This is an application to continue studies of extracellular matrix protein regulation of phagocytosis. The overall hypothesis is that interaction with extracellular matrix is an important mechanism for activation of professional phagocytes, polymorphonuclear leukocytes monocytes, and macrophages, at sites of inflammation. This hypothesis implies signal transduction from plasma membrane receptors which interact with the extracellular matrix. Assays which are well suited to studies of signal transduction in phagocytes as a result of interaction with extracellular matrix proteins have been developed. They involve stimulation of neutrophil phagocytosis and chemotaxis by extracellular matrix proteins. These assays separate ligand-receptor interactions from the possible effects of biomechanical forces that result from cell spreading on the extracellular matrix. In these assays, unique short linear peptide sequences are able to activate the phagocytes through the same receptor as intact matrix proteins. This receptor is immunologically related to the Beta3 Integrin family and is called the Leukocyte Response Integrin. Furthermore, a novel non-receptor membrane molecule is involved in signal transduction from ligand binding to the Leukocyte Response Integrin. This 50kd protein which spans the membrane multiple times is called Integrin Associated Protein. This application proposes to study signal transduction involved in phagocyte activation via Leukocyte Response Integrin in detail. Studies are proposed of the binding of monovalent and oligomeric peptides; of the roles for G proteins, [Ca], phospholipase C, and protein kinase C; and of the specific second messengers required for activation of phagocytosis, respiratory burst, and chemotaxis. For these studies monovalent and oligomeric short peptides will be used which are specific ligands for the Leukocyte Response Integrin. In addition, the role for Integrin Associated Protein will be determined. Its primary sequence will be determined from cDNA cloning; the epitopes for inhibitory and noninhibitory monoclonal antibodies will be mapped; and the phenotype of cell lines specifically created to lack expression of the protein will be investigated. Studies involving transfection of native and mutated IAP cDNA into cells lacking Integrin Associated Protein are proposed as a way to investigate structure- function relationships in this molecule. Taken together these studies will enable rational design of ligand antagonists or other inhibitors of signal transduction from phagocyte interaction with the extracellular matrix. These will be a novel class of agents potentially useful in the pharmacologic regulation of inflammation in many disease states .
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