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. Furthermore, B cells from these mice enter into lymph nodes more easily, target better into lymph node follicles, and move more rapidly than do B cells from wild type mice. 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 as well as other RGS proteins we have developed that express shRNAs that knock-down RGS1, RGS2, RGS3, RGS10, RGS13, RGS14, RGS16, and RGS20 mRNA expression. Introduction of a shRGS13 construct into human B cell lines reduces RGS13 mRNA expression and enhances responses to CXCL13 and more dramatically to CXCL12. To complement these studies we have recently begun to examine B cell function in mice in which Rgs13 has been disrupted. 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. Expression of RGS1-GFP or RGS18-GFP in DCs stimulated with CXCL12 revealed that these RGS proteins significantly impair the migratory capacity of these cells. Dendritic cells prepared from Rgs1-/- mice are also hypersensitive to chemokine stimulation. 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. 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 revealed high levels of RGS1 and RGS13 in the hypo-migratory. We have tested a number of specific inhibitors of signaling molecules 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. Another RGS protein highly expressed in vascular smooth muscle, RGS5, acts as a potent GTPase activating protein for Gi alpha and Gq alpha and attenuates signaling triggered by angiotensin II, endothelin-1, and sphingosine-1-phosphate. 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 these proteins do not possess GAP activity for Ga subunits, with the exception of weak activity of the RGS domain of RGS-PX1 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 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 and characterization of haploid yeast is in progress. In addition an extensive yeast 2-hybrid analysis revealed that the c-terminal portion of RGS-PX2 interacts strongly with several proteins involved in intracellular protein trafficking. Another RGS protein, RGS3 undergoes extensive mRNA splicing. One of the splice variants termed PDZ-RGS3 is widely expressed. A combination of confocal and video time-lapse microscopy revealed that cells overexpressing a PDZ-RGS3 GFP fusion protein failed to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges. The PDZ-RGS3 GFP fusion protein localized at the midbody during the late stages of the cell cycle. Furthermore, we identified an shRNA construct that reduced PDZ-RGS3 expression and its expression resulted in a similar phenotype. We have recently obtained mice with a disrupted Rgs3 allele. To date we have not identified any Rgs3-/- mice suggesting that Rgs3 may be an essential gene for normal development. 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, gamma-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 further facilitate 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. As a model of B cell-DC interactions we examined B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and spleen-derived DCs pulsed with HEL (DC-HEL) in 3-dimensional collagen matrices. Analysis of the live-cell dynamics revealed autonomous movements and random encounters between TgB cells and DC-HEL best described by a """"""""kiss-run and engage"""""""" model that led to formation of micro- and macro-complexes. Control B cells had short-lived interactions and didn?t form macro-complexes with DC-HEL. Antigen localized at contact sites between TgB cells and DC-HEL and both cell types rearranged their actin cytoskeleton toward the contact zone. The TgB cell-DC interaction triggered synchronous Ca2+-transients in both cell types. Thus, B cells productively interact with DCs displaying their cognate antigen to form a stable microenvironment similar to the immune synapse between T cells and DC.
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