Rap1GAP integrates a signaling network between trimeric and monomeric G proteins

G_z is a member of the G_i subfamily of trimeric G proteins, whose primary role in cell physiology is not well understood. In an ongoing effort to elucidate the cellular functions of G_z, a yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Gα_z. One of the molecules uncovered in this screen was Rap1GAP, a previously-identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a downregulator of Rap1 signaling. Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Gα_z and Rap1GAP blocks the ability of RGSs (Regulators of G protein Signaling) to stimulate GTP hydrolysis of the α subunit, and also attenuates the ability of activated Gα _z to inhibit adenylyl cyclase. Co-precipitation assays further revealed that Gα_z, Rap1GAP, and Rap1 could form a stable complex.; In cell studies, activated forms of Gα_z were able to recruit Rap1GAP from a cytosolic location to the plasma membrane. Introduction of constitutively-activated Gα_z, into PC12 cells markedly attenuated the differentiation process of these cells induced by a cAMP analogue. Treatment of PC12 cells expressing wild-type Gα_z with a specific agonist to the α_2A-adrenergic receptor also attenuated cAMP-induced PC12 cell differentiation, demonstrating that receptor-mediated activation of G_z was also effective in this regard. Furthermore, activation of G_z signaling decreased the ability of a cAMP analogue to trigger both Rap1 and ERK activation. Differentiation of PC12 cells induced by NGF is also thought to be a Rap1-mediated process, and G_z activation was found to attenuate this process as well. Rap1 activation, ERK phosphorylation, and PC12 cell differentation induced by either cAMP analogue or NGF treatment were all blocked by either transfection of constitutively-activated Gα _z or receptor-mediated G_z activation. Based on these findings, a model is proposed in which activation of G_z results in recruitment of Rap1GAP to the plasma membrane where it can effectively down-regulate Rap1 signaling.; These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G_z signaling and Rap1 signaling in cells.