Abstract
The family of TGF-β and bone morphogenetic protein (BMP) signaling proteins has numerous developmental and physiological roles. They are made as proprotein dimers and then cleaved by proprotein convertases to release the C-terminal domain as an active ligand dimer. Multiple proteolytic processing sites in Glass bottom boat (Gbb), the Drosophila BMP7 ortholog, can produce distinct ligand forms. Cleavage at the S1 or atypical S0 site in Gbb produces Gbb15, the conventional small BMP ligand, whereas NS site cleavage produces a larger Gbb38 ligand. We hypothesized that the Gbb prodomain is involved not only in regulating the production of specific ligands but also their signaling output. We found that blocking NS cleavage increased association of the full-length prodomain with Gbb15, resulting in a concomitant decrease in signaling activity. Moreover, NS cleavage was required in vivo for Gbb-Decapentaplegic (Dpp) heterodimer-mediated wing vein patterning but not for Gbb15-Dpp heterodimer activity in cell culture. Gbb NS cleavage was also required for viability through its regulation of pupal ecdysis in a type II receptor Wishful thinking (Wit)-dependent manner. In fact, Gbb38-mediated signaling exhibits a preference for Wit over the other type II receptor Punt. Finally, we discovered that Gbb38 is produced when processing at the S1/S0 site is blocked by O-linked glycosylation in third instar larvae. Our findings demonstrate that BMP prodomain cleavage ensures that the mature ligand is not inhibited by the prodomain. Furthermore, alternative processing of BMP proproteins produces ligands that signal through different receptors and exhibit specific developmental functions.
Highlights
IntroductionThe BMP7 prodomain competes with the ligand for binding type II receptors in vitro, but this competition does not reduce signaling activity in the context of cell culture assays [15]
We raised antibodies against specific prodomain epitopes, and with the existing C-terminal directed ␣-GbbC [18] we were able to identify all possible products cleaved from the proGbb precursor protein (Fig. 1A). ␣-GbbN recognizes amino acids 46 – 61, near the N terminus of the prodomain, and was used to identify N-terminal cleavage products. ␣-GbbCore, named after the Core/Arm domain of BMP2 and TGF1 [8, 30], recognizes aa 127–144 and any cleavage products resulting from the removal of the N or C terminus
We found that conditioned media from gbbmS1mS0, which contains Gbb38 and the NH3-NS fragment but no Gbb15, produced phosphorylated Mad (pMad) at 17% of WT levels (p Ͻ 0.001, 95% CI 12–25%) (Fig. 2, A and C), indicating that in this experimental system Gbb38 can induce signaling in Schneider 2 (S2) cells, albeit at lower levels than Gbb15
Summary
The BMP7 prodomain competes with the ligand for binding type II receptors in vitro, but this competition does not reduce signaling activity in the context of cell culture assays [15]. The NS cleavage site has been shown to be necessary and sufficient for the rescue of gbb null lethality [19] In cell culture, both the NS and S1 cleavage sites have been shown to be critical to achieve full BMP signaling activity [18]. Both the NS and S1 cleavage sites have been shown to be critical to achieve full BMP signaling activity [18] Together, these findings indicate that Gbb is an active ligand with functions distinct from Gbb. The suggestion that context-specific requirements for Gbb could be explained by the activities of different ligands produced by alternative processing requires further investigation
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