GPCR's

Question 1

What are GPCR’s

Answer 1

7 transmembrane domains which form alpha helices

Extracellular N-terminus and agonist binds here
G proteins interact with C-terminus

Question 2

G proteins are made up of

Answer 2

A smaller beta and gamma subunit which tend to fuse and act as a monomer
Larger alpha subunit which dictates the preferential interaction

Question 3

G-protein cycle

Answer 3

Upon agonist binding, alpha subunit develops a higher affinity for GTP rather than GDP
Upon GTP binding, the alpha subunit dissociates the beta-gamma subunits which tend to stay within the lipid phase
The alpha subunit goes onto interact with a specific enzyme
Alpha-GTP complex activates a key enzyme; at the same time the alpha subunit hydrolyses GTP back into GDP such that is reassociates with beta-gamma subunit again

Question 4

Alpha subunits: CLASSES?

Answer 4

Gq/11: increased PLC activity

Gs: increased adenylyl cyclase activity

Gi: decreased AC activity

G12/13: regulates GTP exchange factors

Question 5

Beta and Gamma subunit purpose:

Answer 5

Interaction with Kach = Kir3.1 / 3.4 heteromer (GIRK channels)
Direct binding to VDCCs (especially N-type) leads to inhibiton
They stimulate PI-3-kinases
They stimulate Mitogen Activated Protein Kinases
They stimulate Adenylate cyclase

Question 6

Signal adaption

Answer 6

Rather than the linear pathway we are familiar with, instead GPCRs actually exhibit a retrograde signalling pathway that can lead to the receptor turning off (shown in blue)

Question 7

What is desensitisation?

Answer 7

The recruitment of a receptor kinase phosphorylates the receptor which leads to the receptor ‘turning off’ and becoming internalised – this is called desensitization

Question 8

Desensitisation is driven by:

Answer 8

G-protein receptor kinases (GRK1-3)

These have a high affinity for free beta-gamma subunits

They phosphorylate GPCR

The phosphorylated receptor is a target for beta-arrestin

GRKs lead to functional downplay of receptors (evidence: suggests GRK siRNA prevents desensitisatio)

Question 9

What does beta-arrestin do?

Answer 9

Associates with proteins involved with protein trafficking such as clathrin, which leads to the endocytosis of the receptor such that it is removed from the membrane – now there is far less receptor for agonist to activate

Beta-arrestin 1 associates with Non-receptor Tyrosine kinase (c-SRC) such that it activates various tyrosine molecules downstream

Beta-arrestin 2 associates with c-Jun N-terminal kinase 3 (JNK3) which is a kinase implicated in various mitogenic activities

Extracellular signal regulated kinases 1 and 2 (ERK1/2) – also involved in MAPK activation

Question 10

GPCRs might NOT always signal through G proteins

Answer 10

the ability to direct a G protein–coupled receptor to selectively signal through a G protein–mediated pathway or a β-arrestin–mediated pathway is known as biased signaling

Question 11

Production Beta-arrestin biased ligans for COVID

Answer 11

The virus that underlies COVID-19 binds, via its spike protein, to ACE 2 causing it to be internalised such that it replicates and wreaks havoc

ACE 2 converts angiotensin II to angiotensin-(1–7) which can interact with AT receptor and preferentially activates beta arrestin which activates pathways involved in cell survival and changes in cardiac contractility

Hyperactivation of the Ang II type 1 receptor (AT1R) is major contributor to adverse outcomes in patients with COVID-19–related acute respiratory distress syndrome (ARDS) - – could we develop and AT type 1 receptor anatagonist?

Question 12

Functional GPCRs are not always a single polypeptide: calcitonin

Answer 12

Calcitonin family of peptides comprises:
- calcitonin
- amylin
- two calctitonin gene related peptides (CGRP1 & CGRP2)
- adrenomedullin (ADM)

effective modulators of biological activity
receptors are unclones

Question 13

CGRP receptors

Answer 13

Discovery of a “calcitonin receptor-like receptor” (CLR) unresponsive to CGRP stimulation when expressed in cell lines.

Execept in human embryonic kidney (HEK293) cells - cloned CRLR CGRP produced a 60-fold increase in cAMP generation following blocked by CGRP8–37.

HEK293 cells endogenously express a protein that, when coexpressed with CLR, responded to CGRP.

This protein became known as Receptor activity modifying protein or RAMP:

Question 14

RAMP

Answer 14

small (148-175-amino acid) proteins
single predicted membrane-spanning domain
large extracellular domain
short cytoplasmic domain
THEY ALTER RECEPTOR RESPONSES MASSIVELY

They are a chaperone such that they ‘hold hands’ with the receptor protein and helps it to get to the membrane so it can be glycosylated to be fully functional

Question 15

What happens in the absence of RAMP?

Answer 15

In the absence of RAMP, certain receptor proteins like the CLR protein end up being maintained in the endoplasmic rectiulum rather than the cell membrane

Question 16

As well as this, RAMPs dictate the pharmacology of these receptors once they’re in the membrane:

Answer 16

RAMP 1: associated CLR have a different pharmacology to the same receptor associated with RAMP-2

RAMP-1= CGRP
RAMP-2/RAMP-3= AM (Adrenomedullin)

Question 17

Some functional GPCRs are not always a single polypeptide-dimerisation:

Answer 17

In neurones GABA produces rapid responses through GABAA R (ligand-gated ion channel)

and slower responses through GABAB (GPCR)

But expression of the gene encoding a GABAB receptor (GABABR1) = fails to recreate native receptor

However co-expression with GABABR2 in HEK293T cells=

GABABR1 is fully glycosylated

expressed at the cell membrane

binds GABA with a high affinity equivalent to that of the endogenous receptor.

Question 18

Functional GPCRs agonists derived from the receptor protein

Answer 18

There are also protein-activated GPCRs in which enzymes chomp the end off and then the end activates the receptor
Thrombin has long-lasting effects bc it cleaves amino terminus of a small family of GPCRs; the last 6 amino acids are important in activating the receptor – protease activated receptor (PAR)

Question 19

Protease activated receptor

Answer 19

PAR1 th > tryp SFLLRN (h) or SFFLRN (m, r)

PAR2 trypsin SLIGKV (h) or SLIGRL (m, r)

PAR3 thrombin TFRGAP (h) or SFNGGP (m, r)

PAR4 tryp > thr GYPGQV (h) or GYPGKF (m, r)

Question 20

Inverse agonist

Answer 20

The work on inverse agonist has led to the acceptance that many , if not ALL GPCRs are actually active in the absence of ligand.
The ligand tips the balance towards greater turnover / greater coupling

Question 21

Summary

Answer 21

GPCRs can become auto-regulated by the phosphorylation of various amino acids through G-protein receptor kinases (GRK1-3)
This phosphorylated receptor protein can bind beta-arresting and then various things can happen
Translocation into other cellular organelles and out of the membrane
Activation of long-term signalling cascades e.g tyrosine kinases, MAPKs which have very different biological effects than the phospolipase c / cAMP signalling
They can be associated with other proteins; CLR is associated with RAMP which dictates the presence of the main agonist receiving protein in the membrane as well its pharmacology
Type C sub-family of GPCRs form functional dimers with large EC domain (venus-fly trap domain)
There are also protein-activated GPCRs in which enzymes chomp the end off and then the end activates the receptor