This Physician-Scientist Award proposal has as its major goal the training in principles and techniques of cell biology of a pulmonary physician who is dedicated to a career in academic medicine. Phase I of the training, which will focus on isolation and characterization of lectin receptors in cellular slime molds, will be in the laboratory of Dr. Richard Fine, Associate Professor of Biochemistry. Phase II of the training, which will apply techniques learned in phase I to alveolar epithelial cells will involve Dr. Fine and Dr. Jerome S. Brody, Professor of Medicine. The mannose-6-phosphate (M-6-P) receptor is a key element in the internalization of extracellular lysosomal enzymes and also in the correct targeting of newly synthesized lysosomal enzymes to the lysosome containing the M-6-P group. Dictyostelium Discoideum, the cellular slime mold, synthesizes lysosomal enzymes in large quantities, and the synthesized molecules contain the M-6-P marker. We propose to isolate and characterize the M-6-P receptor from D. Discoideum and to produce a monospecific antibody against it. We will then use the antibody to study the biosynthesis and regulation of M-6-P receptor during growth and development. We will also determine the role of coated vesicles, which we have recently purified from D. Discoideum in the transport of the M-6-P receptor and lysosomal enzymes. We will employ a variety of modern biochemical techniques used in membrane receptor isolation, especially affinity chromatography and subcellular fractionation in these studies. We will also employ immunochemical techniques including protein blotting and immune precipitation. These studies should aid in the understanding of lysosomogenesis and membrane trafficking in cells and should be of great value in furthering Dr. Dickey's training in membrane receptor purification and characterization which he will then apply to lung development and lung repair. In this phase of the proposal, Dr. Dickey will isolate and characterize lectin receptors from alveolar type 2 cells and from type 1 like cells which evolve in vitro. Once isolated and purified, these receptors, which bind distinct exogenous lectins in each type of cell will be used to raise monoclonal antibodies. The antibodies will be used as the D. Discoideum to study synthesis and regulation of the receptor during lung growth and repair.
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