The regulation of cellular sensitivity, often termed desensitization, is observed in systems as diverse as yeast to man. In many of these system an agonist-promoted phosphorylation of cell surface receptors appears to play a major role in the desensitization process. For the beta-adrenergic receptor, which is coupled to stimulation of adenylyl cyclase, phosphorylation appears to be mediated by a kinase, the beta-adrenergic receptor kinase or beta-ARK has been purified from bovine brain and recently a cDNA which encodes this enzyme has been isolated. The objectives of this proposal are to isolate, express and characterize other protein kinases which are related to beta-ARK. Initially, cDNA species related to the beta-ARK cDNA will be isolated using low stringency hybridization and/or the polymerase chain reaction (PCR). Isolated cDNAs will e inserted into the mammalian expression vector pBC 12BI followed by transient expression in COS-7 cells. This should establish whether or not an active enzyme is expressed. Overexpression of BARK and other related kinases will be attempted in a baculovirus expression system. The expressed kinases will then be purified using conventional methodology. The purified kinases will be extensively characterized with regard to their substrate specificity. Substrates used will include adenylyl cyclase stimulatory and inhibitory receptors, phospholipase C stimulatory receptors, rhodopsin, phosvitin and synthetic peptides. Finally, mRNAs for the various kinases will be localized by Northern blot hybridization.
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