The long-term goal of the proposed research is to determine how proteolytic maturation of precursor proteins regulates the activity and range of action of cell-celt signaling molecules during mammalian development. ProBMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain and this allows for subsequent cleavage at an upstream nonconsensus furin motif within the prodomain, in Xenopus embryos, BMP-4 synthesized from ectopic precursor in which the upstream site is non-cleavable is initially degraded and signals at shorter range, while that synthesized from a precursor that is simultaneously cleaved at both sites is more active and signals at greater range than does BMP-4 cleaved from native precursor. The proposed studies will test the hypotheses that 1) sequential cleavage of proBMP-4 is essential for proper regulation of endogenous BMP-4 activity and for normal embryonic patterning, 2) tissue-specific cleavage at the upstream site provides a mechanism for tissue-specific regulation of BMP-4 signaling range and 3) it does so, in part, by modulating attachment of mature BMP-4 to the cell surface or extracellular matrix. To de so, we will generate mice carrying targeted point mutations that disrupt or accelerate cleavage at the upstream site without altering the primary cleavage that releases the mature tigand. BMP-4 activity and signaling range wilt be compared in various tissues of wild type and mutant littermates by measuring levels and pattern of expression of BMP-4 target genes and immunoreactive phosphoSmadl, and by crossing mutants with Smad1-response element-LacZ reporter mice. ProBMP-4 processing will be analyzed in various tissues of wild type and mutant mice to directly assay for tissue-specific use of the upstream site. Pro-BMP-4 cleavage will also be analyzed in tissues isolated from furin, PACE4 or PC6B mutant mice to identify convertases that cleave at each site. Finally, cell surface attachment of mature BMP-4 will be compared in wild type and mutant mice. Proper regulation of BMP-4 activity is essential for normal embryonic patterning and for tissue homeostasis in adults. Understanding the molecular mechanisms by which BMP activity is regulated is key to understanding, treating and preventing congenital anomalies and diseases. ? ?
