The long-range goal of this project is to elucidate the molecular basis of catecholamine-induced internalization and down-regulation of B-adrenergic receptors. Understanding of these mechanisms will help form the basis of a more rational use of drugs that must be administered chronically. The results should promote further understanding of changes that occur in the expression of receptors for neurotransmitters, hormones and other signal molecules in various pathologic states. The experimental approach will utilize cultured mammalian cells that express native or modified forms of the B-adrenergic receptor. The relation of the structure of the receptor to its capacity to undergo catecholamine-induced endocytosis and down-regulation will be examined in cell expressing B-adrenergic receptors modified by site-directed and/or domain-directed mutagenesis and isolated by molecular cloning techniques. Specific antibodies raised against portions of the native or mutated protein will be used to visualize the subcellular localization of the receptor utilizing the techniques of immunogold electron microscopy. Experiments will seek answers to the following specific questions: 1) What subcellular structures are associated with internalized receptors? 2) What are the structural determinants of the receptor for internalization and down-regulation? 3) Do Gproteins play a role in catecholamine-induced receptor down-regulation? 4) Where in the cell are down-regulated receptors localized? 5) What is the structural status of down-regulated B-adrenergic receptors? Successful completion of these studies will be pursued using modem techniques of immunology, molecular biology, biochemistry, pharmacology, and electron microscopy.
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