In contrast to the well characterized beta-adrenergic receptor- adenylate cyclase system, the plasma membrane components which couple the alpha 1-adrenergic receptor to intracellular calcium mobilization remain unidentified.
The specific aims of this proposal are to: 1) develop a reliable method to purify large amounts of the alpha 1-adrenergic receptor from the DDT1 MF-2 cell line, 2) obtain polyclonal and monoclonal antibodies against the alpha 1-adrenergic receptor, and 3) using a hamster lambda gtll library, isolate a complementary DNA (cDNA) from which the primary amino acid sequence of the alpha 1-adrenergic receptor will be deduced. DDT1 MF-2 cells are the starting point for receptor purification. We plan to use a combination of affinity chromatography and size exclusion HPLC to obtain sufficient purified receptor for sequencing using a ga phase protein sequenator and for immunization of mice and rabbits to produce anti-receptor polyclonal and monoclonal antibodies. For sequence information, oligodeoxynucleotides will be synthesized. The oligodeoxynucleotides and anti-receptor antibodies will be used to screen a lambda gtll cDNA library already established in our laboratory. The isolated DNA sequence complementary to the alpha 1-adrenergic receptor mRNA will be transfected into 6580 cells, derived from a hamster uterine leiomyosarcoma, which lack alpha 1-adrenergic receptors. Unidirectional 45 Ca+2 efflux and radioligand assays will be performed to determine whether functional alpha 1-adrenergic receptor is inserted into the plasma membrane by this procedure. The long term goals are to use the probes (antibodies and cDNA) generated in this project to study the membrane events associated with alpha 1-adrenergic stimulation, to study regulation of receptor function and to obtain the genomic DNA sequence and structure which codes for the alpha 1-adrenergic receptor. The availability of antibodies and cDNA probes should provide investigators new tools with which to study disease states (e.g. hypertension) which possibly involve the alpha 1-adrenergic receptor.
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