glass bottom boat/Transforming growth factor beta at 60A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - glass bottom boat

Synonyms - Transforming growth factor beta at 60A

Cytological map position - 60A1--60A3

Function - ligand

Keywords - ectoderm, midgut, dorsal closure, imaginal discs

Symbol - gbb

FlyBase ID: FBgn0024234

Genetic map position - 2-[106]

Classification - TGF-beta superfamily

Cellular location - secreted



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Hong, S. H., Kang, M., Lee, K. S. and Yu, K. (2016). High fat diet-induced TGF-beta/Gbb signaling provokes insulin resistance through the tribbles expression. Sci Rep 6: 30265. PubMed ID: 27484164
Summary:
Hyperglycemia, hyperlipidemia, and insulin resistance are hallmarks of obesity-induced type 2 diabetes, which is often caused by a high-fat diet (HFD). However, the molecular mechanisms underlying HFD-induced insulin resistance have not been elucidated in detail. This study established a Drosophila model to investigate the molecular mechanisms of HFD-induced diabetes. HFD model flies recapitulate mammalian diabetic phenotypes including elevated triglyceride and circulating glucose levels, as well as insulin resistance. Expression of glass bottom boat (gbb), a Drosophila homolog of mammalian transforming growth factor-β (TGF-β), is elevated under HFD conditions. Furthermore, overexpression of gbb in the fat body produced obese and insulin-resistant phenotypes similar to those of HFD-fed flies, whereas inhibition of Gbb signaling significantly ameliorated HFD-induced metabolic phenotypes. tribbles, a negative regulator of AKT, is a target gene of Gbb signaling in the fat body. Overexpression of tribbles in flies in the fat body phenocopied the metabolic defects associated with HFD conditions or Gbb overexpression, whereas tribbles knockdown rescued these metabolic phenotypes. These results indicate that HFD-induced TGF-β/Gbb signaling provokes insulin resistance by increasing tribbles expression.
Tian, A., Wang, B. and Jiang, J. (2017). Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration. Proc Natl Acad Sci U S A 114(13): E2699-E2708. PubMed ID: 28289209
Summary:
Many adult organs rely on resident stem cells to maintain homeostasis. Upon injury, stem cells increase proliferation, followed by lineage differentiation to replenish damaged cells. Whether stem cells also change division mode to transiently increase their population size as part of a regenerative program and, if so, what the underlying mechanism is have remained largely unexplored. This study showed that injury stimulates the production of two bone morphogenetic protein (BMP) ligands, Dpp and Gbb, which drive an expansion of intestinal stem cells (ISCs) by promoting their symmetric self-renewing division in Drosophila adult midgut. BMP production in enterocytes is inhibited by BMP signaling itself, and BMP autoinhibition is required for resetting ISC pool size to the homeostatic level after tissue repair. This study suggests that dynamic BMP signaling controls ISC population size during midgut regeneration and reveals mechanisms that precisely control stem cell number in response to tissue needs.
Jordan-Alvarez, S., Santana, E., Casas-Tinto, S., Acebes, A. and Ferrus, A. (2017). The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila. PLoS One 12(9): e0184238. PubMed ID: 28892511
Summary:
Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways.
Anderson, E. N. and Wharton, K. A. (2017). Alternative cleavage of the bone morphogenetic protein (BMP), Gbb, produces ligands with distinct developmental functions and receptor preference. J Biol Chem [Epub ahead of print]. PubMed ID: 28924042
Summary:
The family of TGF-beta and BMP signaling proteins are made as proprotein dimers, 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, while NS site cleavage produces a larger Gbb38 ligand. It was hypothesized that the Gbb prodomain is involved not only in regulating the production of specific ligands but also their signaling output. 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, Gbb38 was found to be produced when processing at the S1/S0 site is blocked by O-linked glycosylation in third instar larvae. These 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.
BIOLOGICAL OVERVIEW

