GPCR's Flashcards

1
Q

What are GPCR’s

A

7 transmembrane domains which form alpha helices

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

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2
Q

G proteins are made up of

A

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

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3
Q

G-protein cycle

A

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

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4
Q

Alpha subunits: CLASSES?

A

Gq/11: increased PLC activity

Gs: increased adenylyl cyclase activity

Gi: decreased AC activity

G12/13: regulates GTP exchange factors

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5
Q

Beta and Gamma subunit purpose:

A

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

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6
Q

Signal adaption

A

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)

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7
Q

What is desensitisation?

A

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

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8
Q

Desensitisation is driven by:

A

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)

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9
Q

What does beta-arrestin do?

A

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

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10
Q

GPCRs might NOT always signal through G proteins

A

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

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11
Q

Production Beta-arrestin biased ligans for COVID

A

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?

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12
Q

Functional GPCRs are not always a single polypeptide: calcitonin

A

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

effective modulators of biological activity
receptors are unclones

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13
Q

CGRP receptors

A

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:

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14
Q

RAMP

A

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

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15
Q

What happens in the absence of RAMP?

A

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

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16
Q

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

A

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)

17
Q

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

A

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.

18
Q

Functional GPCRs agonists derived from the receptor protein

A

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)

19
Q

Protease activated receptor

A

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)

20
Q

Inverse agonist

A

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

21
Q

Summary

A

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