models of pruritus, dermatitis, asthma, and
arthritis. Several other H4 antagonists have also
been entered into clinical trials for these 74

75.
Concept of pharmacopherones
Pharmacoperones or Chaperone
 Small nonpeptide molecules do scaffolding
in order to promote correct folding.
 Regulation of routing of cellular proteins will
provide opportunity for novel drug
development.
75

76.
Permeate plasma membrane
Enter cells
Correct folding
Allowing routing to plasma membrane
How Chaperones Work ?
76
Bind selectively to misfolded proteins

77.
Deorphanisation of GPR55
 GPR55 has been recently deorphanized to
be a receptor for lysophophatidylinositol.
Other GPR55 ligands identified so far are
neither cannabinoids nor bind to the
cannabinoid CB1 and CB2 receptors.
 GPR55 has been implicated in three
therapeutic areas, including the regulation
of energy intake and expenditure,
resorption of bone, and agonist pro-
carcinogensis. 77

78.
The simple dogma that underpins
much of our current understanding of
GPCRs, namely,
one GPCR gene− one GPCR protein−
one functional GPCR− one G protein
−one response
is showing distinct signs of wear.
78
Future Prospects & Conclusions

79.
Future Prospects & Conclusions
 Ever expanding field of research
 Concept is diverting from a linear signaling to
increasingly complex signaling networks
 Next generation platforms for studying
internalisation and heterodimerisation is to
adopt novel, universal β-arrestin
recruitment assays for known and orphan
GPCRs instead of second messenger
signaling assays
79

80.
 Further studies are needed to explore
 Advances in novel forms
 Methods to rescue function of misfolded or
truncated GPCRs
 Complexity demands collaborative
approaches between persons of medicinal
chemistry, analytical pharmacologists &
bioinformatic experts
80
Future Prospects & Conclusions

81.
 GPCRs were once considered highly
tractable targets.
 Current targets much lower success
rates
◦ Low hanging fruit largely picked
◦ Lack of Hits
◦ Hits have high molecular weight
 Poor PK/in vivo activity
 Difficult to optimize
81
Future Prospects & Conclusions

82.
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