Characterizing Gα12 and p114RhoGEF’s specific interaction through site-directed mutagenesis

Ricardo Garcia


G proteins are heterotrimeric proteins that signal for a variety of important processes in the cell. There are four subfamilies of G proteins in mammals, including the G12/13 subfamily that consists of Gα12 and Gα13 and has been implicated in cancerous progression. There are many proteins that interact with Gα12, among these the closely related Rho-specific guanine nucleotide exchange factors p114RhoGEF and AKAP-Lbc.  Although the structural features of the interaction between Gα12 and p114RhoGEF have yet to be determined, we have found that p114RhoGEF and AKAP-Lbc utilize a region closely homologous between these proteins for interaction with Gα12.  Using a PCR-based strategy, we engineered point mutations to create charge-substitutions of amino acids identical in AKAP-Lbc and P114RhoGEF. These constructs were then utilized for protein interaction experiments to determine which constructs showed decreased binding affinity for Gα12.  To examine the specificity of Gα12 interaction for these RhoGEF proteins, we performed G protein driven assays of serum response factor mediated transcriptional activation.  These results showed that a dominant-negative p114RhoGEF preferentially blocked the Gα12-mediated stimulation of serum response factor in comparison to Gα13, suggesting that specific binding to p114RhoGEF plays a role in the Gα12-specific mechanism of this signaling pathway. The long-term purpose of this research is to characterize the structural interaction between Ga12 and P114RhoGEF and use this knowledge to further investigate the role of Ga12-p114RhoGEF binding in different cell types.

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