The adrenergic receptors mediate the effects of the catecholamine hormones on the body and are the sites of action of many clinically important drugs. The functional capacity of these receptors often decreases during the course of prolonged exposure to drugs that activate the receptors, a phenomenon referred to as agonist-induced desensitization. The cellular and molecular mechanisms of desensitization for beta adrenergic receptors are becoming well-established, but knowledge of alpha-1 adrenergic receptor regulation lags considerably behind. This application proposes experiments to determine the mechanisms involved in alpha-1 adrenergic receptor desensitization and to understand differences in regulation for alpha-1 and beta receptors. The emphasis is on studies in DDT1 MF-2 hamster smooth muscle cells, which express both beta and alpha-1 adrenergic receptors. Knowledge of beta receptor desensitization and our previous studies of alpha-1 receptor desensitization in these cells provide the rationale for the specific studies proposed.
Aim 1 : Receptor binding studies, photoaffinity labelling, permeabilized cells, and protein kinase and phospholipase inhibitors, and site-directed mutagenesis will be used to investigate the occurrence and mechanisms of rapid uncoupling of alpha-1 receptors from stimulation of phosphoinositide turnover.
Aim 2 : Further studies will be conducted to document and characterize the two- step nature of alpha-1 receptor sequestration and internalization, including four different assays for sequestration and internalization, studies with endocytosis and kinase inhibitors, site-directed mutagenesis and chimeric receptors, and similar studies to determine if two-step internalization also occurs for beta receptors.
Aim 3 : Studies of the longer-term down-regulation of receptor binding sites will include investigation of apparent differences for beta and alpha-1 receptors, studies with kinase and endocytosis inhibitors, site-directed mutagenesis and chimeric receptors, and characterization of changes in mRNA expression. For each of the three major steps in the desensitization pathway in Aims 1-3, initial basic characterization of the phenomena will guide the site-directed mutagenesis and other studies to determine the detailed molecular mechanisms involved.
Aim 4 : The role of receptor sequestration and internalization and other possible mechanisms in the low affinity binding of agonists to intact cells that is observed for both beta and alpha-1 receptors will also be further investigated, including studies with cells expressing mutated receptors and studies with kinase and endocytosis inhibitors. Together these studies should greatly advance our understanding of alpha-1 receptor desensitization and provide important new insights into the regulation of this important class of receptors.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Pharmacology A Study Section (PHRA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Nebraska Medical Center
Schools of Medicine
United States
Zip Code
Hague, Chris; Chen, Zhongjian; Pupo, Andre S et al. (2004) The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression. J Pharmacol Exp Ther 309:388-97
Prinster, Steven C; Schulte, Nancy A; Collins, Megan R et al. (2003) Up-regulation of alpha1B-adrenergic receptors with defects in G protein coupling: ligand-induced protection from receptor instability. Mol Pharmacol 64:1126-35
Toews, Myron L; Prinster, Steven C; Schulte, Nancy A (2003) Regulation of alpha-1B adrenergic receptor localization, trafficking, function, and stability. Life Sci 74:379-89
Wang, J; Wang, L; Zheng, J et al. (2000) Identification of distinct carboxyl-terminal domains mediating internalization and down-regulation of the hamster alpha(1B)- adrenergic receptor. Mol Pharmacol 57:687-94
Cerutis, D R; Nogami, M; Anderson, J L et al. (1997) Lysophosphatidic acid and EGF stimulate mitogenesis in human airway smooth muscle cells. Am J Physiol 273:L10-5
Bird, K S; Anderson, J L; Toews, M L (1997) Modulation of alpha1B-adrenoceptor expression by agonist and protein kinase inhibitors. Eur J Pharmacol 340:267-75
Wang, J; Zheng, J; Anderson, J L et al. (1997) A mutation in the hamster alpha1B-adrenergic receptor that differentiates two steps in the pathway of receptor internalization. Mol Pharmacol 52:306-13
Zhu, S J; Hatcher, L I; Brown 3rd, J C et al. (1996) Effects of hypertonic sucrose and potassium depletion on the binding properties of beta and alpha 1 adrenergic receptors measured on intact cells. Recept Signal Transduct 6:131-40
Zhu, S J; Cerutis, D R; Anderson, J L et al. (1996) Regulation of hamster alpha 1B-adrenoceptors expressed in Chinese hamster ovary cells. Eur J Pharmacol 299:205-12
Hoffman, J; Whittle, S; Toews, M (1996) Modulation of cyclic AMP accumulation in glial cells by exogenous phospholipase C. J Lipid Mediat Cell Signal 13:51-62

Showing the most recent 10 out of 27 publications