Inhibition of Gαi2 Activation by Gαi3 in CXCR3-mediated Signaling

Brian D Thompson,Yongzhu Jin,Kevin H Wu,Richard A Colvin,Andrew D Luster,Lutz Birnbaumer,Mei X Wu

doi:10.1074/jbc.m610931200

Abstract

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(i2) 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 G alpha(i2) 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 (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) 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 G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) 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.

Highlights

AI050822 and AI070785, Research Scholar Grant RSG-01-178-01-MGO from the American Cancer Society, Senior Research Award 1657 from the Crohn & Colitis Foundation of America, and by the Intramural Research Program of the NIEHS, National Institutes of Health
Heterotrimeric G proteins consist of ␣, ␤, and ␥ subunits that couple to seven transmembrane receptors called G proteincoupled receptors (GPCRs)
The G protein resides attached to the intracellular face of the plasma membrane in an inactive form consisting of the G␣ subunit bound to GDP, a structure that is stabilized by interaction with the ␤␥ dimer

Summary

Introduction

AI050822 and AI070785, Research Scholar Grant RSG-01-178-01-MGO from the American Cancer Society, Senior Research Award 1657 from the Crohn & Colitis Foundation of America, and by the Intramural Research Program of the NIEHS, National Institutes of Health The bacterial toxin has proven to be an excellent tool in dissecting the essential role of G␣i proteins in chemotaxis, proliferation, and differentiation of various cells [3, 4, 6, 7] It cannot distinguish unique properties of individual G␣i/o protein family members. The receptor has the following three known ligands: monokine-induced IFN-␥ (MIG)/CXCL9, IFN-␥-inducible 10-kDa protein (IP-10)/CXCL10, and IFN-␥-inducible T cell ␣-chemoattractant/CXCL11 These three chemokines exhibited discernible biological effects in vivo and in vitro (20 –24), but they all activated exclusively G␣i2 through the CXCR3 receptor, as indicated by a lack of CXCR3-mediated responses in G␣i2Ϫ/Ϫ T cells, regardless of the agonist employed. We found that G␣i3 was negatively involved in the CXCR3-mediated signaling This translated to cellular responses, where a lack of G␣i3 significantly enhanced T cell migration toward CXCR3 agonists. This work begins to unravel an interplay between individual G␣i proteins that has previously been masked by the use of PTX

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