Our laboratory has purified to homogeneity the mammalian alpha-1 adrenergic receptor from rat hepatic membranes. This proten is characterized by a Stoke's radius of 49A, a sedimentation velocity of 7.1S and a principle subunit of 59,000 daltons. Additionally, high-specific activity, high-affinity probes for the alpha-1 receptor have been synthesized and characterized. We propose now to extensively investigate the molecular properties of the receptor, including the detailed determination of its subunit structure by two-dimensional electrophoresis and after peptide cross-linking, the determination of its p1 by isoelectric focusing, the assessment of glycosylation by lectin affinity chromatography, and the construction of peptide maps of the purified receptor-protein. Using the radiolabeled receptor probes, the hydrodynamic properties of the unlabeled receptor will be confirmed, and possible interspecies and intertissue molecular heterogeneity investigated. To complement these studies, polyclonal (elicited) and monoclonal antibodies to the purified receptor and to specific isolated peptide fragments, will be raised. These antibodies will aid in the further pufification of the receptor by immunoadsorbent chromatography, in identifying binding site regions responsible for subtype selectivity, and in the identification of the receptor-protein by a technique independent of its binding activity. To gain insight into the expression and regulation of the receptor-protein, and into its amino acid composition, we further propose to isolate a cDNA cloneencoding for the alpha-1 receptor mRNA. This goal should now be feasible with our ability to effect high-grade purification of the receptor and because of recent developments in protein microsequencing, and in the isolation of rare mRNAs. Finally, preliminary studies directed towards the purification and characterization of the alpha-2 adrenergic receptor from platelet membranes, will be continued using developed affinity chromatography techniques and newly synthesized receptor-specific probes potentially capable of covalent interaction with the receptor-binding site.
Showing the most recent 10 out of 36 publications
