We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. We have focused on the role of RGS proteins in modulating signaling through lymphocyte chemokine receptors. This has led to a series of studies examining lymphocyte chemokine receptor signaling and an assessment of the effects of modulating the signaling pathway on B lymphocyte and to lesser extent T cell trafficking. Rgs1 /- B cells obtained from mice in which Rgs1 has been disrupted by gene targeting have an enhanced response to the chemokines CXCL12 and CXCL13, and fail to desensitize properly following exposure to these chemokines. B cells from the Rgs1 -/- mice enter lymph nodes more easily, better target lymph node follicles, and move more rapidly than do B cells from wild type mice. Activation of the G alpha subunit Gi is required for lymphocyte chemotaxis and the molecular target of RGS1. Humans and mice have three Gi isoforms although G alphai2 (encoded by Gnai2) and Galphai3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have profound defects in chemokine-induced mobilization of intracellular calcium, chemotaxis, and lymph node homing, whereas Gnai2 +/- T and B cells exhibit modest defects. Intravital microscopy revealed sluggish Gnai2 -/- CD4 T cell and B cell motility. The Gnai2-/- B cells failed to properly access lymph node follicles and the Gnai2-/- T cells largely remain in the region surrounding the high endothelial venules failing to properly access the T cell zone. We have crossed the Gnai2 targeted mice with the Rgs1 targeted mice. Analysis of the double heterozyge, Rgs1 +/- Gnai2 +/- indicates that the ratio between Gnai2 expression and Rgs1 expression regulates responsive of B lymphocytes to chemokines. To complement these studies we have established G alphai1-Yellow fluorescent protein (YFP), Galpha i2 YFP, and Galpha i3 YFP expressing B cell lines. Analysis of these cells indicates that Gialpha1 and Gialpha2 function to trigger chemotaxis while Gialpha3 does not. We have recently acquired Gnia3-/- mice and Gnai2 flox mice. Both are on mixed genetic backgrounds and we have begun backcrossing these mice with C57Bl/6 mice so that the mice have a defined genetic background for comparision to control mice. To delineate the G beta/gamma subunits used by chemokine receptors we have focused on Gnb1, Gnb2, and Gng5; which all each prominently expressed in lymphocytes. We are using a combination of overexpression and knock-down studies to determine their relative importance in mediating chemokine receptor signaling. Germinal center B lymphocytes strongly express the RGS protein, RGS13. We have functionally characterized the Rgs13 -/- mice. B cells obtained from immunized Rgs13 -/- mice have heightened responses to chemokines and the Rgs13 -/- mice generate antibody responses with increased IgG affinity. They have a two fold decrease in serum IgA compared to control mice. However, we had expected a more robust phenotype in these mice and are exploring other potential roles for Rgs13 in germinal center function. To facilitate these studies and for in vivo imaging of germinal center B cells we have generated mice with GFP (green fluorescent protein) introduced into the RGS13 locus. These mice have just become available. Two other RGS proteins, RGS10 and RGS19 are strongly expressed in lymphocytes. We have obtained RGS10 deficient mice and should receive RGS19 deficient mice soon. Three different isoforms of RGS10 exist and differ in their intracellular localization and their activities in modulating GPCR signaling. The strong expression of two of the isoforms within the nucleus of transfected cells as well as in primary lymphocytes suggests that RGS10 may have other effects on lymphocyte function in addition to the regulation of GPCR signaling. Analysis of the RGS10 mice has been delayed due because of problems with the colony and we now expect the mice to be available at the end of this year. We also generated GFP fusion constructs with mouse and human RGS19. We have found that both predominantly localize in the cytosol with some plasma membrane expression. Signaling studies using knock-down and overexpression indicate that RGS19 only marginally modulates lymphocyte chemokine receptor signaling suggesting other intracellular roles for RGS19. In addition to chemokine receptors, another group of GPCRs have emerged as important regulators of lymphocyte trafficking. These receptors all bind the phospholipid sphingosine 1-phosphate (S1P). The S1P receptors function at the level of vascular endothelial cells to regulate lymph node egress by controlling access to the medullary sinus and directly on lymphocytes to promote lymph node exit. In addition, S1P receptors function in the positioning of B cells in the marginal zone of the spleen. We have shown that RGS1 potently impairs signaling through several different S1P receptors. Using a series of S1P analogues and receptor inhibitors we have shown that three different S1P receptors termed S1P1, S1P3, and S1P4 function to regulate lymphocyte responses to S1P. S1P1 functions predominantly to slow lymphocyte migration, while S1P3 and S1P4 act as chemoattractant receptors. While most of our studies have focused on RGS proteins in lymphocytes we have had an interest in Rgs5, an RGS protein found at high levels in vascular smooth muscles and in pericytes. Mice deficient in Rgs5 are lean and hypotensive. The underlying molecular mechanism by which Rgs5 regulates blood pressure remains to be defined.
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