RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of G alpha(o)

In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA. enc oding a protein that interacts with both Rap1 and Rap2, but not with Ras; t he use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of t he recently discovered family of RGS ('regulators of G-protein signalling') proteins that stimulate the GTPase activity of the GTP-binding alpha subun it of heterotrimeric G-proteins (G alpha). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf- 1 kinases. RGS14 is distinguished from other RGS proteins by its marked pre ference for G alpha(o) over other G alpha subunits: RGS14 binds preferentia lly to G alpha(o) in isolated brain membranes, and also interacts preferent ially with G alpha(o) (as compared with G alpha(il)) to stimulate its GTPas e activity. In adult mice, RGS14 expression is restricted to spleen and bra in. In situ hybridization studies showed that it is highly expressed only i n certain areas of mouse brain (such as the CA1 and CA2 regions of the hipp ocampus), and that this pattern closely resembles that of Rap2, but not Rap 1, expression. Double in situ hybridization experiments revealed that certa in cells in the hippocampus express both RGS14 and G alpha(o), as well as b oth RGS14 and Rap2, showing that the interaction of RGS14 with G alpha(o) a nd Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulatio n of signalling pathways downstream from G-protein-coupled receptors involv ing heterotrimeric proteins of the G(i/o) family and those involving the Ra s-related GTPase Rap2.