This application attempts to relate adrenergic receptor molecular structure to hormone/drug interaction and evoked physiological response. The principle investigator has demonstrated that in the prostate the predominant alpha 1 adrenergic receptor subtype is alpha 1c and suggested that it plays a role in the pathogenesis of benign prostatic hyperplasia. The hypothesis is that specific regions of human alpha 1c receptor are involved in its overall function.
In specific aim 1, the applicant proposes to elucidate regions of the human alpha 1c receptor involved in subtype specific ligand binding. The proposed approaches involve chemical modification by alkylating agents (chloroethylchlonidine) and photoaffinity ligands (125I-azidoprazosin), and subsequent enzymatic and chemical cleavage and peptide sequencing. An additional approach to identify such amino acids will involve site-directed mutagenesis.
In specific aim 2, the applicant cloned and sequenced 1.5-2 kb of the 5'untranslated region of the human alpha 1c receptor to determine transcription initiation sites and regulatory elements in this region. The applicant proposes to investigate the effects of transcription regulation by hormones, second messengers and protein synthesis inhibitors.
In specific aim 3, mechanisms of desensitization and protein regulation will be studied by creation of alpha 1c receptor tagged with an epitope for a commercially available antibody, to study receptor phosphorylation; to evaluate a host of protein kinases and their ability to phosphorylate alpha 1c receptor; to elucidate the specific amino acid residues involved in alpha 1c receptor phosphorylation.
