Mutation of Class‐Distinctive Residues in Gα13 to Identify Determinants of RH‐RhoGEF Binding Selectivity

Abstract

Gα13 belongs to the G12/13 subfamily of heterotrimeric guanine nucleotide‐binding proteins that play a signaling role in cell growth and tumorigenic pathways, cytoskeletal rearrangements, and metastatic invasion. A structural bioinformatics analysis [Temple et al., 2010, PLOS Comput. Biol., 6(10): e1000962] 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 our mutant constructs from native Gα13 in cultured human embryonic kidney cells, we introduced a myc epitope tag to all Gα13 mutants, positioned within the αB–αC loop of the helical domain. Installation of this epitope tag was non‐disruptive to SRF signaling by Gα13, and allowed us to differentiate its recombinant and native forms in protein‐protein interaction experiments. While characterization of these Gα13 mutants is ongoing, we have identified 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) RhoGEFs. 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.Support or Funding InformationWe acknowledge support from the UNC Lineberger Comprehensive Cancer Center (University Cancer Research Fund), and the North Carolina GlaxoSmithKline Foundation.