The alpha1-adrenergic receptor plays a critical role in sympathetic neuro-transmission mediating responses involved, for example, in circulatory homeostasis, metabolism and central nervous system function, such as vasoconstriction, hepatic glycogenolysis and alterations in locomotor activity. Recent evidence suggests that alpha1-adrenergic receptor signalling occurs via a unique mechanism involving membrane polyphosphoinositide breakdown. Activation of this receptor-linked pathway leads to cellular calcium mobilization and may involve coupling via an as yet uncharacterized guanine nucleotide-binding regulatory protein. We have previously purified the mammalian alpha1-adrenergic receptor to homogeneity and investigated its biophysical and structural properties, both directly and after covalent labeling of the receptor with a specific high-affinity, radioiodinated photoaffinity probe that we developed. The major focus of this application is the isolation of a cDNA clone for the receptor using molecular biology strategies as well as more traditional approaches based on amino acid sequence data determined from internal peptides liberated from the purified receptor protein. The receptor cDNA will then be used to clone the receptor's gene and to perform more detailed investigations of receptor expression and receptor structure-function relationships. These studies will involve the use of molecular biology techniques, reconstitution studies and anti-receptor antibodies. Anti-receptor antibodies will be developed using peptide antigens based on various amino acid sequences determined from the elucidated primary structure of the receptor. Additionally, we propose to test the hypothesis that a guanine nucleotide-binding regulatory protein couples the alpha1-adrenergic receptor to polyphosphoinositide breakdown. Initially, studies will be performed to rigorously define the involvement of such a regulatory protein in alpha:- receptor functioning, and to gain mechanistic insights in receptor- regulatory protein interactions. With the availability of this data, we propose to purify this regulatory protein and to evaluate its molecular characteristics using affinity labeling and reconstitution techniques. When completed, these studies should provide important insights into the biochemical and molecular mechanisms of signal transduction by the alpha1-adrenergic receptor. Using the tools of protein and immunochemistry, as well as molecular biology, new approaches will have been developed for understanding alpha1-adrenergic receptor expression, and for defining the kinetics and stoichiometry of its interaction with cellular effector proteins.
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