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

RGS (regulators of G-protein signalling) modulate signalling by acting as GAPs (GTPase-activating proteins) for a subunits of heterotrimeric G-proteins. RGS14 accelerates GTP hydrolysis by G_iα family members through its RGS domain and suppresses guanine nucleotide dissociation from G _iα1 and G_iα3 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. 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, 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 G_iα1 in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated G_iα1 and prenylated G_βγ is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free G _α and suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of G _βγ-mediated signalling pathways.