The a-adrenergic receptors (a1ARs) are one of three major types of adrenergic receptors that together mediate the diverse effects of the catecholamines epinephrine and norepinephrine on various tissues throughout the body. The a1ARs play a critical role in mediating sympathetic nervous system responses, with particular importance in regulating cardiovascular function. Because of their physiological importance, the a1AR are the targets of numerous clinically useful drugs. These drugs not only activate or inhibit a1AR responses acutely; they also initiate adaptive changes that regulate responsiveness of these receptors to subsequent or continued drug exposure. A better understanding of these regulatory processes may allow for pharmacological manipulation of receptor regulation as a rational approach to improving therapeutic use of drugs targeted at these receptors. This project focuses on elucidating the cellular molecular mechanisms involved in a key aspect of drug-induced regulation for a1ARs, their agonist-induced internalization from the cell surface into intracellular vesicles. Previous studies and preliminary data strongly suggest involvement of two separable steps in a1AR internalization, and initial sequestration within the plasma membrane followed by endocytosis into intracellular vesicles. These sequestration and endocytosis steps will be assessed and characterized for the a1BAR subtype in mammalian cells grown in culture, including cells that normally express a1BARs and cells artificially expressing a1BARs that have been altered by site-directed mutagenesis. Immunofluorescence and electron microscopy will be used to morphologically identify the compartments involved in sequestration and endocytosis. Agents and conditions that can selectively alter either the sequestration or endocytosis steps will be identified. These tools will then be used together with mutated receptors to determine the role of each step in regulating various aspects of receptor function. Finally, various enzyme inhibitors and molecular biology techniques will be used to identify the receptor sequences, accessory proteins, and specific molecular modifications involved in a1BAR internalization. These advances in our understanding of the sequestration of endocytosis steps of a1BAR internalization are likely to apply to other members of the G-protein-coupled receptor family as well.
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