The exocytic secretion of lung surfactant requires fusion of lamellar bodies with the plasma membrane in alveolar type II cells. Although several processes regulate the alveolar pool of surfactant, the secretion is the most important, as only it can acutely upregulate the level of functionally active surfactant. The mechanism of membrane fusion between lamellar bodies (distinct membrane-enclosed organelles) and plasma membrane has remained poorly investigated. Our studies suggest a role for lung annexin A7 in membrane fusion during surfactant secretion. Annexin A7 (protein and mRNA) is present in type II cells. Annexin A7 binds to specific protein in lamellar body and plasma membrane, promotes Cadependent fusion between these two fractions in vitro, and increases surfactant secretion in permeabilized type II cells. The goal of our ongoing project is to understand the regulation of membrane fusion during surfactant secretion through the structure-function analysis of annexin A7. Our studies with proteins containing deletions and point mutations show that the NH2-terminus can regulate the membrane binding and fusion properties of annexin A7. We plan to extend these structure-function studies to demonstrate that secretagogues of lung surfactant cause phosphorylation of annexin A7 (Specific Aim 1).
In Specific Aim 2, we will identify specific phosphorylation sites by MS analysis of the in vivo and in vitro phosphorylated annexin A7. The in vivo phosphorylation of these sites will be verified in subsequent studies.
In Specific Aim 3, we will demonstrate that surfactant secretagogues cause annexin A7 relocation to specific membranes in type II cells. This relocation, regulated by protein structure and by site-specific phosphorylation, can be correlated with surfactant secretion (Specific Aim 4). The in vivo studies with the type II cells and the in vitro studies with the recombinant proteins will extensively utilize immunoprecipitation and Western blot analysis techniques following one or 2D-gel electrophoresis. The annexin A7 relocation will be determined by immunofluorescence and biochemical techniques. We will utilize adenovirus-mediated transfection and expression of GFP-annexin A7 fusion proteins and determine annexin A7 relocation by confocal microscopy. The proposed studies are important in understanding the mechanism and annexin A7 regulation of membrane fusion during surfactant secretion, which is essential for normal lung function. PROJECT NARRATIVE: Lung surfactant is essential for lung function of gas-exchange since it prevents lung collapse. Its deficiency causes respiratory distress that is well documented in the premature newborn infant. The secretion of active surfactant from lung cells occurs following fusion of surfactant storage vesicles with the cell membrane. We have been conducting structure-function analysis of a cell protein that is postulated to promote such membrane fusion. We propose to extend our studies on the structural regulation of (this) protein function. The overall goal of our studies is to define the regulatory steps in the fusion process so that efficient alternative molecules can be tested to enhance fusion and the secretion processes.
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