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

摘要

RGS (regulators of G-protein signalling) Modulate signalling by acting as GAPs (GTPase-activating proteins) for a Subunits Of heterotrimeric G-proteins. RGS 14 accelerates GTP hydrolysis by G(i alpha) family members through its RGS domain and suppresses guanine nucleotide dissociation from G(i alpha 1) and G(i alpha 3) Subunits through its C-terminal GoLoco domain. Additionally, RGS 14 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. 379, 627-632]. In the present study, we tested whether Rap binding regulates RGS14's biochemical activities. We found that RGS14 activity towards heterotrimeric G-proteins, its either a GAP or it 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 G(i alpha 1) in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated G(i alpha 1) and prenylated G(beta gamma) is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free G(alpha) and Suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of G(beta gamma)-mediated signalling pathways.