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 Gαo and Gαi subunits through its N-terminal RGS domain, and to down-modulate signalling from receptors coupled to Gi. 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 Gαi. In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate Gi signalling, we generated RGS14 mutants invalidated in each of its domains, as well as truncated molecules, and assessed their effects on Gi signalling via the βγ pathway in a stable cell line ectopically expressing the Gi-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 Gi. Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on Gi 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 Gi activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Gαi–GTP via its RGS domain to negatively regulate signalling downstream of Gi.

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