9510380 Thomsen These studies will address the biochemical and embryological function of a set of proteases that regulate, by an unknown mechanisms, the activity of growth factors in the transforming growth factor beta (TGF-beta) superfamily. The proteases under investigation are BMP-1 and closely related factors which belong to a group of metalloendoproteases, the astacins (named after the first isolate of the group, a crayfish digestive protease). Genetic studies in Drosophila suggest that an astacin related protease called tolloid affects the activity of decapentaplegic (dpp), a ligand in the TGF-beta family. Members of the TGF-beta family have a wide variety of developmental functions in embryos of vertebrates and invertebrates. In the development of the amphibian Xenopus, TGF-beta related factors (e.g. activin, Vg-1 and bone morphogenetic or BMPs) induce mesodermal tissues and influence body pattern formation. There is also evidence that the activity of these growth factors can be regulated at the level of protein processing. Proteases such as tolloid and a BMP-1 might proteolytically process and thus activate TGF-beta related proproteins, or indirectly affect the activity of TGF-beta ligands by proteolytically inactivating a ligand inhibitor. In Xenopus embryos, BMP-2 and BMP- 4 induce ventral mesoderm (e.g. erythroid blood) and antagonize the development of dorsal mesodermal tissues. Ectopic expression of BMP-1 in Xenopus embryos similarly antagonizes the development of dorsal mesoderm, perhaps by activating BMP ligands which then exert a ventralizing affect. This grant will focus on the physical and biological characterization of Xenopus BMP-1 and perhaps other related astacins. The studies will a) describe the primary cDNA structure and the expression patterns of Xenopus BMP-1 and potentially other astacins, b) determine the embryological activities of BMP-1 and other astacin related factors by ectopic expression of wild-type (functional) and mutat ed dominant negative (functionally competitive) versions of the proteins in embryos, and c) attempt to understand the interaction of astacins and the TGF- beta related BMPs at the biochemical level, by measuring protease activity and testing for the formation of protein complexes. The studies are anticipated to reveal how astacins regulate TGF-beta ligand activities and whether this is a mechanism to regulate mesoderm induction and embryonic pattern formation in vertebrates. ***
