Modulation of rap activity by direct interaction of Gαo with Rap1 GTPase-activating protein

We used the yeast two-hybrid system to identify proteins that interact directly with G alpha(o). Mutant-activated G alpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that G alpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with G alpha(o) and G alpha(i) but not with G alpha(s) or G alpha(q). Rap1GAP interacts more avidly with the unactivated G alpha(o) as compared with the mutant (Q205L)-activated G alpha(o). When expressed in HEK-293 cells, unactivated G alpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin, When unactivated G alpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-G alpha(o). Expression of unactivated G alpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of G alpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.