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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS019583-05
Application #
3399688
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1982-09-01
Project End
1988-02-29
Budget Start
1986-03-01
Budget End
1987-02-28
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Guarino, R D; Perez, D M; Piascik, M T (1996) Recent advances in the molecular pharmacology of the alpha 1-adrenergic receptors. Cell Signal 8:323-33
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Perez, D M; Hwa, J; Gaivin, R et al. (1996) Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor. Mol Pharmacol 49:112-22
deS Senanayake, P; Denker, J; Bravo, E L et al. (1995) Production, characterization, and expression of neuropeptide Y by human pheochromocytoma. J Clin Invest 96:2503-9
Piascik, M T; Guarino, R D; Smith, M S et al. (1995) The specific contribution of the novel alpha-1D adrenoceptor to the contraction of vascular smooth muscle. J Pharmacol Exp Ther 275:1583-9
Riek, R P; Handschumacher, M D; Sung, S S et al. (1995) Evolutionary conservation of both the hydrophilic and hydrophobic nature of transmembrane residues. J Theor Biol 172:245-58
Piascik, M T; Smith, M S; Soltis, E E et al. (1994) Identification of the mRNA for the novel alpha 1D-adrenoceptor and two other alpha 1-adrenoceptors in vascular smooth muscle. Mol Pharmacol 46:30-40
De Young, M B; Keller, J C; Graham, R M et al. (1994) Brefeldin A defines distinct pathways for atrial natriuretic factor secretion in neonatal rat atrial and ventricular myocytes. Circ Res 74:33-40
Perez, D M; Piascik, M T; Malik, N et al. (1994) Cloning, expression, and tissue distribution of the rat homolog of the bovine alpha 1C-adrenergic receptor provide evidence for its classification as the alpha 1A subtype. Mol Pharmacol 46:823-31
Noda, K; Saad, Y; Graham, R M et al. (1994) The high affinity state of the beta 2-adrenergic receptor requires unique interaction between conserved and non-conserved extracellular loop cysteines. J Biol Chem 269:6743-52

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