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G-Protein-Coupled Receptors: Dr. Prashant Shukla Junior Resident Dept of Pharmacology
G-Protein-Coupled Receptors: Dr. Prashant Shukla Junior Resident Dept of Pharmacology
Historical Background
GPCR - Basics
2
Introduction
4
GPCRs
The G protein-coupled receptor (GPCR)
superfamily comprises the largest and
most diverse group of proteins in
mammals.
7
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
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)
10
Historical background
11
Common Experimental Tools used to
Study GPCRs
12
GPCR- basics
Structure
Classification
Signal molecules/ Ligands
Physiological role
G proteins
Mechanism of action
13
GPCR- Basic structure
Extracellular loops
EXTRACELLULA
R
Plasma
membrane
CYTOSOL
Intracellular loops
14
GPCR- Basic structure
15
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
GPCR: Classification
17
GPCR
Tree
18
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.
22
Structure of G Protein
G proteins, also known as guanine
nucleotide-binding proteins, involved in
transmitting signals and function as
molecular switches.
23
G protein complexes are made up of
20 alpha (α) γ subunit
6 beta (β)
α subunit
12 gamma (γ) subunits.
24
Types of G Proteins
25
G protein cycle
Basal state
Activated state
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GPC Receptors
G Receptors Signaling Pathway
Protein
GS Beta adrenergic receptors, Increase CAMP
glucagon, histamine, serotonin Excitatory effects
Gi Alpha2 adrenergic receptors, Decrease CAMP
mAchR, opioid, serotonin Cardiac K+ channel
open- decrease heart rate
Gq mAchR, H1, α1, Vasopressin PLC- IP3 , DAG
type 1, 5HT1C Increase Cytoplasmic Ca
27
G Protein Mediated
Pathways
Secondary messenger Systems
Involved In Signal Transduction:
Adenylate cyclase cAMP mediated
pathway
Phospholipase mediated pathway
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cAMP Mediated Pathway
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Gs cAMP Dependent Pathway
hormone Inhibitor
EXTRACELLULA
R
RS Ri
AC
GDP
GTP
GDP
CYTOSOL
GTP ATP
AT P
Inactive
Protein protein
cAMP kinase
31
Gq Protein Coupled Receptor
EXTRACELLULA
R
CYTOSOL
32
Gt Protein Coupled Receptor
Gt PCR: involved in photo transduction.
33
Signal Amplification through G
proteins
34
Regulation of GPCRs
Turning GPCRs Off
A cell must also be able to stop
responding to protect overstimulation
37
38
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
Homologous desensitization
41
Heterologous desensitization
Based on feedback regulation of
receptors by the second-messenger-
regulated kinases.
43
GPCR as drug targets…
45
Diseases associated with G-
proteins
Abnormal G protein signalling can result
by
2. Gene mutations
Loss of function mutations
Gain of function mutations
Two types –
Loss-of-function : Block signalling in
response to the corresponding agonist(s)
Hormone resistance, mimicking hormone deficiency
48
Diseases caused by Loss of function
Mutation
FSH Hypergonadotropic AR
Ovarian failure
Ca2+ Hypocalciuric AD
sensing hypercalcaemia
Ca2+ sensing Neonatal hyperthyroidism AR
GHRH G H deficiency AR
GnRH Central hypogonadism AR
Endothelin-B Hirschsprung disease Complex
Melanocortin Extreme obesity Co-dominant
4
PTH/PTHrP Chondrodysplasia AR
49
Diseases caused by Gain of function
Mutation
51
Diseases due to GPCR misfolding
Disease/ Pharmacoperones
GPCR
Abnormality
53
POLYMORPHISMS.... Challenges Ahead
56
GPCR types No. of members Orphan receptors
Glutamate- 22 Two third (15)
class GPCRs
Rhodopsin- 701 63
class GPCRs
Adhesion- 33 Majority
class GPCRs
Frizzled/ taste 36 (11 frizzled and None among
GPCRs 25 taste) frizzled ; Most
taste
Secretin-class 15 None
GPCRs
57
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
Tools for de-orphanization
High -throughput screening
GPCR over expressing cells
59
Search available orphans that have an effect
on a specific therapeutic area
61
GPCR Screening
Cell-based screens performed with calcium-
sensitive or membrane-potential-sensitive
dyes
Pharmacoperones or Chaperone
75
How Chaperones Work ?
Permeate plasma membrane
Enter cells
Correct folding
76
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.
80
Future Prospects & Conclusions