Guanine-nucleotide binding proteins, commonly referred to as G proteins, are molecules involved in transmission of a signal to downstream pathways in cells. G proteins are regulated by activation via GTP. When bound, the activated G protein is able to interact with secondary messengers and trigger a cellular response. The G12 subfamily of G proteins includes Ga12 and Ga13, which diverged from a single ancestor protein in evolutionary history. These two proteins have been shown to drive pathways leading to proliferation and migration in certain types of cancer cells, but also drive pathways that are critical to basic functioning of the cell such as cytoskeleton rearrangement and membrane transport. In this project I have used a comparative evolutionary analysis to pinpoint and mutate amino acids in Ga13 that may be critical for its unique signaling properties, and then completed a variety of protein analyses to determine impaired function. These data were compared side by side with Ga12 to identify differences between the two proteins. For all class-distinctive mutants of Ga13 tested, none showed impaired ability to drive serum response, suggesting that these amino acids are not critical for Ga13 stimulation of Rho. Ga13 wildtype also showed close to the same response as the activated form, Ga13QL whereas Ga12QL had a significantly higher response than did the wildtype form. This suggests that Ga13 wildtype can bind and drive this Rho pathway equally as well as Ga13QL. However, a side-by-side pull down assay shows that Ga13WT has a lesser binding affinity for Rho than Ga12WT. This information suggests that serum response is being driven by different pathways in the two proteins. Further experimentation will be done to test known effectors of Ga12 and Ga13 for binding to activated and wildtype forms of each. If one is found to have a significantly higher affinity for Ga13WT, then further work will be done to silence this effector in cells and preform additional serum response assay to determine its impact on this pathway.
