Cells adhere to extracellular matrix molecules via receptors located on the cell surface. These receptors serve not only to anchor the cell to the ECM, but also as focal points for cytoskeletal organization. They are important to cell migration and as such are involved in such processes as wound healing, metastasis, and embryonic development. Avian integrin is such an ECM receptor. Structurally it is a heteromer consisting of both alpha and beta subunits. Its precise subunit composition has yet to be determined. The work proposed for the next 5 years will take advantage of the existence of antibodies specific for avian integrin, and the fact that the gene for the beta subunit of avian integrin has been cloned. We propose a series of structure/function studies in which the gene for the beta subunit will be cloned into mammalian cell expression vectors, transfected into mammalian cells, and the avian subunit specific antibodies used to evaluate its ability to combine with mammalian alpha subunits to form an active receptor. Once this system is established, a series of oligonucleotide-specific, site-directed mutagenesis experiments will be performed to determine the regions on the beta subunit responsible for ECM binding, subunit assembly, and cytoskeletal organization. To determine the regions of the molecule required for selection of the appropriate alpha subunit within the cell, chimeric beta subunits will be constructed containing different portions of beta subunits from other members of the integrin superfamily. The chimeric subunit will be evaluated again using our avian specific antibodies, for its ability to combine with alpha subunits from different subfamilies of the avian super family. The role of the cytoplasmic domain and transmembrane domains in cytoskeletal organi will be evaluated by injecting synthetic peptides corresponding to different portions of these domains, as well as specific antibodies into single cells and following their effect on cell shape and cytoskeleton assembly by fluorescent cinematography. Finally, antibodies specific for the alpha subunit of avian integrin will be produced in order to determine if avian integrin is a mixture of ECM receptors with a common beta subunit, or a single promiscuous heterotrimer. These same antibodies will also be used to follow integrin expression during embryonic development with a special emphasis on hematopoiesis and T-cell maturation using quail-chick chimeras.
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