Understanding the Mechanism of G⍺i Protein Activation

<p style="font-weight: 400;">The heterotrimeric G protein complex, that acts downstream of G-Protein-Coupled Receptors (GPCR), consists of three subunits - G&alpha;, and heterodimeric G&beta;&gamma;. When G&alpha; is bound to GDP, the protein is inactive and remains bound to G&beta;&gamma;. Upon agonist binding, GPCR activation occurs, which promotes structural changes in G&alpha;, and leads to G&alpha; activation by exchanging bound GDP with GTP. The GDP to GTP exchange stabilizes three dynamics Switches of G&alpha; protein that leads to the release of G&alpha;-GTP (active form) from G&beta;&gamma;, and activation of their respective downstream pathways. There are four isoforms of G&alpha;, two of which regulate adenylyl cyclase.&nbsp; The G&alpha;s isoform activates adenylyl cyclase while G&alpha;i inhibits it. In both of these isoforms, an allosteric network of residues (activation network) have been proposed to mediate switch dynamics and conformation, and subsequent G&alpha; protein activation leading to regulation of adenylyl cyclase activity. Interestingly, a point mutation (R201C) in the activation network of G&alpha;s is a disease-causing mutation while the equivalent mutation in G&alpha;i (R178C) is not reported to cause any disease. Based on these observations, we propose that residue R178 of G&alpha;i plays a distinct role in regulation the activation network of Gai and Gas. To test this hypothesis, we generated an R178C mutation in G&alpha;i and applied using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy approaches to elucidate its involvement in an &lsquo;activation network&rsquo; required for adopting the active signaling state. Our preliminary data suggests that, unlike G&alpha;s, the R178C mutation in G&alpha;i is not able to stabilize the &lsquo;activation network&rsquo; in its GDP-bound form and thus cannot adopt the active state to cause disease. Future plans involve further characterizing differences in how the R178C mutation rewires the &lsquo;activation network&rsquo; in G&alpha;s and G&alpha;i to delineate the differences in their activation networks.