We have discovered a protein family known as Regulators of G-protein Signaling (RGS) that impair signal transduction through pathways that involve seven-segment trans-membrane receptors and heterotrimeric G proteins. Such receptors, when activated following the binding of a ligand such as a hormone or chemokine, trigger the G alpha subunit to exchange GTP for GDP; this causes the dissociation of G alpha and G beta gamma subunits and downstream signaling. RGS proteins bind G alpha subunits and function as GTPase activating proteins (GAPs), thereby deactivating the G alpha subunit and facilitating their re-association with G beta gamma. 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 infiltrate 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 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 with a lack of directional persistence. To complement these studies we have expressed Galphai1-Yellow fluorescent protein (YFP) in a B cell line and have begun to establish G alphai2-YFP and G alphai3-YFP expressing cell lines as well. These cell lines will be used to examine the roles of the G alphai proteins in chemotaxis and lymphocyte polarization. Toll-like receptor (TLR) signaling, a powerful regulator of human and mouse B cells, is known to regulate RGS protein expression. We found that lipopolysaccharide (LPS) stimulation, which engages TLR4, enhances mouse B cell expression of CXCR5 and CCR7, increases the ratio of Gai2 to RGS1, and increases the percentage of B cells responding in chemotaxis assays. When transferred into recipient mice the LPS-activated B cells preferentially localize to the center of the lymph node follicle, where they form clusters of highly polarized cells. Cell tracking studies provided evidence of B cell-B cell interactions and directed cell migration. Dye dilution studies revealed extensive proliferation of the LPS stimulated cells in the recipient mice. Blocking lymph node ingress indicates that LPS activated B cells exit lymph nodes more slowly that do non-stimulated B cells. Germinal center B lymphocytes strongly express another RGS protein, RGS13. To study the role of RGS13 as well as RGS1 in human B cells we expressed shRNAs that reduced RGS13 and RGS1 expression individually or together in human B cell lines. Reducing RGS13, and to a lesser extent RGS1 expression in a Burkitt?s lymphoma cell line enhanced responsiveness to two chemokines, CXCL12 and CXCL13, while reducing both mRNAs more dramatically augmented the responses. The double knock-down (KD) cells responded better to re-stimulation with CXCL12 or CXCL13 after a primary stimulation with CXCL12 than did the control cells. The double KD cells also exhibited a greater propensity to polarize and developed multiple small lamellipodia following chemokine exposure. To complement these studies we have begun to functionally characterize 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. These mice also have a reduction in B1a B cells in the peritoneum, but an increase in B1b cells. To facilitate the isolation of Rgs13-expressing cells and the in vivo imaging of germinal center B cells we are generating mice with GFP (green fluorescent protein) introduced into the RGS13 locus. Two other RGS proteins, RGS10 and RGS19 are strongly expressed in lymphocytes. We have obtained RGS10 deficient mice and have begun a gene targeting project to develop RGS19 deficient mice. 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. We also generated constructs with mouse and human RGS19-GFP and found that both predominantly localize in the cytosol with some plasma membrane expression. Signaling studies to assess the potential role of RGS19 in lymphocyte chemokine receptor signaling are in progress. 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. Current studies are directed at understanding the role of RGS1, other RGS proteins, and the different G alpha i isoforms in regulating S1P signaling in endothelial cells and lymphocytes. Many of the effectors of Gi signaling responsible for directed cell migration and lymphocyte polarization remain unknown and known effectors often have ill defined roles in B cell trafficking. We found that pharmacologic inhibitors of phosphoinositide 3-kinases (wortmannin, WMN), Bruton?s tyrosine kinase (LFM-A13), and Jun kinases (SP600125) all significantly impair CXCL12-induced mouse B cell chemotaxis and that of a human B lymphoma cell line. Each of the inhibitors impaired the homing of transferred B cells to peripheral lymph nodes. Intravital imaging of control and inhibitor treated mouse B cells in the inguinal lymph node high endothelial venules demonstrated varying reductions in the number of firmly adherent B cells with WMN being the most effective. To complement these studies we have developed a methodology to image primary B cells within lymph nodes without a requirement for lymph node entry. We have found that labeled B cells will enter into the lymph node follicles of 300 micron lymph node sections maintained in vitro. Treatment of the cells with pertussis toxin prior to transfer inhibits the entry into the follicle. Treatment of the cells or the section prior to transfer with either the BTK inhibitor or WMN failed to alter the motility of B cells within the lymph node follicle; however, the JNK inhibitor profoundly inhibited B cell motility.? 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, hypotensive, and develop cardiac hypertrophy.
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