Simultaneous cleavage of both sites of proBMP4 leads to loss of activity in mice, perhaps due to disrupted interactions with the ECM

BMPs are generated as latent precursors that are proteolytically activated by proprotein convertases (PCs). ProBMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1), and then at an upstream site within the prodomain (S2). Sequential cleavage of proBMP4 is driven in part by the presence of optimal (RXKR) and minimal (RXXR) PC consensus cleavage motifs at the S1 and S2 sites, respectively. When the S2 site of proBMP4 is mutated to an optimal consensus motif, the two sites are cleaved rapidly and stochastically rather than sequentially. Ectopic expression of this precursor (BMP4S2K) in Xenopus embryos generates a ligand with enhanced activity, suggesting that ordered cleavage restricts ligand activity. To test this hypothesis, we generated mice carrying a knockin point mutation that introduces an optimal consensus cleavage motif at the S2 site. Rather than generating a gain of function allele as predicted, BMP4S2K is a severe hypomorphic allele. Most BMP4S2K homozygotes die by E12.5, and they display reduced BMP-reporter activity. Equivalent levels of proBMP4 and cleaved prodomain are present in wild type and mutant embryos, indicating that the point mutation does not interfere with expression or cleavage of proBMP4. Surprisingly, pulse chase analysis suggests that both sites of proBMP4 are cleaved extracellularly. Because the prodomain of BMP4 binds to the extracellular matrix (ECM) protein, fibrillin, and cleavage at the S2 site dissociates the mature ligand from the prodomain, we hypothesize that simultaneous cleavage of both sites in proBMP4S2K causes premature release, or prevents the mature ligand from being deposited into the ECM, thus leading to loss of local signaling activity.