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Characterization of the Non-Receptor GEF, RIC8 with Genetic, Chemical, and Biochemical Analyses

  • Author(s): Schacht, Patrick Carl
  • Advisor(s): Borkovich, Katherine A
  • et al.
Abstract

RIC8 plays a critical role in the regulation of G protein signaling. In this study, a specific chemical inhibitor of the RIC8-GNA-1 interaction is identified. In addition to disrupting the interaction in vitro, compound F2342-0045 elicits many of the defects associated with loss of ric8 or gna-1. A suppressor screen identified a novel suppressor of delta-ric8, predicted to be a ham-5 mutant by mapping and sequencing. Suppressors of delta-ric8, delta-pde-1 and delta-rgs-1, were also identified by crosses with G protein related mutants further implicating RIC8 in G&alpha and cAMP regulation. Systematic mutagenesis of the RIC8 protein revealed multiple structure-function relationships. The N-terminus of the protein is essential for binding to GNA-1, while the C-terminus is dispensable for this action. Mutations of conserved residues along the front face of the C-terminus generally inhibit the GEF activity of the RIC8 protein towards GNA-1 and GNA-3 alike, while mutations in the central third of the protein have less effect on GEF activity. Residues that cluster together on the top face of the C-terminus show varied but potent effects, either inhibiting or enhancing GEF activity by 50% or more. These data combine to suggest that the N-terminus of RIC8 is involved in binding of G&alpha proteins, while the C-terminus is involved in GEF activity.

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