EXCEED THE SPACE PROVIDED. Alveolar type II cells synthesize and store lung surfactant in lamellar bodies prior to secretion in the alveolar space. The synthesis, secretion, clearance, and degradation of surfactant components tightly regulate the alveolar pool of surfactant. In this context, the secretion of lung surfactant is probably most important, as it is the only means to acutely upregulate the alveolar pool of functionally active surfactant. The lamellar bodies are distinct organelles that are enclosed by a limiting membrane. The mechanism of membrane fusion between lamellar body and plasma membrane, an obligatory step during surfactant secretion, has remained poorly investigated. A role for lung synexin (annexin VII), a member of the 'annexins' family of proteins, in membrane fusion during surfactant secretion has been suggested. Synexin is present in type II cells, it bind_ to specific protein in lamellar body and plasma membrane, promotes in vitro fusion between these twc fractions in a Ca2+-dependent manner, and increases surfactant secretion in permeabilized type II cells. Additionally, surfactant secretagogues increase synexin binding to type II cell membranes, and several inhibitors of synexin activity inhibit surfactant secretion. The overall goal of this project is to understand the regulation of membrane fusion during surfactant secretion through the study of structure-function analysi_ of synexin. Using deletion and substitution mutation constructs of rat synexin cDNA, recombinant protein and peptides will be expressed, purified, and characterized to determine the specific domains and amino acid residues that regulate self-association, membrane binding, membrane fusion, and secretion of lunl surfactant. Phosphorylation-dependent regulation of synexin function will be studied using commercialb available protein kinase enzymes that are implicated in surfactant secretion. Synexin contains severaJ putative phosphorylation sites. Recombinant proteins with deletions and substitution mutations will be utilized for identification of specific domains and sites that are phosphorylated with these enzymes. The site- specific phosphorylation in the regulation of synexin function will be verified with wild type and mutan| synexin proteins using in vitro models of membrane binding, and surfactant secretion in permeabilized type II cells. The proposed studies will provide important information about the mechanism and regulation oi synexin action in membrane fusion during surfactant secretion. PERFORMANCE SITE ========================================Section End===========================================
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