Intro to GPCRs

Question 1

Different examples of GPCR stimuli

Answer 1

NTs, neuropeptides, calcium, light, relaxin (for labour).

Question 2

General structure of GPCRs

Answer 2

N-terminus, 7 TM domains, 3 EC loops, 3 IC loops, C-terminus

Question 3

Classes of GPCRs and structural characteristics of them

Answer 3

A: rhodopsin, e.g., D, AR, opioids, rhodopsin. Short

B1: secretin, e.g., glucagon, CGRP, PTH. Medium EC domain.

B2: adhesion, e.g., latrophilin-1. Large EC domain with GAIN and adhesion domains

C: glutamate, e.g., mGluRs and GABAB. Large venus fly trap EC domain.

F: frizzled, e.g., frizzled-1, smoothened. Large EC domain

Question 4

Subunits of G-proteins and their isoforms

Answer 4

Ga is membrane associated. it has GTPase activity. active when bound GTP. has 4 subfamilies: Gas, Gai/o, Gaq/11, Ga12/13

GBy: is am obligate heterodimer that is also membrane associated. 6 GB members. 12 Gy members

Question 5

G-protein cycle

Answer 5

Binding of an agonist induces a conformational change. This initates a conformational change in Ga, enabling GTP binding (exchange of GDP).

The Ga-GTP subunit dissociates from GBy. Both Ga-GTP and GBy activate effector proteins.

GTP in Ga is hydrolysed to GDP and the Ga-GDP reassociates with GBy

Question 6

Signalling cascades of the different Ga isoforms

Answer 6

Gas stimulates adenylate cyclase, increasing cAMP - activates PKA.

Gaq activates PLCgamma, signalling through DAG (activates PKC) and IP3 (mobilises intracellular calcium stores)

Gai/o inhibits adenylate cyclase reducing cAMP and thus PKA activity.

Ga12/14 activates RhoA, which through Rho effectors induces changes to the cytoskeleton.

Question 7

Desensitisation and internalisation of GPCRs

Answer 7

GBy subunit interacts with G-protein receptor kinases (GRKs), which phosphorylate the receptor. can also be done by other kinases.

This increases the affinity of the receptor for B-arrestin. B-arrestin prevents G-protein binding - desensitisation.

B-arrestin can also drive receptor internalisation through interactions with Clathrin coated pits, which transport the receptor to endosomes for storage, or to lysosomes for degradation (down regulation).

Question 8

Way to measure GTP binding experimentally

Answer 8

Utilising GTPyS, a non-hydrolysable form of GTP. It can be tagged radioactively or fluorescently.

It accumulates in active G-proteins, and then separation by filtration can identify the extent of GTPyS that bound to Ga, and the amount that flows through the filter unbound.

Question 9

Examples of uses of BRET/FRET

Answer 9

For PPIs, protein-protein proximity (recruitment), and to identify conformational changes in a single protein.

A miniG could be used to measure recruitment of G-proteins. it has no GTPase activity.

Ga and GBy could be tagged to measure their dissociation.

Interactions between GBy and a high affinity tether can measure dissociation of GBy.

B-arrestin recruitment can be measured with a tagged B-arrestin.

cAMP can be measured using a cAMP sensor. The sensor will have both the BRET donor and acceptor, releasing a BRET signal when no cAMP is bound. Upon cAMP binding, the sensor undergoes a conformational change that prevents the BRET from occurring.

Same thing can be done with Ca2+ sensors, except in these, calcium binding causes a heightened BRET signal.