Heterotrimeric G proteins transduce a variety of signals from membrane-bound receptors to intracellular effectors. Members of the Rho family of low-molecular-weight GTPases control the organization of the actin cytoskeleton and are involved in a variety of cellular functions. The objective of this proposal is to understand the mechanism of regulation of Rho family GTPases by heterotrimeric G protein-mediated signaling pathways. Among heterotrimeric G proteins, Galpha12 and Galpha13participate in cell transformation and embryonic development, and Rho appears to be involved in their signaling pathways. Thus, the immediate goal of this proposal is to understand biochemical mechanisms of regulation of the activity of Rho family members by Galpha12 and Galpha13. Rho family GTPases are activated by guanine nucleotide exchange factors (GEFs). We have demonstrated that Galpha12 and Galpha13 interact with and regulate the activity of a novel Rho GEF, p115. Galpha13 stimulates the Rho GEF activity of p115. In contrast, Galpha12 inhibits activiation of p115 by Galpha13. Furthermore, RhoGEF acts as a GTPase activating protein (GAP) for both Galpha12 and Galpha13. The regulation of p115RhoGEF will be further characterized in detail. The GAP assay will be improved using the selected mutants of the Galpha proteins, and the domain of p115 responsible for GAP activity will be defined. In addition, the molecular mechanism of differential regulation of p115 by Galpha12 and Gaalpha13 will be investigated. Galpha12 can activate Rho in vivo. However, Galpha12 does not stimulate the RhoGEF activity of p115. We will thus attempt to find a RhoGEF that is activated by Galpha12 by affinity chromatography using immobilized Galpha12. The physiological significance of regulation of RhoGEF activity by Galpha12 and Galpha13 will also be investigated in vivo. The involvement of the G12, G13-RhoGEF pathways in axonal growth and guidance will be characterized.
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