Transforming growth factor beta at 60A, known in the literature as 60A, is a member of the TGFbeta superfamily that bears a closer resemblence to vertebrate osteogenic protein (OP-1 or BMP-7) than to BMP-4 or to BMP-4's homolog in Drosophila, Decapentaplegic. Although the gene will be referred to here as Tgfbeta-60A, it is more correctly termed glass bottom boat (gbb). Other close relatives of Tgfbeta-60A in vertebrates are BMP-5 and BMP-6. It is likely that the two evolutionary lineages, one giving rise to Dpp and its homologs, and a second giving rise to Tgfbeta-60A and its homologs, diverged well before the divergence of insects and mammals (Wharton, 1991).

Little about Tgf-beta-60A was known beyond its expression pattern because mutants were unavailable, but a 1998 paper by Chen changed all that. Tgfbeta-60A expression had been previously found to be pronounced in mesodermal cells and in cells of the stomadeal and posterior midgut invaginations, foregut and hindgut cells and in the endodermal cells of the anterior and posterior midgut (Doctor, 1992). Chen sought out dominant enhancer mutations magnifying the effects of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. Hypomorphic alleles result in partial loss of function of a gene, and dominant enhancers worsten the phenotypic effects of these hypomorphic alleles. Enhancing mutations were found in Mad, Medea, punt and thick veins, all of which are known components of the dpp signaling pathway and in Tgfbeta-60A. Phenotypic analysis of Tgfbeta-60A single mutants and tkv 6;Tgfbeta-60A double mutants revealed both dpp-independent and dpp-dependent functions for Tgfbeta-60A. Tgfbeta-60A mutants lack the first constriction of the embryonic midgut and Antennapedia expression in parasegment 6, indicating that 60A is required for the formation of the first constriction, possibly through regulating Antp expression. This function is independent of dpp signaling, since mutations in dpp or its receptors only disrupt the formation of the second but not the first constriction. This also suggests that there is either redundancy or that a different receptor system is responsible for mediating Tgfbeta-60A signaling to pattern the first constriction. It would be interesting to see if AtrI (Childs, 1993), a type I receptor resembling the type I activin receptor, and STK-D (Ruberte, 1995), a type II receptor in Drosophila, both of unknown function, are mediators of 60A signaling at the site of the first constriction (Chen, 1998).

The fact that Tgfbeta-60A mutations are dominant enhancers of a sensitized dpp pathway implicates Tgfbeta-60A in potentiating dpp signaling (for review, see Raftery, 1999). This is most obvious in the visceral mesoderm of the midgut where dpp signaling is required to regulate homeotic gene expression and to maintain its own expression through a positive feedback mechanism. Although dpp signaling in the visceral mesoderm appears intact in Tgfbeta-60A mutants, a requirement for Tgfbeta-60A is revealed in tkv 6 Tgfbeta-60A double mutants. When dpp signaling is attenuated through a mutant tkv receptor, eliminating Tgfbeta-60A function reduces the signaling to below threshold level. The derepression of Sex combs reduced in the anterior midgut and the loss of expression of dpp target genes (wingless, Ultrabithorax and dpp) in the visceral mesoderm and labial in the endoderm are consistent with inadequate dpp signaling. A similar requirement for Tgfbeta-60A is observed during dorsal closure of the embryonic ectoderm. The enhanced phenotypes of the adult appendages closely resemble those of the Tgfbeta-60A hypomorphic mutants, suggesting that Tgfbeta-60A activity is also required for imaginal disc patterning. It is interesting that the imaginal discs are more sensitive than are mesoderm or endoderm to the reduction of Tgfbeta-60A function, as a 50% reduction in Tgfbeta-60A function is sufficient to produce a phenotype in a tkv 6 genetic background. This may reflect a differential threshold requirement for dpp signaling in different tissues (Chen, 1998).

