The Gα12/Gα13 subfamily of trimeric G proteins are implicated in various physiological processes and disease states, including tumorigenesis. These proteins reside in the cytoplasm and transmit information from membrane-bound receptors to the cell interior. Earlier studies suggest Gα13 is phosphorylated on a threonine side chain at position 203 within the 377-amino acid protein. In this project I focused on this specific threonine, mutating it to mimic the phosphorylated and dephosphorylated states by creating negatively charged and hydrophobic substitutions, respectively. Using directed PCR-based mutagenesis, Thr-203 was converted to glutamic acid, aspartic acid, and alanine in three separate DNA constructs, which were then expressed in human kidney cells. These Gα13 mutants were tested for protein binding through pull-down experiments, gel electrophoresis, and immunoblotting. Preliminary results suggest that these mutations disrupt binding to multiple RhoGEFs and these disruptions may be selective to each mutant. These mutants were also tested for ability to stimulate SRE-mediated transcription, a cell growth pathway linked to cancer. The alanine-substituted Gα13 showed a sharp decrease in ability to drive this response. Because this threonine is conserved in Gα12, I am currently generating similar mutants in this protein to examine effects on its protein binding and signaling properties.
