Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity.
Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.
Ladds
Graham
G
Division of Clinical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK. Graham.ladds@warwick.ac.uk
Goddard
Alan
A
Hill
Claire
C
Thornton
Steven
S
Davey
John
J
D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010675:Pheromones / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D020710:RGS Proteins / Q000235:genetics / Q000502:physiology*; D012568:Schizosaccharomyces / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction
D010675:Pheromones; D021122:Protein Subunits; D020710:RGS Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11