Tgfbeta-60A may form functional heterodimers with Decapentaplegic. In a signaling system with multiple interacting dimeric ligands, the interpretation of any single mutant phenotypes must consider the effect of losing both homomeric and possible heteromeric ligands. Therefore, the functions of the dpp pathway may be a composite input from Dpp homodimers, and Dpp/Scw and Dpp/Tgfbeta-60A heterodimers. Alternatively, Tgfbeta-60A homodimers may function in an additive fashion with Dpp homodimers at sites of overlapping expression. However, the loss-of-function phenotypes of dpp are as severe as the loss-of- function phenotypes of its downstream components, such as tkv or Mad, suggesting that there is very little signaling, if any at all, from Tgfbeta-60A homodimers in dpp-dependent events. Therefore, it is unlikely that Tgfbeta-60A homodimers play a significant role in dpp-dependent processes. Rather, it is thought that Dpp/Tgfbeta-60A heterodimers form at sites of overlapping expression and participate with Dpp homodimers in multiple signaling events. The broad distribution of Tgfbeta-60A proteins provides an opportunity for forming Dpp/Tgfbeta-60A heterodimers. Unlike scw null mutations, no obvious disruption of dpp signaling is observed in Tgfbeta-60A null mutants, suggesting that Dpp/Tgfbeta-60A heterodimers are not as limiting as Dpp/Scw heterodimers, but function in partially redundant manner with Dpp homodimers (Chen, 1998).

The BMP Ligand Gbb gates the expression of synaptic homeostasis independent of synaptic growth control

Inhibition of postsynaptic glutamate receptors at the Drosophila NMJ initiates a compensatory increase in presynaptic release termed synaptic homeostasis. BMP signaling is necessary for normal synaptic growth and stability. It remains unknown whether BMPs have a specific role during synaptic homeostasis and, if so, whether BMP signaling functions as an instructive retrograde signal that directly modulates presynaptic transmitter release. This study demonstrates that the BMP receptor [(Wishful thinking (Wit)] and ligand (Gbb) are necessary for the rapid induction of synaptic homeostasis. Evidence is provided that both Wit and Gbb have functions during synaptic homeostasis that are separable from NMJ growth. However, further genetic experiments demonstrate that Gbb does not function as an instructive retrograde signal during synaptic homeostasis. Rather, the data indicate that Wit and Gbb function via the downstream transcription factor Mad and that Mad-mediated signaling is continuously required during development to confer competence of motoneurons to express synaptic homeostasis (Goold, 2007).

These data advance understanding of BMP signaling at the Drosophila NMJ in several important ways. First, it was demonstrated that BMP signaling is essential for the rapid, protein-synthesis-independent, induction of synaptic homeostasis identified at this NMJ. Because expression of UAS-wit in motoneurons restores synaptic homeostasis in the wit mutant and because suppression of Mad-mediated signaling in neurons blocks synaptic homeostasis, it is concluded that BMP signaling acts upon the motoneuron to enable the rapid induction of synaptic homeostasis. Next, it was shown that the requirement for BMP signaling during synaptic homeostasis is separable from BMP-dependent support of synaptic growth and baseline neurotransmission. Finally, the temporal and spatial requirements for BMP signaling was dissected. The data support the conclusion that Mad-mediated signaling is required constitutively, downstream of the Wit receptor, in order to maintain the competence of motoneurons to express homeostatic plasticity. Further, the data argue that Gbb is not the retrograde signal that directly acts upon the presynaptic motoneuron terminal to homeostatically modulate presynaptic release (Goold, 2007).

It has been hypothesized that Gbb could function as a homeostatic retrograde signal at the Drosophila NMJ. According to this model, Gbb would be released in proportion to the perturbation of postsynaptic muscle excitation in a glutamate receptor mutant and, thereby, instruct the degree of homeostatic compensation expressed by the presynaptic motoneuron terminal. In favor of this model, homeostatic compensation observed in a glutamate receptor mutant is blocked by the wit mutation. This study present two lines of evidence that are consistent with the necessity of BMP signaling for homeostatic compensation. First, it was confirmed that the rapid induction of homeostatic compensation following application of a use-dependent glutamate receptor antagonist, Philanthotoxin (PhTx) is blocked by null mutations in both wit and gbb. Furthermore, it was shown that muscle-specific rescue of the gbb null mutation is sufficient to restore the rapid induction of homeostatic compensation (Goold, 2007).

