GPCRs Flashcards
GPCR structure
3 EC loops for ligand binding, 3 IC loops for G proteins, allosteric modulators, other second messengers
Agonist binding alters relative positions of TM3 and TM6
No single active conformation, there are many, which may give rise to biased signalling
Many exist in dimers, which requires cholesterol in a lipid raft
Alpha subunit
4 types (s AC, i AC, q PLCbeta, 12 p115-RhoGEF
8 human genes
40 kDa
Beta subunit
5 bladed propellor arrangement
7 human genes
38 kDa
Gamma subunit
12 human genes
8 kDa
Modifications to alpha/beta/gamma subunits
alpha and gamma are altered for insertion into the cell membrane - myristoylation on the amino group of the terminal glycine, palmitoylation
Betagamma come as a pair, don’t really swap partners. Functional significance of different permutations unknown.
Was previously thought that alpha subunits share betas from a pool, but fusing them together did not prevent translocation or activation, and FRET based studies have shown they don’t move apart far on activation
GPCR interaction with G protein
We don’t know whether a G protein stays associated with a receptor even when receptor is inactive, but G proteins definitely bind preferentially to certain conformations (link to ligand bias)
But we have more conformations that bias away from Arrestin binding that towards.
Rosenbaum et al 2011 - the same conformation was produced in B2AR from agonist and inverse agonist application!
Single-molecule imaging has shown actin fibres criss-crossing the membrane, that create ‘hot-spots’ for receptor-G protein interactions. They’ll transiently bind when they bump into each other, but only form a lasting interaction when a ligand is bound.
Inhibitors of Galphai
RGS proteins - have 120 aa RGS box, GTPase Activating Proteins
GPR proteins - 20-30 aa GPR motif, Guanine Nucleotide Dissociation Inhibitors
Guanine Exchange Factors
They will only bind inactive G proteins (i.e. won’t bind Gi-protein-GTP), which ensures directionality
Girdin aka GIV was the first GEF to have its GEF sequence found
Ric-8a, Ric-8b, Get3 and various others are also GEFs but we haven’t found a conserved motif for it.
Ric-8a is essential for normal RTK function, but is also a chaperone protein for betagamma, so how much is its role is due to GEF function vs chaperone function?
Girdin - binding detail
Originally identified in yeast studies
Mainly binds alpha i, binds alpha s a bit, doesn’t bind alpha q at all
Has a motif to bind to the G protein, and another to bind to the receptor, so can cause G protein signalling from all sorts of receptors
–SH2 domain, part of receptor binding domain, shows structural plasticity, existing in a disordered state until recruited by an activated RTK
Short aliphatic helix of GEF motif binds between switchII and alpha3 helix of alpha subunit, which explains why it can’t bind active G protein (because switch II has moved to occlude the site)
This binding site also may mean it displaces betagamma. How much of its effects are actually due to betagamma signalling downstream?
-but FRET studies have suggested betagamma doesn’t need to dissociate to be active, so maybe this is a moot point -
G proteins with non GPCRs
Both beta2 and alpha7 nAChRs can physically associate with alpha i, q, 12, and betagamma.
nAChR can coimmunoprecipitate with Gprin1 and Gai.
Gprin1 controls axon growth. nAChR and Gprin1 form a complex that activates GAP-43, which binds cytoskeleton and promotes growth cone growth
When ACh binds, the growth cone breaks down
Applying betagamma increases nAChR channel open probability 5 fold (perhaps every subunit in the pLGIC can bind betagamma?)
Girdin can bind all sorts of different receptors, and mediate their signalling via G proteins, but we only know its mechanism of binding to RTK
Yevenes et al 2003 - betagamma can bind GlyR via the M3-M4 loop of its alpha subunit. Binding is phosphorylation-independent, but affected by pertussis toxin. Binding increases affinity for Glycine, and may affect amplitude of current
Girdin - in disease
GEF motif has been specifically implicated in increasing metastasis in invasive cancer, and can be used as a predictor of survival for colorectal or breast cancer (Liu et al 2012).
Girdin downregulates antifibrotic pathways (by activating Gi, to reduce cAMP, PKA, CREB) and upregulates profibrotic pathways (e.g. PI3K, FoxO1), thus increasing liver fibrosis. GIV expression is undetectable in hepatocytes, but high in hepatic stellate cells.
In nephrotic syndrome, Girdin is protective, because it reduces apoptosis of podocytes, via the prosurvival pathway PI3K, in response to VEGF.
3 basic properties of G protein signalling
Gain (there’s always amplification)
Convergence (many transmitters and receptors will activate the same subset of G protein),
Divergence (one transmitter can activate various receptors, one receptor can work via different G proteins..?)
GPCRs and the genome
3% of genome is GPCRs, most of them olfactory
Olfactory receptors, and others, are part of the rhodopsin family, characterised by a lysine in TM7 for attachment of chromophore.
Other families include Frizzled (24), Adhesion (24), GABA (15), and Secretin (15).
Classification is based on agonist specificity and subtle structural differences
Peptides must bind to loops, monoamines can go down into TM domain, proteases can cleave to activate
GPCRs in healthcare
50-70% of drugs target GPCRs, though much of the phylogenetic tree is uninterrogated - we have not yet found many drugs that will bind
Many diseases are caused by mutations in GPCRs, e.g. single amino acid substitution in V2 vasopressin mutation causes diabetes insipidus
Hard to find specific drugs that bind ligand pocket of GPCRs because of homology between them. Allosteric sites are less conserved, and allosteric modulators modulate physiological responses rather than binary activate or inactivate, so may have fewer side effects. E.g. Cmpd-15 is the first allosteric ‘beta-blocker’.
Allosteric modulators that bind at the intracellular side of two chemokine receptors have been found, may be of use in treating chronic inflammatory disorders.
5-HT7 and 5-HT1A are both important in depression, and homodimeris of 5-HT1A are resistant to 5-HT-mediated internalisation, so maybe pattern of dimerisation is important in pathogenesis.
Homologous and heterologous desensitisation
Can cause G protein independent signalling
Heterologous desensitisation - PKA phosphorylates activated receptor, changes preference of B2AR from Gs to Gi, an there’s a negative feedback loop AC, cAMP, PKA. Homologous desensitisation - Then GRK can phosphorylate, and recruit arrestin (a new signalling scaffold), then clathrin mediated endocytosis
Signalling can happen from the intracellular compartment, via G proteins or other pathways, e.g. via c-Src and the MAPK pathway
Different GPCRs go through different internalisation pathways. All pass through early endosome, but e.g. GPER (binds steroids) ends up in the perinuclear compartment - transcriptional effects??
Gate-keepers inc PRABs determine whether the receptor gets recycled from endosome or broken down
There’s also caveolin-dependent and clathrin and caveolin independent. There’s recycling from the endosome, sequence dependent or in bulk.