Inhibition of Gαi2 Activation by Gαi3 in CXCR3-mediated Signaling

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express Gαi2 and Gαi3, two members of the Gαi/o protein family, but whether these two Gαi proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct Gαi2 and Gαi3 effects on chemokine receptor CXCR3-mediated signaling. Our data showed that Gαi2 was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of Gαi2 abolished CXCR3-stimulated migration and guanosine 5′-3-O-(thio)triphosphate (GTPγS) incorporation. In sharp contrast, T cells isolated from Gαi3 knock-out mice displayed a significant increase in both GTPγS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPγS incorporation was blocked by Gαi3 protein in a dose-dependent manner. Gαi3-mediated blockade of Gαi2 activation did not result from Gαi3 activation, but instead resulted from competition or steric hindrance of Gαi2 interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only Gαi2 activation induced by a CXCR3 agonist but also the interaction of Gαi3 to the CXCR3 receptor. These findings reveal for the first time an interplay of Gαi proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the Gαi/o protein family.