Despite these compelling genetic data, several experiments now argue against the possibility that Gbb functions as an instructive, retrograde signal that directly modulates presynaptic release during synaptic homeostasis. First, it was found that although muscle-specific rescue of the gbb null mutation is sufficient to restore synaptic homeostasis, so is neuron-specific rescue of the gbb null mutation. Thus, homeostatic compensation can occur even in the absence of muscle-derived Gbb. These data argue against a model in which Gbb functions as the instructive retrograde signal that directly modulates presynaptic release during synaptic homeostasis (Goold, 2007).

Next, it was demonstrated that homeostatic signaling is blocked by expression of DN-Glued in neurons, which disrupts retrograde axonal transport. In this experiment, Gbb signaling at the NMJ should, in theory, persist. Furthermore, it was established that an intact motor axon is not required for the rapid induction of synaptic homeostasis. Thus, it can be concluded that trans-synaptic Gbb signaling from muscle to nerve is not sufficient for the rapid induction of synaptic homeostasis (Goold, 2007).

Given that Wit and Gbb are necessary for synaptic homeostasis, how do they participate in the process if Gbb is not the instructive retrograde signal? This study demonstrates that Mad is necessary for synaptic homeostasis, and evidence is providied that Mad-mediated signaling is required in the motoneuron. In addition, neuronal expression of UAS-Gbb restores homeostatic compensation in the presence of the DN-Glued transgene. These results suggest that the reason DN-Glued disrupts synaptic homeostasis is because it interferes with the retrograde axonal transport of P-Mad downstream of the Wit receptor. This is consistent with the prior demonstration that neuronal expression of Gbb can restore nuclear P-Mad in the presence of UAS-DN-Glued. Because the induction of synaptic homeostasis does not require the motoneuron soma, it is concluded that Gbb does not function as an acute, retrograde signal. Rather, Gbb may be a muscle-derived signal that acts developmentally to confer the competence of motoneurons to express synaptic homeostasis. Thus, the identity of the homeostatic retrograde signal at the NMJ remains unknown. It remains possible that other TGF-β superfamily signaling molecules could function at the NMJ in this capacity, including myoglianin and maverick, though it has been shown that synaptic homeostasis is intact in the baboon receptor mutant (Goold, 2007).

There are several possible ways in which BMP signaling could confer competence for motoneurons to express homeostatic plasticity. One possibility is that the BMPs control a transcriptional program that is necessary for synaptic homeostasis. For example, BMPs are potent regulators of cell fate during embryonic development. Perhaps the ability of motoneurons to express synaptic homeostasis is related to the maintenance of their cellular or electrical identity. An alternate possibility is that BMPs control the expression of essential presynaptic proteins that are required for synaptic homeostasis. For example, it has been shown in other systems that target-dependent TGF-β signaling can modulate neuronal ion channel expression. It has been demonstrated that CaV2.1 calcium channels are required for synaptic homeostasis at the Drosophila NMJ. However, it is considered unlikely that BMPs control synaptic homeostasis through the regulation of CaV2.1 channel expression because there is not a strong correlation between altered baseline synaptic transmission and the expression of synaptic homeostasis. Furthermore, overexpression of a GFP-tagged CaV2.1 calcium channel (cacophony-GFP) is unable to restore synaptic homeostasis when coexpressed with UAS-dad. Finally, BMP signaling could influence the expression of synaptic homeostasis by targeting the rate of spontaneous miniature release. Spontaneous release events that persist in the absence of evoked neurotransmission are sufficient to induce homeostatic compensation at the Drosophila NMJ. However, no strong correlation is found between baseline mEPSP frequency and whether or not a mutant NMJ is able to express synaptic homeostasis. Although the wit mutants show a severe decrease in mEPSP rate compared to wild-type, the expression of UAS-dad or UAS-DN-Glued both block synaptic homeostasis without severely impairing baseline mEPSP rate. Ultimately, continued forward genetic investigation of homeostatic signaling may be required to identify the BMP-dependent mechanisms that control the expression of synaptic homeostasis (Goold, 2007).

