Novel Activity of RGS14 on Goα and Giα Nucleotide Binding and Hydrolysis Distinct from Its RGS Domain and GDI Activity

The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for Giα and Goα and a guanine nucleotide dissociation inhibitor (GDI) for Giα. This GDI activity is isolated to a region of the protein distinct from the RGS domain that contains an additional G protein-binding domain (RBD/GL). Here, we report that RGS14 missing its RGS domain (R14-RBD/GL) binds directly to Go and Gi to modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. In brain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14) bind Goα-GDP, Giα-GDP, and also Gβγ. When reconstituted with M2 muscarinic receptors (M2) plus either Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulates the steady-state GTPase activities of both G proteins, whereas R14-RBD/GL has little or no effect. R14-RBD/GL potentiates RGS4 GAP activity in membrane-based assays by increasing the apparent affinity of RGS4 for Giα and Goα, suggesting a cooperative interaction between the RBD/GL domain, RGS4, and Gα. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assays with purified Giα or Goα, suggesting that membranes and/or receptors are required for this activity. When these findings are taken together, they indicate that regions of RGS14 outside of the RGS domain can bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Gα nucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDI activity.