GPCR's Flashcards
What are GPCR’s
7 transmembrane domains which form alpha helices
Extracellular N-terminus and agonist binds here
G proteins interact with C-terminus
G proteins are made up of
A smaller beta and gamma subunit which tend to fuse and act as a monomer
Larger alpha subunit which dictates the preferential interaction
G-protein cycle
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
Alpha subunits: CLASSES?
Gq/11: increased PLC activity
Gs: increased adenylyl cyclase activity
Gi: decreased AC activity
G12/13: regulates GTP exchange factors
Beta and Gamma subunit purpose:
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
Signal adaption
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)
What is desensitisation?
The recruitment of a receptor kinase phosphorylates the receptor which leads to the receptor ‘turning off’ and becoming internalised – this is called desensitization
Desensitisation is driven by:
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)
What does beta-arrestin do?
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
GPCRs might NOT always signal through G proteins
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
Production Beta-arrestin biased ligans for COVID
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?
Functional GPCRs are not always a single polypeptide: calcitonin
Calcitonin family of peptides comprises:
- calcitonin
- amylin
- two calctitonin gene related peptides (CGRP1 & CGRP2)
- adrenomedullin (ADM)
effective modulators of biological activity
receptors are unclones
CGRP receptors
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:
RAMP
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
What happens in the absence of RAMP?
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