Biochemical characterization of RGS14: RGS14 activity towards G-protein α subunits is independent of its binding to Rap2A

摘要

pRGS (regulators of G-protein signalling) modulate signalling by acting as GAPs (GTPase-activating proteins) for α subunits of heterotrimeric G-proteins. RGS14 accelerates GTP hydrolysis by Gsubiα/sub family members through its RGS domain and suppresses guanine nucleotide dissociation from Gsubiα1/sub and Gsubiα3/sub subunits through its C-terminal GoLoco domain. Additionally, RGS14 binds the activated forms of the small GTPases Rap1 and Rap2 by virtue of tandem RBDs (Raf-like Ras/Rap binding domains). RGS14 was identified in a screen for Rap2 effectors [Traver, Splingard, Gaudriault and De Gunzburg (2004) Biochem. J. b379/b, 627–632]. In the present study, we tested whether Rap binding regulates RGS149s biochemical activities. We found that RGS14 activity towards heterotrimeric G-proteins, as either a GAP or a GDI (guanine nucleotide dissociation inhibitor), was unaffected by Rap binding. Extending our biochemical characterization of RGS14, we also examined whether RGS14 can suppress guanine nucleotide exchange on Gsubiα1/sub in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated Gsubiα1/sub and prenylated Gsubβγ/sub is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free Gsubα/sub and suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of Gsubβγ/sub-mediated signalling pathways./p