To date, active TGF-β and BMP family dimeric ligands are known to consist of two peptides processed from the C-terminal 100 to 110 amino acids of two larger proproteins. All ligands adopt a similar “inverted clasped hands” or “butterfly” fold because of the high conservation in this domain, especially of the hallmark cysteine residues. Here, we report the identification of a BMP ligand form containing the highly conserved C-terminal domain contiguous with a portion of the less conserved prodomain. This large ligand (Gbb38), one of two forms produced by gbb
, arises from proconvertase processing at a previously unrecognized furin consensus sequence (NS) at residue 123 of the 455–amino acid Gbb proprotein. A second ligand, Gbb15, is produced by cleavage at the canonical S1 site located at residue 322, which is N-terminal to the conventional ligand domain. Both Gbb38 and Gbb15 are active, with Gbb38 exhibiting a higher level of signaling activity both in cell culture () and in vivo ( and and fig. S6
). The ability of Gbb38 and Gbb15 to signal is influenced by the tissue domain in which they are expressed. When expressed in either the dorsal or the anterior compartment of the wing imaginal disc, Gbb38 and Gbb15 induce ectopic signaling commensurate with their relative signaling abilities as measured in cell culture (, , and ). However, when expressed in the posterior compartment, their ability to elicit ectopic signaling is impaired (). This finding is particularly interesting in light of our previous observations that Gbb produced in the anterior compartment behaves differently from that generated in the posterior compartment (5
). On the basis of the fact that Dpp is produced solely in the anterior compartment, one could envision that the compartmental difference in the behavior of Gbb may be explained by the action of Dpp-Gbb heterodimers formed only in the anterior compartment. However, we found that Gbb38 induced signaling from clones lacking Dpp (fig. S6, C and D
) and that Gbb15 induced a significant increase in the amplitude of the anterior phosphorylated Mad peak when expressed in the posterior compartment (). Furthermore, the expression of wild-type gbb
) in the posterior compartment, which produces both Gbb38 and Gbb15 (), induced considerable ectopic phosphorylated Mad in all cells of the disc in both posterior and anterior compartments (). Thus, the differential response to expression of gbb
cleavage mutants in various wing disc domains cannot be explained solely by the presence of Dpp. Given that both Gbb38 and Gbb15 are produced by posterior cells (), the factors responsible for this compartment-specific behavior are likely to be involved in the reception or integration of their independent signals.
In addition to compartment-specific behavior, we also found that Gbb38 promoted signaling in cells far from where it is produced (, and fig. S6, C and D
), as did wild-type Gbb (, and 5, B, E, J, and N, and fig. S6, A and B
). However, Gbb15 in general did not because it induced high signaling in the notum and hinge of the anterior compartment only when it was expressed in the anterior compartment (). Tissue-specific processing at the conventional linker domain cleavage sites can regulate the amount of BMP4 and Dpp ligand produced and thus influence signaling strength and ligand function in Xenopus
). In the case of the larger Gbb38 ligand, the presence of considerable additional “pro-domain” sequences not found in the conventional ligands adds a new set of parameters to be considered in understanding the mechanisms that regulate TGF-β signaling. The additional protein domains Gbb38 could allow for or block interactions with extracellular factors that regulate ligand movement, such as the heparan sulfate proteoglycans Dally and Dally-like protein (Dlp), Pentagone (Pent) and Larval Translucida (Ltl), and different combinations of receptors (50
). In this vein, the existence of an active large ligand form is of particular interest in light of the structure of the TGF-β1 prodomain (34
). Cleaved prodomains are thought to assist in intracellular ligand folding and secretion, but for some family members, the ligand remains noncovalently associated with its two prodomains after secretion. In most cases, this association confers latency (inactivity) as a result of structural changes, with ligand activation requiring proteolytic cleavage of the prodomain in the latent complex or a force-dependent mechanism through integrin interactions (such as is the case for TGF-β, BMP2, and GDF8) (34
). The prodomain-BMP7 complex is not latent, and the prodomains are displaced upon ligand binding to the BMP type II receptor (56
). In this case, ligand activation does not require extracellular matrix molecules or proteases, an observation that may hold true for AMH as well (56
). Despite the apparent differences in TGF-β and BMP family members and their extracellular associations with their prodomains, structural studies indicate that prodomains in TGF-β and BMP family ligands are expected to share the same cysteine knot fold (58
). In the case of TGF-β1, the prodomain dimer envelops the C-terminally derived ligand dimer to prevent ligand activation, with a straitjacket motif created by the overlapping N-terminal 45 residues of the prodomain (). If processing occurs at the NS site in Gbb, the N-terminal 90 amino acids would be removed, eliminating the straitjacket domain and the “latency lasso” that encircles the residues critical for receptor binding. As our analyses have shown, the removal of this N-terminal–most domain must allow for receptor binding of the Gbb ligand without requiring any further processing and subsequent signaling. Indeed, the 73 amino acids at the extreme N terminus of the GDF8 prodomain confer its inhibitory effect over GDF8 (53
). Although the structural conformation of the large ligand is not yet known, it is conceivable that the butterfly structure of the C-terminally derived ligand domain will fold on the basis of the conserved cysteine knot structure, with the receptor interaction faces unimpeded by the linker domain (). Given the more robust signaling elicited by Gbb38 than Gbb15 and the therapeutic uses of BMPs, it will be interesting to resolve the structure of a large ligand form.
Fig. 6 Model summarizing the signaling capacity and range of action for products produced by TGF-β and BMP family members. (Top) Linear schematic of TGF-β/BMP protein domains (prodomain, linker, and ligand domains), NS and linker cleavage sites (more ...)
