We have discovered a protein family termed Regulators of G-protein Signaling (RGS) that impair signal transduction through pathways that use seven 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. RGS1 over-expressing B lymphocytes fail to migrate in response to the chemokine CXCL12. Conversely, RGS1 -/- B cells obtained from mice in which the RGS1 gene has been disrupted by gene targeting have an enhanced chemotaxic response to CXCL12 and fail to desensitize properly following exposure to chemokines. Likely as consequence the RGS1 -/- mice have impaired immune responses, altered lymphoid tissue architecture, an excessive germinal center response, and improper trafficking of plasma cells. We have also demonstrated that germinal center B lymphocytes and thymic epithelial cells strongly express another RGS protein, RGS13. To study the role of RGS13 in the setting of RGS1 deficiency we have developed high titer lentivirus constructs that express shRNAs that knock-down RGS13 mRNA expression. Bone marrow reconstitution studies and transgenic mice expressing this construct are in progress. Dendritic cells (DCs) provide a useful model for studying chemoattractant receptor signaling and the role of RGS proteins in regulating chemoattractant responses. Immature DCs expressed RGS2, RGS10, RGS18, and RGS19. Toll receptor signaling resulted in the induction of RGS1, RGS16, and RGS20 and the downregulation of RGS14 and RGS18. RGS10 and RGS19 levels remained unchanged. Expression of RGS18-GFP in DCs plated on fibronectin suggested that RGS18 localized to focal adhesions. In vivo imaging of RGS1-GFP or RGS18-GFP transfected DCs stimulated with CXCL12 revealed that these RGS proteins significantly impair the migratory capacity of these cells. In order to better understand the mechanisms underlying B-lymphocyte migration, sublines of a B cell line refractive or hyper-migratory to either CXCL12 or CXCL13 were developed. Chemokine receptor levels on all the cell lines are similar to the parental cell. Cell lines refractive to chemotactic signaling tended to be universally refractive to many chemotactic stimuli. The Ca++ responses following chemokine stimulation in the refractive line were inhibited while an increased response was observed in the hyper-responsive lines. Comparisons of the gene expression patterns, determine by gene chip analysis, between the parental, refractive and hyper-migrational lines has been done. Interestingly, the refractive lines had significantly increased levels of RGS1 and RGS13 compared to the hyper-migratory lines. These cells will be useful tools in examining the underlying mechanism regulating migration. We have tested a number of specific inhibitors of signaling moleucles on B-lymphocyte chemotaxis. These studies have revealed potential roles for PI-3 kinase, P38 kinase, JAK kinases, and Rho kinase in B cell migration. To further facillitate our studies of B cell migration we have developed a number of new imaging tools that allow us to study B cell migration and the interaction of B cells and dendritic cells in more detail. We have used a 3-D culture system where the cells are cultured in a collagen matrix. Using standard micro-injectors we can introduce chemoattractants into the collagen matrix at the site of our choosing. Using this methodolgy we have shown that the chemokine stimulated RGS1 -/- B cells have a dramatic increase in their migratory velocity as compared to wild type B cells. We have also observed that dendritic cells significantly augment the migratory capacity of B cells. Co-culture experments with antigen pulsed dendritic cells and B cells revealed that the presence of antigen results in a marked increase in B cell-dendritic cell interactions. In situ hybridization revealed a striking and extensive expression pattern of RGS5 in the arterial walls of E12.5-E17.5 mouse embryos. The distribution and location of the RGS5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E14.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of RGS5 in their vascular plexi and small arteries. We also showed that RGS5 acts as a potent GTPase activating protein for Gi alpha and Gq alpha and that it attenuates angiotensin II, endothelin-1, sphingosine-1-phosphate, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation. To confirm the physiologic importance of RGS5, mice in which the RGS5 gene has been disrupted have been developed. We have just begun to analyze these mice. We also identified and cloned a cDNA for murine RGS-PX1 as well as two other related proteins termed RGS-PX2 and RGS-PX3. All three proteins possess a similar overall structure with an n-terminal hydrophobic region, a PX-associated region (PXA), an RGS domain, a PX domain, and 2 coiled-coiled domains. The RGS domains of RGS-PX2 and RGS-PX3 did not possess GAP activity for Ga subunits, while the RGS domain of RGS-PX1 had very weak activity for Gs. Using lipid overlay assays we identified the specific phospholipids that interact with the PX domains of these proteins. The production of GFP fusion proteins allowed us to determine their intracellular localization. A RGS-PX protein homolog was found in fission yeast and Drosophila and two homologs in C. elegans. To facilitate the understanding of the role of RGS-PX proteins in mammalian cells, the fission yeast RGS-PX isoform was disrupted. Initial characterization of haploid yeast is in progress. RGS14, a larger member of the RGS family, contains an RGS, Rap-interacting, and GoLoco domain. Using RGS14-specific antibodies we found that RGS14 co-localized with a centrosome marker, g-tubulin in centrosomes in a cell cycle-dependent manner. Further studies revealed that RGS14 is a nuclear-cytoplasmic shuttling protein. Time-lapse video microscopy showed that cells over-expressing RGS14 failed either to enter mitosis or to complete mitosis. Prolonged over-expression of RGS14 resulted in formation of multinucleated cells containing supernumerary centrosomes as well as formation of micronuclei, a hallmark of unequal chromosome segregation. RGS14 may play a role in proper positioning of centrosomes/spindle via heterotrimeric G-proteins. To characterize the functional role of RGS14 we have designed shRNAs that knock-down RGS14 mRNA expression. Studies are in progress to functionally characterize the consequences of reducing RGS14 expression. These studies are leading to better understanding of the roles of GPCR signaling in lymphocyte function and defining the funcitonal roles of RGS proteins in immune cells as well as other cell types.
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