This project continues my laboratory's studies of cellular actions of alpha-adrenergic receptors using a clonal isolate of Madin Darby Canine kidney (MDCK) cells. Our laboratory has shown that in these cells alpha- adrenergic receptors link to one or more guanine nucleotide binding (G) protein and in turn to multiple phospholipases, including one or more phospholipases alpha2, C and D. Regulation of phospholipase alpha2 by alpha1-adrenergic receptors occurs in part via protein kinase C, in part, we believe, by specific isoforms of this enzyme. The current studies are designed to test hypotheses related to alpha1 adrenergic receptor structure, G protein linkage, phospholipase regulation, and protein kinase C isoforms in MDCK-D1 cells. Studies of alpha1-adrenergic receptor structure will emphasize characterization and expression of a unique truncated alpha1b- adrenergic receptor, the cDNA for which we have recently isolated. In addition, we will use MDCK-D1 cell alpha1b- adrenergic cDNA that we have isolated to transfect MDCK-D1 cells in order to define quantitative relationships between receptor expression and response. Studies of G proteins will utilize photolabelling and immunoprecipitation as well as antisense """"""""knockout"""""""" with full length cDNA and isolation of stable transfectants to define G-alpha proteins that link to alpha1b receptors. Studies of phospholipases will involve biochemical characterization of phospholipase D and phospholipase A2 and protocols designed to define the role of MAP kinase in protein kinase C- mediated regulation of the phospholipases.
The final aim i s to assess the role of isoforms of protein kinase C in cell regulation, especially in regulation of phospholipases. The methods to be used in these latter studies include stable transfection with antisense cDNA to generate cells null with respect to individual C-kinase isoforms. Other approaches will involve microscopy with fluorescently labelled phorbol ester and immunological probes for particular isoforms. Taken together, the studies proposed should provide new information regarding alpha-adrenergic receptor action, G proteins, phospholipase regulation, and the role of protein kinase C isoforms in regulation of cellular function. Given the importance of alpha1-adrenergic receptors and protein kinase C for a wide variety of cellular functions, the results may have importance to disease settings such as cancer and several types of cardiovascular, renal, and metabolic disorders.
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