Molecular and Functional Profiling of Gαi as an Intracellular pH Sensor

Heterotrimeric G proteins (Gα, Gβ and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. Previously, human Gαi and its yeast homolog Gpa1 have been reported to function as intracellular pH sensors, yet the pH sensing capabilities of Gαi and the underlying mechanism remain to be established. Herein, we identify a pH sensing network within Gαi, and evaluate the consequences of pH modulation on the structure and stability of the G-protein. We find that changes over the physiological pH range significantly alter the structure and stability of Gαi-GDP, with the protein undergoing a disorder-to-order transition as the pH is raised from 6.8 to 7.5. Further, we find that modulation of intracellular pH in HEK293 cells regulates Gαi-Gβγ release. Identification of key residues in the pH-sensing network allowed the generation of low pH mimetics that attenuate Gαi-Gβγ release. Our findings, taken together, indicate that pH-dependent structural changes in Gαi alter the agonist-mediated Gβγ dissociation necessary for proper signaling.

异源三聚体G蛋白（Gα、Gβ和Gγ）在G蛋白偶联受体（GPCRs）的下游发挥作用，介导调节各种生理过程和人类疾病状况的信号通路。此前，人类Gαi及其酵母同源物Gpa1已被报道可作为细胞内pH传感器，但Gαi的pH传感能力及其潜在机制仍有待确定。在此，我们在Gαi中鉴定出一个pH传感网络，并评估pH调节对G蛋白结构和稳定性的影响。我们发现，在生理pH范围内的变化显著改变Gαi - GDP的结构和稳定性，当pH从6.8升高到7.5时，该蛋白质经历从无序到有序的转变。此外，我们发现调节HEK293细胞内的pH可调节Gαi - Gβγ的释放。对pH传感网络中关键残基的鉴定使得能够产生减弱Gαi - Gβγ释放的低pH模拟物。我们的研究结果共同表明，Gαi中依赖于pH的结构变化改变了适当信号传导所必需的激动剂介导的Gβγ解离。