Abstract

Gα13 belongs to the Gα12/13 subfamily of heterotrimeric guanine nucleotide-binding proteins, which play a signaling role in cell growth and tumorigenic pathways, cytoskeletal rearrangements, and metastatic invasion. A structural bioinformatics analysis1 identified a set of “class-distinctive” residues in Gα13 which correspond to a different residue conserved in the other G protein subfamilies: Gs, Gi, and Gq. Using this information, we created a panel of Gα13 point mutants that replace each class-distinctive residue with its putative ancestral form. In order to distinguish mutant constructs from native Gα13 in cultured human embryonic kidney cells, a myc epitope tag was introduced to all Gα13 mutants, positioned within the αB–αC loop of the helical domain. Installation of this epitope tag was non-disruptive to Serum Response Factor signaling by Gα13 and allowed for differentiation of the recombinant and native forms in protein-protein interaction experiments. While characterization of these Gα13 mutants is ongoing, there is a class- distinctive Phe at position 234 for which ancestral substitution appears to cause selective uncoupling of protein binding within the Gα13-responsive, RGS-homology (RH) RhoGEF. These findings shed light on the mechanism of Gα13 interaction with the individual RH-RhoGEFs - p115RhoGEF, PDZ-RhoGEF, and leukemia-associated RhoGEF - and further suggest that class-distinctive Gα13 mutants may reveal binding determinants for additional effector proteins.

How to Cite