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 deficient B cells obtained from mice in which the RGS1 gene has been disrupted by gene targeting have an enhanced chemotaxic response to SDF-1. In addition these 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 Gs induced cAMP production. These findings are relevant to the olfactory system. Odorants activate the Gs 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 also found that RGS3 profoundly inhibits the activation of pathways triggered by Gbeta gamma subunits. This is independent of RGS3's GAP activity and likely depends upon the binding of RGS3 to free beta gamma subunits. We have continued our studies of RGS14. We have also identified a panel of proteins with which RGS14 interacts. One of those molecules is ninein, a protein of unkown function that resides in the centrosome. RGS14 and ninein co-localize as assessed by confocal microscropy in the centrosome. A number of signals have been identified that shift RGS14 from the cytosol into centrosomes. The role of RGS14 in centrosome function is being investigated. The murine RGS13, RGS3, and RGS5 genes have been isolated and gene targeting of the mouse RGS3 and RGS5 loci is in progress. An analysis of the murine RGS3 gene locus has led to the identification of several novel RGS3 splice variants including two variants that arise from the adjacent C2PA gene. We have isolated and sequenced three human cDNAs that correspond to these splice variants. One of the splice variants has been expressed in mammalian cells as a GST-fusion protein and co-immunoprecipitating proteins are being identified. We have carried out a series of biochemical studies of RGS5, which indicate that RGS5 is a potent Gqalpha GAP and functions in the regulation of cardiovascular function. Specific antibodies to RGS1, RGS2, RGS3, RGS5, RGS10, RGS13, RGS14, and RGS18 have been produced. Using these antibodies and in situ hybridization for specific RGS mRNAs, we are examining the developmental exrpession of the RGS family.
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