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 G alpha(12), and G alpha(i3), two members of the G alpha(i/o). protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that Gai, was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTP gamma S incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTP gamma S incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of Gai, activation did not result from Gai3 activation, but instead resulted from competition or steric hindrance of Gai, interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of Gai3 to the CXCR3 receptor. These findings reveal for the first time an interplay of Gai 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 G alpha(i/o) protein family.