Inhibition of regulator of G protein signaling function by two mutant RGS4 proteins

Regulators of G protein signaling (RGS) proteins limit the lifetime of activated (GTP-bound) heterotrimeric G protein α subunits by acting as GTPase-activating proteins (GAPs). Mutation of two residues in RGS4, which, based on the crystal structure of RGS4 complexed with Giα1-GDP-AlF4−, directly contact Giα1 (N88 and L159), essentially abolished RGS4 binding and GAP activity. Mutation of another contact residue (S164) partially inhibited both binding and GAP activity. Two other mutations, one of a contact residue (R167M/A) and the other an adjacent residue (F168A), also significantly reduced RGS4 binding to Giα1-GDP-AlF4−, but in addition redirected RGS4 binding toward the GTPγS-bound form. These two mutant proteins had severely impaired GAP activity, but in contrast to the others behaved as RGS antagonists in GAP and in vivo signaling assays. Overall, these results are consistent with the hypothesis that the predominant role of RGS proteins is to stabilize the transition state for GTP hydrolysis. In addition, mutant RGS proteins can be created with an altered binding preference for the Giα-GTP conformation, suggesting that efficient RGS antagonists can be developed.

Genetic interactions with Rap1 and Ras1 reveal a second function for the Fat facets deubiquitinating enzyme in Drosophila eye development

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.