Alpha1-adrenergic receptors play a critical role in sympathetic neurotransmission. They mediate a variety of responses including those involved in central nervous system functions, circulatory homeostasis and metabolism, such as alterations in locomotor activity, vascular smooth muscle contraction and glycogenolysis, respectively. Recent evidence suggest that alpha1-receptors are a heterogenous group of distinct but related membrane glycoproteins and are members of a superfamily of receptors that have a common structural motif. They are all composed of single polypeptide chains containing 466 to 560 amino acids with seven hydrophobic domains that most likely represent alpha-helical membrane spanning regions. Alpha1-Adrenergic receptors also interact with a heterogenous group of effectors and are coupled to these effectors via guanine nucleotide-binding regulatory proteins (G-proteins). In most instances receptor activation results in membrane polyphosphoinositide breakdown, although the G-protein involved in this signalling pathway has not been defined. During the present funding period a cDNA clone encoding the alpha1b-adrenergic receptor has been expressed and the ligand-binding properties of the receptor characterized; a cDNA clone encoding a previously undescribed putative subtype of the alpha1-adrenergic receptor has been isolated; a computer model of the alpha1b-receptor has been developed, and thermodynamic studies have been undertaken to evaluate receptor conformation. Additionally, a unique 74 kDa G-protein (Gh) that functionally couples to the alpha1-adrenergic receptor has been identified, characterized, and purified, and considerable progress has been made in isolating a cDNA clone encoding this protein. To gain further insights into the molecular mechanisms involved in signal transduction by alpha1- adrenergic receptors, we propose now to continue efforts to clone and sequence the genes and cDNAs for these proteins. We also propose to undertake a multifaceted approach to understanding receptor structure and function. This involves gene synthesis, site-directed mutagenesis, combinatorial cassette mutagenesis, thermodynamic analyses, macromolecular modeling, and Fourier-transform infrared difference spectroscopy. Each of these approaches should provide unique but complementary information aimed at addressing the following issues: i) which residues are critical for the formation of the ligand binding pocket, and for receptor interactions with agonists and antagonists; ii) which residues are responsible for subtype selectivity; iii) what is the degeneracy of the message encoded by the amino acid sequence that underlies receptor shape and function; iv) what are the major domains and amino acid determinants of receptor G-protein interactions; and v) what is the molecular basis for the actions of agonists and antagonists. Finally, we propose to further test the hypothesis that Gh mediates alpha1-adrenergic receptor signalling. These studies will be aimed at understanding, in more detail, the kinetics and stoichiometry of receptor-G protein interactions and at isolating a cDNA clone for Gh. With the availability of a cDNA for Gh, efforts will be directed at developing a null phenotype for Gh, and at over-expressing this protein to obtain larger quantities for the more detailed evaluation of the functional properties of receptor mutants.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS019583-11
Application #
3399691
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1989-07-01
Project End
1994-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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Graham, R M; Perez, D M; Hwa, J et al. (1996) alpha 1-adrenergic receptor subtypes. Molecular structure, function, and signaling. Circ Res 78:737-49
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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