The RGS (regulator of G-protein signalling) and GoLoco domains of RGS14 co-operate to regulate Gi-mediated signalling

RGS (regulator of G-protein signalling) proteins stimulate the intrinsic GTPase activity of the α subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 has been shown previously to stimulate the GTPase activities of Gα_o and Gα_i subunits through its N-terminal RGS domain, and to down-modulate signalling from receptors coupled to G_i. It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Gα_i. In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate G_i signalling, we generated RGS14 mutants invalidated in each of its domains, as well as truncated molecules, and assessed their effects on G_i signalling via the βγ pathway in a stable cell line ectopically expressing the G_i-coupled M₂ muscarinic acetylcholine receptor (HEK-m2). We show that the RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of G_i. Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on G_i signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. We therefore propose that RGS14 combines the inhibition of G_i activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Gα_i–GTP via its RGS domain to negatively regulate signalling downstream of G_i.