We have discovered a protein family termed RGSs 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 chemokine receptors and that they can inhibit chemotaxis. RGS1 expressing B lymphocytes fail to migrate in response to the chemokine SDF- 1. Conversely, RGS1 -/- B cells obtained from mice in which the RGS1 gene has been disrupted by gene targeting have an enhanced chemotaxic response to SDF-1 and fail to desensitize properly following exposure to chemokines. Likely as consequnce the RGS1 -/- mice have impaired immune respones and altered lymphoid tissue architecture. We have also shown that certain RGS proteins can directly inhibit the activation of adenylyl cyclase, thereby providing a mechanism by which these proteins can inhibit RGS induced cAMP production. These findings are relevant to the olfactory system. Odorants activate the RGS family member Golf, which leads to activation of adenylyl cyclase type III (AC III) and the production of cAMP. RGS2 potently inhibits AC III mediated cAMP production. Olfactory neurons express both RGS2 and RGS3 and the microinjection of an antibody to RGS2 into olfactory neurons profoundly enhances odorant induced signal transduction. We have found that the RGS3 gene is situtated close to another coding region termed C2PA. Together the C2PA and RGS3 coding regions combine to produce several distinct proteins including PDZ-RGS3, a recently described protein that interferes with the CXCL12 signaling in cerebellar neurons. CXCL12 is a chemokine important for the recruitment of these neurons during embryonic development. The combined loci are now collectively referred to as the RGS3 gene. We have fused the coding region for green fluorescent protein to 5 of the RGS3 isoforms and are studying their intracellular location. We have also isolated several interacting proteins with the C2PA portion of RGS3 by analyzing co-immunoprecipitating proteins by mass spectroscopy. We have confirmed their interaction by direct co-immunoprecipitation experiments. We are now studying the functional importance of the interaction between the RGS3 isoforms and these newly identified interactors. We have continued our studies of RGS14 and its potential role in centrosome function. We have documented endogenous RGS14 expression in centrosomes in both normal cells and in cell lines. Centrosomes organize the mitotic spindle during cell division. We have found that overexpression of RGS14 leads to profound defects in cytokinesis, the splitting of cells following DNA replication and chromosomal segration, thereby causing multinucleated cells. Current studies are focused on analyzing the consequences of reducing RGS14 levels in cells. The murine RGS13, RGS3, RGS18, and RGS5 genes have been isolated and gene targeting of the mouse RGS3 and RGS5 loci is in progress. Initial targeting attempts have been unsuccessful and re-designed targeting constructs are now being tested. We have carried out a series of biochemical studies of RGS5, which indicate that RGS5 is a potent Gqalpha GAP and takes part in the regulation of cardiovascular function. In addition, we have shown that RGS5 is a novel marker for pericytes and vascular smooth muscle cells. The absence of RGS5 expression in PDGF-B and PDGFR-beta null embryos correlated with known sites of pericyte loss in these animals. We have shown that another RGS protein, RGS13, is an excellent marker for germinal center B lymphocytes. RGS13 inhibits both Gqalpha and Gialpha mediated signaling and inhibits chemotaxis in response to the B lymphocyte chemokines, CXCL12 and CXCL13. RGS18 is strongly expressed in megakaryocytes and platelets and at lower levels in monocytes and immature dendritic cells. Upon differentiation to mature dendritic cells RGS18 levels dramatically decline suggesting that enhanced mobility of mature dendritic cells may be related to the fall in RGS18 levels. We have made GFP fusion proteins with RGS1, RGS2, RGS3, RGS4, RGS5, RGS13, RGS14, and RGS18. These constructs are being used to make permanent cell lines to study the intracellular localization of these proteins following stimulation by a panel of different agonists. Recently two human genes, RGS-PX1 and RGS-PX2, which encode GAPs for Gsalpha have been identified. These are complicated proteins that possess a number of domains besides their RGS-like domain. We have isolated two murine genes that are orthologues of the human genes and a third gene, RGS-PX3. Coding regions for both human and mouse RGS-PX3 have been identified. We have recently completed a study of their expression in various mouse tissues. We have also identifed a yeast protein that shares considerable homology with the RGS-PX proteins. We plan to study the function of this protein in yeast and attempt to complement yeast deficient in RGS-PX with mammalian genes. In addition a C. elegans RGS-PX has also been identified. The role of this gene in C. elegans will be approached by using RNAi to inhibit its expression. In order to pursue another approach to identifying molecules important in regulating GPCR signaling, we have selected B cell lines that are either hyper- or hypo-responsive to chemokine stimulation. By repetitively selecting those cells that responded or did not respond in chemotaxis assays we have developed B cell lines in which the majority of the cells move in response to CXCL12 or in which very few of the cells move. Using a similar approach we have developed B cell lines hyper- or hypo-responsive to CXCL13. Using gene chips we have analyzed the gene expression in the hyper and hypo-responsive cells. Interesting both RGS1 and RGS13 were significantly upregulated in the hypo-responsive cell lines as compared to the hyper-responsive lines. The expression of a number of other interesting genes were also modified. We are currently verifying these changes by Northern blot and RT-PCR.
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