Vellano_2011_Biochemistry_50_752

RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Galphai/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Galphai1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Galphai1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Galphai1 in cells, suggesting formation of a functional protein complex with Galphai1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Galphai1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Galphai1-GDP complex to form a stable Ric-8A-Galphai complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Galphai1-GDP complex to stimulate both the steady-state GTPase activity of Galphai1 and binding of GTP to Galphai1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Galphai1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Galphai1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Galphai1 signaling.