G [alpha] o/i targeting of direct interactors for degradation by the ubiquitin-dependent proteasome system: A mechanism for signal integration

One mechanism used by cells to respond to extracellular stimuli is the G protein coupled receptor. These transmembrane spanning receptors couple to members of the heterotrimeric G protein family, which are capable of being activated and further propagating the extracellular signal. The G_o protein belongs to the G_i family of G proteins, which prototypically inhibit specific isoforms of adenylyl cyclase although the α subunit of G_o cannot. In fact no direct effector molecules have been identified for the α subunit of G_o; however several downstream biological effects have been attributed to Gα_o signaling including the induction of neurite outgrowth and modulation of calcium channel function. In order to identify putative effector proteins for Gα_o I screened a chicken embryonic dorsal root ganglia libary using the yeast two-hybrid method. I found that Gα_o interacted with several proteins including RGSZ1, a new RGS protein (RGS-17), a novel protein of unknown function (IP6/GRIN3), Rap1GAP, and a putative tyrosine phosphatase Paladin. The interactions between several of these proteins and Gα_o were further characterized. Initially I found that Rap1GAPII selectively interacts more avidly with the inactive Gα_o as compared to the mutant (Q205L) activated Gα_o. When wild type Gα_o was expressed the amount of activated Rap1 was greatly increased, however this effect was not observed with the Q205L-Gα_o. Expression of inactive Gα _o stimulated MAP-kinase activity in a Rap1GAP dependent manner. These results identify a novel function of Gα_o, which in its resting state can sequester Rap1GAPII and thus regulate Rap1 activity. The mechanism used to inhibit Rap1GAPII activity was quite surprising. I found that expression of Gα_o/i results in the reduced protein stability of its direct interactors, two of which are GTPase activating proteins for Rap1 and G_z. Decreases in the level of these regulators results from the Gα_o/i mediated targeting for ubiquitination and proteasomal degradation. This effect was found to occur for three Gα_o/i interacting proteins, Rap1GAPII, RGSZ1, and Paladin. Targeting of these regulatory molecules for degradation would allow for the alteration of signaling thresholds and thus enable long-term regulation and integration between signaling pathways.