3 Adrenoceptors (pARs) signaling is finely regulated to control cardiovascular function in response to sympathetic nervous system input (norepinephrine) and to adrenaline (epinephrine) from the adrenal gland. In animal hearts, two highly homologous pARs (P1AR and P2AR) are stimulated by catecholamines to enhance contractility and heart rate. Chronic stimulation of cardiac (3ARs by elevated circulating catecholamines as well as decreased cardiac (31AR density is clinically associated with heart failure. The primary functions attributed to GPCRs are ligand binding and G-protein coupling. However, the specific and diversified functional properties of GPCRs in vivo involve kinetic receptor conformational changes for binding different partners in signaling complexes, and the localization of receptor signaling complexes in cell type- specific manner. We hypothesize that norepinephrine and epinephrine can activate distinct signaling pathways for both (31AR and (32AR in cardiac myocyte. We have chosen the neonatal cardiac myocyte isolated from the PAR gene deficiency mice as a model system to study the functional roles of ligand- receptor interactions in differentiated cells. The goals of this proposal are (1) to characterize (31 AR and |32AR subtype-specific signaling by different agonists in neonatal myocytes, (2) define the cellular and biochemical properties of (31 AR and P2AR under norepinephrine and epinephrine stimulation in cardiac myocytes, (3) to characterize the subcellular distribution of (31ARand (32AR, and other signaling molecules under norepinephrine and epinephrine stimulation in cardiac myocyte, (4) to identify receptor interaction domains and their targeted proteins, and to characterize the receptor signaling complex formation and stability upon agonist stimulation. A better understanding of agonist-induced PAR conformationchange for the organization of signaling complexes may facilitate new drug designs and suggest new clinical applications in treating various cardiovascular diseases.
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