EXCEED THE SPACE PROVIDED. Atrial natriuretic peptide (ANP) plays a major role in the regulation and maintenance of the cardiovascular system and its aberrations may lead to hypertension, cardiac hypertrophy, congestive heart failure, and other cardiovascular diseases. The actions of ANP are mediated by cell membrane receptors coupled to guanylate cyclase (GCase). The goal of our research is to elucidate the mechanisms of ANP-receptor interaction and transmembrane signal transduction. The ANP receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain (BCD), a single transmembrane sequence, and an intracellular domain (ICD) consisting of a kinase-homologous regulatory domain and a guanylate cyclase catalytic domain. The mechanism of signal transduction by the ANP receptor remains largely unknown.It has been suggested that GCase activation by ANP may be mediated by ligand-induced receptor dimerization. However, the ANP receptor has been shown by us and others to dimerize in the absence of the hormone, indicating that dimerization alone is not sufficient for receptor activation. Thus, we hypothesize that ANP binding induces, in addition to dimerization, a conformational change in the extracellular domain which is transduced across the cell membrane to the intracellular region and causes activation of the GCase catalytic activity. Recently, we have found that ANP binding to its receptor is chloride concentration dependent. This finding suggest a possible chloride mediated feedback control of the ANP receptor playing a role in salt-fluid homeostasis. To understand the mechanisms of signaling and its control by chloride, we propose 1) to elucidate the mechanism of receptor-ANP interaction and the ANP-induced conformational changes responsible for signal transduction by a combination of site-directed mutagenesis and crystallographic determination of the structures of the BCD bound with ANP and with the inactiveanalog AP-I, 2) to elucidate the mechanism of chloride-control of ANP binding by characterizing the chloride-binding site by mutagenesis, analyzingchloride effects on the BCD conformation and dimerization by biophysical analyses, and determining the crystal structure of the BCD lacking chloride, and 3) to express and purify the ICD and to characterize its activities and molecular behavior to understand the mechanism of ICD action in ANP receptor signaling. These studies will provide us a more accurate understanding of the mechanisms of ANP-receptor interaction and signaling, chloride-control of the ANP receptor, and the mechanism of GCase activation in response to ANP binding signal. Such knowledge will give us a better understanding of the physiological actions of ANP that, in turn, will promote development of more reliable diagnostic methods and more effective therapies for the cardiovascular diseases. PERFORMANCE SITE ========================================Section End===========================================
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