Novel Activity of RGS14 on GoR and GiR Nucleotide Binding and Hydrolysis Distinct from Its RGS Domain and GDI Activity†

The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for GiR and GoR and a guanine nucleotide dissociation inhibitor (GDI) for GiR. 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 GoR-GDP, GiR-GDP, and also Gâγ. 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. R14RBD/GL potentiates RGS4 GAP activity in membrane-based assays by increasing the apparent affinity of RGS4 for GiR and GoR, suggesting a cooperative interaction between the RBD/GL domain, RGS4, and GR. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assays with purified GiR or GoR, 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 GR nucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDI activity. Heterotrimeric G proteins (GRâγ) transduce signals from cell-surface receptors to intracellular signaling pathways to mediate the actions of many neurotransmitters and hormones at target cells (1-4). After agonist binding and receptor activation, G proteins are activated such that GR-guanosine triphosphate (GTP)1 and a Gâγ complex act alone or in concert to regulate the action of target effector proteins. GR subunits are GTPases that act as molecular switches, and the lifetime of GR-GTP dictates the lifetime of the signaling event. The protein regulators of G protein signaling (RGS proteins) act as GR GTPase activating proteins (GAPs) to limit the duration of the linked GR signaling event (5). Mammalian RGS and RGS-like proteins comprise a family of more than 30 distinct proteins, which serve to modulate and/or integrate receptor and linked G protein signaling (6, 7). All family members share a conserved 130 amino acid RGS domain that binds to activated GR subunits and confers GAP activity. In addition, RGS proteins can directly interfere with effector binding to GR to serve as effector antagonists (8, 9). A hallmark property of smaller simple RGS protein family members and isolated RGS domains in particular is their preferential binding to activated GR subunits, whereas inactive GDP-bound forms of GR have much lower affinity and bind RGS proteins poorly or not at