Abstract

Gα12 and Gα13 are alpha subunits of heterotrimeric guanine nucleotide-binding proteins, and drive signal transduction pathways responsible for cellular growth, cytoskeletal alterations, and oncogenic development. Both proteins bind and activate a specific group of Rho-directed guanine nucleotide exchange factors (RhoGEFs) in order to stimulate several cellular responses, but the structural features that are distinct between Gα12 and Gα13 in this mechanism are not known. Invertebrates possess a single homolog of mammalian Gα12/Gα13, which retains the ability to trigger cytoskeletal rearrangements but is incapable of signaling to SRF (serum response factor), a transcriptional activator that plays a role in Gα12- and Gα13-mediated tumor progression. Previous research in our lab revealed a highly-conserved 36-amino acid region that contained only 10 (for Gα12) and 9 (for Gα13) divergent residues when compared to the Caenorhabditis elegans homolog. Although invertebrate substitution of this region eliminated SRF signaling for both Gα12 and Gα13, disruption of RhoGEF binding was observed only for the Gα13 mutant. To understand the Gα13-specific structural determinants of this interaction, we bisected its 36-residue region and found the N-terminal invertebrate substitutions were crucial to abolishing RhoGEF binding and SRF signaling. However, single-point mutations in this region were inconsequential, leading to our current strategy of constructing multi-point invertebrate substitutions to subdivide the Gα13 N-terminal region and identify its critical residues for SRF signaling and RhoGEF interaction. We have identified two mutants of Gα13, each containing three invertebrate substitutions, that show a dramatic loss of SRF signaling. Currently, co-precipitation assays using immobilized RhoGEF domains are being conducted to observe the impact of these mutations on binding to RhoGEFs and other known Gα13 target proteins.

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