Alternative cleavage of a bone morphogenetic protein (BMP) produces ligands with distinct developmental functions and receptor preference

Abstract

TGF-β and Bone Morphogenetic Protein (BMP) family proteins are made as proprotein dimers, which are cleaved by proprotein convertases to release the active C-terminal ligand dimer. Multiple proteolytic processing sites in Glass bottom boat (Gbb), the Drosophila BMP7 ortholog, can produce distinct forms of active ligand. Cleavage at the S1 or atypical S0 site produces Gbb15, the conventional small BMP ligand, while cleavage at the NS site produces the larger Gbb38 ligand (1, 2). Here, we found that blocking NS cleavage increased association of the full length prodomain with Gbb15 resulting in a concomitant decrease in signaling activity. NS cleavage is required in vivo for Gbb-Decapentaplegic (Dpp) heterodimer-mediated wing vein patterning but not in cell culture to enable Gbb15-Dpp heterodimer activity. Gbb NS cleavage is also required in vivo for the regulation of pupal ecdysis and viability that is dependent on the type II receptor Wishful thinking (Wit). We found that the ability of Gbb38 to signal requires the expression of either Wit or the type I receptor, Saxophone (Sax). Finally, we discovered that the production of Gbb38 in 3rd instar larvae results when processing at the S1/S0 site is blocked by O-linked glycosylation. Our findings demonstrate that BMP prodomain cleavage can ensure that the mature ligand is not inhibited by the prodomain. Furthermore, alternative processing of BMP proproteins produces ligand types that signal preferentially through different receptors and exhibit specific developmental functions.Bone Morphogenetic Proteins (BMPs), members of the TGF-β family of signaling proteins, have numerous developmental and physiological roles (3)(4)(5). Like other TGF-β family members, BMPs are synthesized as large 400-500 amino acid proproteins that form dimers linked by a Cterminal disulfide (6). The 110-140 amino acid ligand domain is proteolytically cleaved from the C-terminus by a proprotein convertase (PC) such as Furin. After secretion, the C-terminally derived ligand dimer binds and activates a complex of type I and type II transmembrane serine/threonine kinase receptors. In the active complex, the type II receptor phosphorylates the type I receptor that in turn phosphorylates downstream receptor-mediated Smad (RSmad) signal transducers that act as transcription factors. While the vast majority of research in the field has focused on the activity of the ligand, there is a growing appreciation of the regulatory functions of BMP and TGF-β family prodomains (6)(7)(8). In general, it has been proposed that prodomains are important for proper folding, dimerization, and secretion of the mature ligand. A comparison of prodomains between different members of the TGF-β/BMP family shows a lower sequence conservation, in contrast to the high sequence conservation observed between their ligand domains. The low degree of prodomain sequence conservation might mean that 1 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder...