We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering G alpha i nucleotide exchange. Humans and mice have three Gi isoforms although G alpha i2 (encoded by Gnai2) and G alpha i3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have severe defects in chemokine-receptor signaling while Gnai2 +/- T and B cells exhibit modest defects. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells failing to properly enter the T cell zone. We have crossed the Gnai2 targeted mice with the Rgs1 targeted mice. Analysis of the double heterozygote, 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 alpha i1-Yellow fluorescent protein (YFP), G alpha i2 YFP, and G alpha i3 YFP expressing B cell lines. Analysis of these cell lines indicates that G alpha i1 and G alpha i2 function to trigger chemotaxis while G alpha i3 does not. Furthermore, in contrast to the Gnia2-/- B cells, Gnai3-/- B cells exhibit enhanced chemokine receptor signaing when compared with wild type B cells. Mice in which the the Gnai2 gene is flanked with CRE recombination signals are being back-crossed on to C57/Bl6. Once back-crossed they will be used delete Gnai2 expression only in B lymphocytes and to generate mice with B cells that lack Gnai2 and Gnai3 expression. The G beta/gamma complex is an essential component of the receptor-G protein system required for chemotaxis of leukocytes. To delineate the G beta/gamma subunits used by chemokine receptors we have focused on Gnb1, Gnb2, and Gng5; which are 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. It has been proposed that component(s) of the receptor-G protein system may accumulate at the front of polarized cells, accounting for increased responsiveness to chemoattractants at the anterior. To investigate the molecular basis of chemotactic properties of mammalian cells, we are examining the distribution of heterotrimeric G proteins and RGS proteins in living cells during chemotaxis. A confocal microscope-based cell migration assay has been established to visualize localization of fluorescent protein-tagged G protein signaling components in differentiated HL-60 cells. This assay will be further developed for primary lymphocyte cell migration and to monitor the interaction of the signaling components during chemotaxis using FRET. Germinal center B lymphocytes strongly express the RGS protein, RGS13. To facilitate our studies of RGS13 and to provide a potential resource for in vivo imaging endogenous germinal center B cells we have generated mice where GFP (green fluorescent protein) has been targeted into the Rgs13 locus under the control of the RGS13 regulatory sequences. High levels of RGS13-GFP have been detected in B220+IgD- B cells, which further increase following immunization. We have also observed modest expression in transitional B cells. GFP+Rgs13-/- B cells respond two fold better in chemotaxis assays than do GFP+Rgs13+/- B cells indicating that Rgs13 limits the responsiveness of germinal center B cells to chemokines. A similar comparison of transitional B cells indicates that Rgs13 also acts to limit the chemotaxis of transitional B cells. Two weeks following immunization wild type and Rgs13-/- mice have a similar percentage of splenic germinal center B cells, however, the there is a 50% increase in the number of cells in S phase of the cell cycle in the Rgs13-/- versus wild type B220+IgD- B cells. Serum IgM and IgG levels are similar between wild type and Rgs13-/- although serum IgA levels are depressed in the Rgs13-/- mice. Two other RGS proteins, RGS10 and RGS19 are strongly expressed in lymphocytes. We have obtained RGS10 deficient mice and recently RGS19 deficient mice. Three different isoforms of RGS10 exist, which 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 not only affect GPCR signaling, but have other functions as well. We have identified a shRNA target sequence for RGS10 and demonstrated that its expression reduces RGS10 expression in Jurkat T cells. We plan to examine the response of control and RGS10 knock-down cell lines to a panel of GPCR ligands. Rgs10-/- mice are viable and born at a normal Mendelian frequency. Preliminary assessment of the Rgs10-/- mice revealed a significant reduction in marginal zone B cells and a decrease in mature T cells in the thymus. No significant difference in responses between wild type and Rgs10-/-splenic B cells to a panel of chemokines has been found. Further experiments to assess the consequences of the lack of RGS10 are in progress. We have generated GFP fusion constructs with mouse and human RGS19. Both predominantly localize in the cytosol with some plasma membrane expression. Signaling studies using knock-down and overexpression indicate that RGS19 marginally modulates lymphocyte chemokine receptor signaling suggesting other intracellular roles for RGS19. GFP has been inserted into the RGS19 locus and appropriated targeted mice identified. Rgs19-/- mice are also born with a normal Mendelian frequency. Flow cytometry revealed that GFP is well expressed in both B and T cells. Analysis of B and T cell subsets has not revealed any significant disturbances following loss of Rgs19. Chemotaxis experiments comparing wild type and RGS19-/- lymphocytes indicate modest increases to a panel of chemokines with the KO cells. Further studies are in progress to assess whether there are immune defects in the KO animals. 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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