Alpha-2-adrenergic receptors (alpha-2-AR) are cell surface receptors which upon binding catecholamines signal to the interior of the cell via G proteins. Alpha-2-AR are expressed in virtually every organ system and are known to play important roles in cardiovascular, pulmonary, renal, hepatic, metabolic, and central nervous system functions. Alpha-2-ARs have also been implicated in a number of pathologic processes and are the targets for pharmacologic agents in the treatment of a number of diseases. The long-term objective of this project is to understand the relationships between the molecular structures of these receptors and their functions. These goals will be carried out primarily by site-directed mutagenesis of the cDNAs encoding for the wild-type alpha-2-AR subtypes (alpha-2C10, alpha-2-C4, and alpha-2-C2), followed by recombinant expression in mammalian cells. This allows for directly comparing a given function between wild-type and mutated receptor and delineating a structure/function relationship.
In Specific Aim 1, the role of G protein coupled receptor kinases in the phosphorylation of alpha-2-ARs during short-term agonist promoted desensitization will be studied.
In Specific Aim 2, the phosphorylation domains of the alpha-2-AR will be mapped by assessing functional short-term agonist promoted desensitization and receptor phosphorylation in wild-type and alpha-2-ARs with mutated residues in regions that we suspect are sites for phosphorylation.
In Specific Aim 3, the molecular features of the alpha- 2-AR subtypes which are responsible for the differences in agonist- promoted desensitization and phosphorylation observed between the three subtypes will be determined.
Specific Aim 4 explores the mechanism of receptor sequestration (internalization) and downregulation, two key events which occur during long-term agonist promoted regulation of alpha- 2-ARs. Here, the subcellular events of receptor trafficking and processing will be examined using immunoelectron microscopy.
In Specific Aim 5, the molecular determinants of alpha-2-AR sequestration and downregulation will be studied using site-directed mutagenesis of regions suspected to be involved in these processes including sites for palmitoylation, glycosylation, and phosphorylation, and regions involved with G protein coupling.
In Specific Aim 6, the domains of the alpha-2- AR responsible for coupling to G1 and G2 will be delineated by both deletion and chimeric substitution mutagenesis.
In Specific Aim 7 the molecular determinants of the observed differences in G2 coupling between the three alpha-2-AR subtypes will be determined. The results of these studies will help to determine at a fundamental level how alpha-2-AR carry out their signal transduction, how they are regulated, and how they can be modulated by therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL053436-01A1
Application #
2231356
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1995-08-01
Project End
1999-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221