Although we found that the signaling ability of cleavage mutants that exclusively produced Gbb38 (gbbmS1
) or Gbb15 (gbbmNS
) depended on the domain in which they were expressed, the expression of wild-type gbb
led to increased BMP signaling in all cells of disc with the exception of the medial wing pouch, which appears to be under tight regulation. Although the phosphorylated Mad gradient in the medial wing pouch is sensitive to the loss of gbb
and other signaling components, it is resistant to their increases most likely in part due to a negative feedback mechanism involving Dad, an inhibitory Smad (5
). In addition, the phosphorylated Mad gradient scales with disc size in the medial wing pouch (48
), an effect that we did not observe in other regions of the disc upon overexpression of gbb
or the gbb
cleavage mutants. Furthermore, we found that scaling was disrupted by wild-type gbb
expression, which produced an overgrown disc with relatively minor changes to the phosphorylated Mad gradient ( and and fig. S6
). Future studies will be important to resolve the molecular basis of these domain-specific signaling responses and the inability of either Gbb38 or Gbb15 to recapitulate the effects of wild-type gbb
, but it is clear that the different Gbb ligands exhibit distinct effects.
The inability of either Gbb38 or Gbb15 to recapitulate wild-type gbb
when expressed under endogenous gbb
control (fig. S4
) indicates that to attain full Gbb signaling, processing must be possible at both the S1 and the NS sites. The simplest explanation for this observation is that wild-type signaling requires the presence of both Gbb38 and Gbb15 ligands either as homodimers or as Gbb38:Gbb15 heterodimers. An alternative explanation is that to produce a canonical active Gbb ligand (presumably Gbb15), processing must occur at both sites either sequentially or simultaneously. Our data do not favor the latter alternative because Gbb38 alone exhibits robust signaling and both forms are present endogenously with differential enrichment in various tissues, suggesting that the cell type or tissue environment may influence proGbb processing (). Instead, we envision that the production, maturation, and stability of the array of active ligands under normal conditions depends on the presence of both NS and S1 cleavage sites and that the reception and integration of the various combinations of ligands occurs in a domain-specific manner.
The differential requirement for different sets of ligands could explain the apparent specificity of the NS mutation in hBMP4 (hBMP4-R162Q) associated with CLP (28
). The associated failure in the fusion of the medial nasal and maxillary processes during orofacial development is a fairly specific temporal and spatial disruption of hBMP4 signaling. BMP4 is required for mouse lip and palate fusion (60
) as well as many other fundamental roles in early development including axis determination and the specification of different germ layers (3
). Given that BMP4 is essential for embryonic development, this relatively minor defect suggests that either processing at the NS site of hBMP4 occurs only in a specific set of cells, at a specific time in development, and individuals carrying the hBMP4R162Q
allele fail to produce the proper hBMP4 ligand type(s) necessary to elicit the proper magnitude of BMP signaling required for lip and palate fusions, or compensatory mechanisms, such as BMP redundancy and feedback regulation, restore BMP signaling activity to its normal magnitude in other tissues of patients with CLP. Bmp4S2G/S2G
mice also survive to adulthood with severe defects in only some tissues, such as the testes, with other tissues or organs that require BMP4 signaling, such as the limb and kidney, developing normally (11
). It is conceivable that ligand produced by cleavage at NS and S2 sites does not affect the magnitude of signaling but rather the signaling between specific sets of cells. Consistent with this possibility, we found that although the S1 site is required for hBMP4 signaling, we could not detect a difference in the amount of signaling induced by mutations in S2 and NS, as well as hBMP4-R162Q in transfected cells (fig. S3
), where the production or reception of a putative large hBMP4 ligand form may not occur, especially over the basal extent of autocrine signaling. Instead, it will be of greater interest to test for the effect of the CLP mutation hBMP4-R162Q on BMP signaling specifically in the orofacial primordia because this tissue may be most sensitive to a large form of the hBMP4 ligand or a prodomain-ligand complex with a shorter prodomain segment. BMP4 plays an instrumental role in the morphological differences in the beaks of Darwin’s finches, as well as in jaw development of the rapidly evolving species of cichlid fishes occupying Lakes Victoria, Malawi, and Tanganyika of the East African Rift valley (63
). Although the existence of a large BMP4 ligand has yet to be determined, it is intriguing to consider that among the East African THMV (Tropheini, Haplochromine, Lake Malawi, and Lake Victoria) group of cichlids, the prodomain of BMP4 contains a high number of amino acid substitutions not seen in the signal peptide or ligand domain (66
). Either the regulatory function of the prodomain in a BMP4 latent complex could be critical for modulating ligand activity during jaw development or amino acid changes in a putative large BMP4 ligand could alter its signaling abilities in orofacial development of cichlids.
In conclusion, our studies have identified a proconvertase cleavage site that is essential for the full activity of the Drosophila
BMP5/6/7 ortholog Gbb, as well as for that of several human BMPs. Given the evolutionary and functional conservation of alternative processing in the N-terminal region of the proprotein, it is of interest to note that despite lower sequence conservation between BMP prodomains, they are thought to share a folded core domain (58
). TGF-β and BMP family members that are processed to yield both large and small ligand forms may exhibit an expanded range of signaling activities because of differences in ligand structural motifs or interaction faces. Thus, the posttranslational regulation of BMP ligands is more intricately controlled than previously anticipated. A role for proconvertase processing as a mechanism to control both quantity and quality of ligand production is now more strongly supported by the identification of the NS cleavage site critical for signaling activity, by its evolutionary conservation, and by the fact that mutations in this site appear to disrupt human development (). Our results raise the possibility that context-dependent behaviors reported for various TGF-β and BMP ligands could be explained by the action of such alternate ligand forms.