9631601 Blobel The proposed studies aimed at understanding the role of the membrane-anchored cellular disintegrin protein metargidin (metalloprotease-RGD-disintegrin), which contains the integrin binding sequence RGD in its disintegrin domain, in cell-cell and cell-matrix intera6tions. Cellular disintegrins are a family of cellular proteins that are closely related to soluble snake venom integrin ligands and metalloproteases. Metargidin is the first known cellular disintegrin protein to contain the RGD sequence in a position analogous to RDG-containing snake venom disintegrins. Since snake venom disintegrins are high affinity integrin ligands, the presence of the RGD sequence in metargidin strongly suggests that it is also an integrin ligand. Since most known cellular disintegrins contain a membrane anchor, they may function as membrane anchored integrin ligands and thus mediate important cell-cell interactions. The cellular disintegrin metargidin also contains a metalloprotease domain, and is therefore likely to have an additional role as a protease. This project focuses on the biochemical characterization of the disintegrin domain and the metalloprotease domain of metargidin. There are two specific aims. The first is to determine whether the metargidin disintegrin domain binds integrins. This will be done using affinity purified metargidin, and metargidin protein domains expressed in COS-7 cells. As sources of RGD binding integrins, platelets and cell lines with known integrin receptors will be employed. The second aim is to determine whether the metalloprotease domain is functional. Again, purified metargidin and expressed domains will be used in protease assays. If the protease is active, the potential regulatory function of the pro-domain through a cysteine switch mechanism will be investigated by site directed mutagenesis. It is anticipated that the proposed studies will shed light on the structure and function of this intriguing protein, will serve as a model for the st udy of other cellular disintegrins, and will contribute to the understanding of how members of this new protein family contribute to cell-cell interactions. ***
