Normal Thymocyte Egress, T Cell Trafficking, and CD4+ T Cell Homeostasis Require Interactions between RGS Proteins and Gαi2

Abstract

Adaptive immunity depends upon mature thymocytes leaving the thymus to enter the bloodstream and the trafficking of T cells through lymphoid organs. Both of these requires heterotrimeric Gαi protein signaling, whose intensity and duration are controlled by RGS proteins. Here, we show that RGS protein/Gαi2 interactions are essential for normal thymocyte egress, T cell trafficking, and homeostasis. Mature thymocytes with a Gαi2 mutation that disables RGS protein binding accumulated in the perivascular channels of thymic corticomedullary venules. Severe reductions in peripheral naïve CD4+ T cells and Tregs occurred. The mutant CD4+ T cells adhered poorly to HEVs and exhibited defects in lymph node entrance and egress. The kinetics of chemokine receptor signaling were disturbed including chemokine induced integrin activation. Despite the thymic and lymph node egress defects, sphingosine-1-phosphate signaling was not obviously perturbed. This study reveals how RGS proteins modulate Gαi2 signaling to facilitate thymocyte egress and T cell trafficking.

Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins

“…RGS proteins decrease the activity of G proteins by accelerating GTP hydrolysis and, thereby, accelerate G protein inactivation but also its activation [ 56 – 58 ], which appears to be essential for chemotaxis [ 8 , 37 ]. It was shown that immune cells, harboring the Gα i2 G184S mutant, which causes resistance to RGS proteins, exhibit major alteration in chemotaxis [ 8 ]. The active state of the G184S Gα i2 mutant is prolonged, a condition mimicking the effects of SseI that persistently activates G i proteins.…”

Unrestrained Gαi2Signaling Disrupts Normal Neutrophil Trafficking, Aging, and Clearance

“…Third, following ligand exposure the effective concentration of heterotrimeric Gαi2 available for GPCR activation declines. This can adversely affect the kinetics of the signal transduction pathways (Hwang et al, 2017). Cells including neutrophils that lack an individual RGS protein typically exhibit enhanced GPCR signaling (Balenga et al, 2014;Chan et al, 2018;Kehrl, 2016;Xie et al, 2016).…”

“…Cells including neutrophils that lack an individual RGS protein typically exhibit enhanced GPCR signaling (Balenga et al, 2014;Chan et al, 2018;Kehrl, 2016;Xie et al, 2016). Furthermore, studies of the G184S mice have largely reported augmented GPCR-linked Gαi signaling (Neubig, 2015), yet past studies examining leukocyte chemoattractant receptors have largely reported diminished signaling (Cho et al, 2012;Hwang et al, 2015;Hwang et al, 2017). In the present study, we found that neutrophils, which carry the Gαi2 G184S mutation exhibit a complex phenotype with features of both hyperactive and hypoactive Gαi signaling, which overlap with the phenotypes noted with Gαi gain-or a loss-of function mutations.…”

“…Chemotaxis assays were performed using a transwell chamber (Costar) as previously described (Hwang et al, 2017). BM cells or splenocytes were immunostained for neutrophils with fluorochrome-conjugated antibodies against CD11b, Ly6C, and Ly6G, washed twice, re-suspended in complete RPMI 1640 medium and added in a volume of 100 μl to the upper wells of a 24-well transwell plate with a 5 µm insert.…”

Unrestrained Gαi2 Signaling Disrupts Neutrophil Trafficking, Aging, and Clearance

“…Chemotaxis assays were performed using a transwell chamber (Costar) as previously described ( 21 ). Where indicated the murine microvascular SVEC4-10EE2 endothelial cell line cells were cultured on the transwell insert until 90% confluence prior to use in the assay.…”