Continued studies will focus primarily on tenascin - the giant hexabrachion molecule in the extracellular matrix of tumors, healing wounds and specific embryonic tissues. We are already making transgenic mice that overexpress tenascin. We propose to make others that will be defective in hexabrachion assembly (monobrachion mice), and mice with depletion in tenascin secretion. We will determine the effects of overexpression, underexpression or defective hexabrachions on embryonic development, tumors and wound healing. A second project is a search for the tenascin gene in Drosophila and C. elegans. These species have highly developed genetic maps and large numbers of mutants, offering a powerful new approach to understanding the functions of tenascin. We will continue our studies of the cell biology and biochemistry of tenascin, to identify and characterize cell surface receptors for different domains of tenascin. A key tool for this project is the library of bacterial expression proteins that we have recently developed, providing large quantities of defined segments of the hexabrachion arm. We will use a similar approach to identify ECM molecules that bind to tenascin. Another continuing project is to characterize a new form of laminin that we identified during the purification of tenascin from cell culture supernatant. This new protein appears to have a variant A chain, a unique tissue distribution, and a cell adhesion activity quite different from any known laminin. Finally, we propose to expand our collaborative projects on the x-ray crystallography of tenascin. Our approach is to crystallize one domain at a time, using our PCR approach to make precisely defined bacterial expression proteins. The two FN-III domains that we have tried so far, the RGD domains from tenascin and fibronectin, have given excellent crystals, and high resolution x-ray diffraction studies are in progress. Additional domains are now proposed for future studies. An atomic resolution structure of the entire hexabrachion is a feasible goal.
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