RGS molecule expression in murine B lymphocytes and ability to down-regulate chemotaxis to lymphoid chemokines

Ag-mediated changes in B lymphocyte migration are important for normal immune function, yet the mechanisms by which these changes occur are poorly defined. Because chemokines direct many lymphocyte movements, molecules that regulate signaling by G protein-coupled chemokine receptors are likely to participate in Ag receptor-induced changes in cell migration. In this study, we have investigated the expression pattern and activity in murine B cells of members of the regulators of G protein signaling (RGS) family of molecules. We present the sequence of mouse RGS1 and describe a novel short isoform of RGS3 that we term RGS3s. Following in vivo activation by Ag, B cells rapidly up-regulate expression of RGS1 and RGS2 while simultaneously decreasing expression of RGS3 and RGS14. Anergic hen egg lysozyme autoantigen-binding B cells are also shown to have slightly elevated RGS1 and RGS2 expression. CD40 signaling, by contrast, fails to cause rapid up-regulation of RGS1 or RGS2. Using a transient transfection approach in a mature B cell line, 2PK3, we demonstrate that RGS1 and RGS3s are effective inhibitors of chemotaxis toward the lymphoid tissue chemokines stromal cell-derived factor-1, B lymphocyte chemoattractant, and EBV-induced molecule 1 ligand chemokine, whereas RGS2 has a minimal effect on migration to these chemokines. Together these findings support the conclusion that Ag-mediated changes in RGS molecule expression are part of the mechanism by which Ag receptor signaling regulates B cell migration within lymphoid tissues. The findings also suggest important roles for additional G protein-mediated events in B cell activation and tolerance.     

Follicular dendritic cell regulation of CXCR4-mediated germinal center CD4 T cell migration

Follicular dendritic cells (FDCs) up-regulate the chemokine receptor CXCR4 on CD4 T cells, and a major subpopulation of germinal center (GC) T cells (CD4(+)CD57(+)), which are adjacent to FDCs in vivo, expresses high levels of CXCR4. We therefore reasoned that GC T cells would actively migrate to stromal cell-derived factor-1 (CXCL12), the CXCR4 ligand, and tested this using Transwell migration assays with GC T cells and other CD4 T cells (CD57(-)) that expressed much lower levels of CXCR4. Unexpectedly, GC T cells were virtually nonresponsive to CXCL12, whereas CD57(-)CD4 T cells migrated efficiently despite reduced CXCR4 expression. In contrast, GC T cells efficiently migrated to B cell chemoattractant-1/CXCL13 and FDC supernatant, which contained CXCL13 produced by FDCs. Importantly, GC T cell nonresponsiveness to CXCL12 correlated with high ex vivo expression of regulator of G protein signaling (RGS), RGS13 and RGS16, mRNA and expression of protein in vivo. Furthermore, FDCs up-regulated both RGS13 and RGS16 mRNA expression in non-GC T cells, resulting in their impaired migration to CXCL12. Finally, GC T cells down-regulated RGS13 and RGS16 expression in the absence of FDCs and regained migratory competence to CXCL12. Although GC T cells express high levels of CXCR4, signaling through this receptor appears to be specifically inhibited by FDC-mediated expression of RGS13 and RGS16. Thus, FDCs appear to directly affect GC T cell migration within lymphoid follicles.     

RGS13 regulates germinal center B lymphocytes responsiveness to CXC chemokine ligand (CXCL)12 and CXCL13

Normal lymphoid tissue development and function depend upon directed cell migration. Providing guideposts for cell movement and positioning within lymphoid tissues, chemokines signal through cell surface receptors that couple to heterotrimeric G proteins, which are in turn subject to regulation by regulator of G protein signaling (RGS) proteins. In this study, we report that germinal center B lymphocytes and thymic epithelial cells strongly express one of the RGS family members, RGS13. Located between Rgs1 and Rgs2, Rgs13 spans 42 kb on mouse chromosome 1. Rgs13 encodes a 157-aa protein that shares 82% amino acid identity with its 159-aa human counterpart. In situ hybridization with sense and antisense probes localized Rgs13 expression to the germinal center regions of mouse spleens and Peyer's patches and to the thymus medulla. Affinity-purified RGS13 Abs detected RGS13-expressing cells in the light zone of the germinal center. RGS13 interacted with both Gialpha and Gqalpha and strongly impaired signaling through G(i)-linked signaling pathways, including signaling through the chemokine receptors CXCR4 and CXCR5. Prolonged CD40 signaling up-regulated RGS13 expression in human tonsil B lymphocytes. These results plus previous studies of RGS1 indicate the germinal center B cells use two RGS proteins, RGS1 and RGS13, to regulate their responsiveness to chemokines.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.