Molecular Cell Biology

Prof. D. Karunagaran
Department of Biotechnology
Indian Institute of Technology Madras

Module 7
Cell Signaling Mechanisms
Lecture 2
GPCR Signaling








Receptors - G protein coupled receptors (GPCRs) conserved in many
species.
Seven transmembrane (7TM)-receptors.
Also called serpentine receptors (Like a snake).
Coupled to heterotrimeric guanine nucleotide binding proteins (G proteins)
Heterotrimeric - refers to three different proteins G, G and G
There are also some monomeric G proteins such as Ras (H-Ras, N-Ras and
K-Ras) that do not couple with GPCRs
GPCR signalling contains many druggable targets
There are over 800 GPCRs in the human genome.
About 150 of these GPCRs have no known ligands orphan receptors.
More than 300 can potentially serve as targets for various disorders
5060% of all current therapeutic agents are targeting GPCRs directly or
indirectly
Structure of a typical GPCR
All GPCRs contain seven transmembrane alpha-helical
Domains, each composed of 2535 amino acid residues.
The highly hydrophobic helices
three extracellular and three intracellular loops,
amino (N-) terminus extracellular
carboxyl (C-) terminus intracellular














Classification of seven transmembrane receptors
Class Ligands
Class A Rhodopsin family

Ligand anchors between the gaps of
transmembrane units
Rhodopsin/cone opsin/melanopsin
Epinephrine/nor epinephrine
Acetyl choline
Histamine
Serotonin
Thromboxane
Prostaglandin
Melatonin
Class B Secretin family
Binding of ligand occurs at the N-
terminus (60-80 residues)
Secretin
Glucagon
PTH
GnRH
CRF
Class C Glutamate and GABA family
Ligand gets trapped between two
distinct lobes at the N-terminus
Glutamate
GABA
Taste stimuli (sweet)
Frizzled class Wnt
Hedgehog
Taste stimuli (bitter)
Adhesion family Chondroitin sulfate
Unidentified signals

Gonadotrophin releasing hormone GnRH; Corticotropin release factor CRF
neuropeptide.



Classifying the ligands
Biogenic amines
Noradrenaline, dopamine
5-HT, histamine,
Acetylcholine
Amino acids and ions
Glutamate, calcium
GABA
Lipids
LPA, SIP, Prostaglandins, leukotrienes
Peptides and proteins
Chemokines, angiotensin, thrombin, endothelin, bradykinin
Others
Light, odorants, nucleotides


Class Second messenger
G
s

-adrenergic receptor, glucagon
receptors, serotonin receptors
cAMP
Adenylyl cyclase activation
G
i

1-adrenergic receptor
cAMP
Adenylyl cyclase inhibition
G
q

2-adrenergic receptor
IP3, DAG
Phospholipase C activation
G
12

LPA receptor
RhoGEF mediated activation
of Rho
G proteins
Numerous members - 16 G subunits, five G subunits, and 14 G subunits
Most if not all have GTPase activity.
The Ga subunits are 3952 kDa proteins divided into four families based on
their sequence similarity: Gi/o, Gs, Gq/11, and G12/13.
Beta subunits 43kDa; Gamma subunits 7.5-10 kDa.
Each G subunit consists of two domains, a GTPase domain and an alpha
helical domain.
In between these two domains is a cleft where the guanine nucleotide binds
Lipid modification of a Cys residue near the N-terminus of most G subunits
allows for binding to membrane.
C-terminus of all G subunits appears to be important for interaction with
the receptor.
G is needed for the binding of the G subunit to the receptor, for the
formation of high-affinity agonist binding, and for receptor catalyzed
activation of G protein
G is also attached to the membrane by a lipid modification of the Gg
subunit and together the G dimer acts as a scaffolding protein.
Following receptor activation, both G and G act as signaling molecules.
Activation of a GPCR results in the release of GDP from G. Until GTP
binds, a high-affinity complex is formed between the receptor and G protein.
The exchange of GDP for GTP leads to dissociation of the G dimer from
the G subunit, and both can initiate their own intracellular signaling
responses
A single GPCR can couple to one or more of G proteins
G-proteins and second messengers
G proteins
Activation of Gq may lead to activation of phospholipase C, an enzyme that
catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to
1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).
IP3 interacts with receptors on intracellular calcium stores resulting in
cytosolic release of calcium while DAG can activate protein kinase C
isoforms.
Other G proteins modulate a large number of second messenger systems
including cyclic AMP, cyclic GMP, calmodulin, and kinases.



























Adenylyl cyclase









Proteins Regulated by cAMP-Development Phosphorylation (by PKA)





















Glycogen synthase
Phosphorylase b kinase
subunit
subunit
Pyruvate kinase (rat liver)
Pyruvate dehydrogenase complex
(type L)
Hormone sensitive lipase
Phosphofructokinase-2/fructose
2,6

bisphosphatase
Tyrosine hydroxylase
Histone H1
Histone H2B
Pathway / process regulated
Glycogen synthesis
Glycogen breakdown
Glycolysis
Pyruvate to acetyl-CoA
Triacylglycerol mobilization and fatty
acid oxidation
Glycolysis / gluconeogenesis
Synthesis of L-dopa, dopamine,
Norepinephrine and epinephrine
DNA condensation
DNA condensation
Ligand induced activation of signal-transducing G proteins associated with
GPCRs

The G dimer acts as a molecular switch that can be
turned on and off via the GTPase cycle




















Desensitization




Ligand mediated activation of GPCRs is terminated by G protein-coupled
receptor kinases (GRKs)
GRKs phosphorylate the receptor
This results in a simultaneous association with arrestins
This complex now interacts with clathrin and the clathrin adaptor AP2
This drives receptor internalization into endosomes.
This is a functional process of receptor desensitization.
Association of arrestins to activated GPCRs can also lead to the initiation of
distinct arrestin-mediated signaling pathways
GPCRs may then be taken to lysosomes, where they are ultimately degraded,
or to recycling endosomes for recycling back to the cell surface
(resensitization).


GPCR Signaling can Generate Multiple Circuits
A simple way is to recruit different G proteins from a single ligand-receptor
interaction
Non-G protein-signaling is also known to occur - G protein-independent
signaling
GPCR may associate with numerous membrane and intracellular proteins
that will potentially alter ligand affinity, ligand selectivity, signaling,
cytoskeletal and extracellular matrix interactions, and receptor internalization.
GPCRs may also form homo- or heterooligomerization to induce
transactivation of other receptors or lead to signal modification. GPCR
phosphorylation, acetylation, palmityolation, ubiquitinylation, and
myristoylation also modify receptor functional properties.
Clearly, all these can generate numerous circuits which have to be
integrated.

Study Questions

1. What are GPCRs?
2. Wrie a note on the second messengers involved in GPCR signaling
3. Most of the G-proteins possess a) No enzyme activity b) Cyclase activity
c) ATPase activity d) GTPase activity
4. Match the following
Desensitisation AP2
LPA PKC
Clathrin Rho
DAG GRKs
5. Receptors without a known ligand are called ---------------