BMP signaling is required for NMJ growth, baseline neurotransmission, and NMJ stability in addition to being required for synaptic homeostasis. It is a challenge, therefore, to determine whether BMP signaling has a specific function during synaptic homeostasis versus a more general role during synapse development. This study presents several lines of evidence that BMP signaling may have a separable function during synaptic growth versus synaptic homeostasis. First, it was demonstrated that synaptic homeostasis can occur at BMP mutant synapses that show severely impaired synaptic growth. For example, the gbb hypomorphic mutant has a decrease in bouton number that is just as severe as the gbb null mutant, but the gbb hypomorphic mutant shows normal homeostatic compensation. As another example, animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in bouton number but normal homeostatic compensation. Thus, it is concluded that normal BMP-dependent synaptic growth is not required for the expression of synaptic homeostasis (Goold, 2007).

It was also possible to dissociate BMP-dependent baseline transmission from both synaptic growth and synaptic homeostasis. (1) Muscle-specific rescue of the gbb null mutation significantly restores synaptic growth and rescues synaptic homeostasis, but baseline transmission remains at levels observed in the null mutant. (2) Motoneuron-specific rescue of the wit mutation (OK371-GAL4) similarly rescues bouton number and synaptic homeostasis, although baseline transmission remains severely impaired. (3) Animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in baseline transmission but normal homeostatic compensation. (4) Results were obtained that show the converse effect. When UAS-dad is expressed for 1.5 days at the end of larval development, both synaptic homeostasis and baseline transmission are significantly impaired, but synaptic bouton numbers remain wild-type. From these data it is concluded that impaired synaptic homeostasis is not a secondary consequence of BMP-dependent functional NMJ development. It also appears that there may be distinct effects of BMP signaling on the anatomical versus functional development of the NMJ. One possibility, consistent with BMPs being a classical morphogen, is that different levels of the ligand could initiate specific transcriptional programs with distinct effects on bouton number, baseline transmission, and homeostatic plasticity. It is also possible that the site of action of BMP signaling will play an important role in specifying signaling outcome (Goold, 2007).

It has been speculated that synaptic homeostasis might function, over the course of development, to ensure that the muscle cell is normally depolarized by the NMJ. How can one explain the observation that csp and syx/+ mutations have decreased baseline neurotransmitter release but normal acute synaptic homeostasis in response to PhTx application, or other genotypes explored in this study that show impaired baseline transmission and normal acute synaptic homeostasis? It has been demonstrated that the acute induction of synaptic homeostasis is independent of evoked neurotransmission. Thus, synaptic homeostasis may not function to modulate the absolute amplitude of evoked neurotransmitter release. Rather, synaptic homeostasis might be a rapid system to offset acute perturbations of postsynaptic receptor function. In this case, developmental programs that specify NMJ anatomy and active zone addition would achieve the reproducible development of the NMJ. Alternatively, the mechanisms of acute homeostatic compensation following PhTx application may be separable, either temporally or molecularly, from the other potential mechanisms that monitor and homeostatically control evoked EPSP amplitudes (Goold, 2007).

The data also suggest a possible link between the expression of homeostatic plasticity and the mechanisms of neuromuscular degenerative disease. Genetic mutations that impair retrograde axonal transport have been shown to cause familial amyotrophic lateral sclerosis. It has also been shown that, in Drosophila and mice, mutations that disrupt dynein-dynactin complex function lead to neuromuscular synapse degeneration. It is hypothesized that impaired retrograde axonal transport deprives motoneurons of muscle-derived trophic support leading to motoneuron degeneration. This study has demonstrated that impaired retrograde axonal transport blocks the expression of homeostatic plasticity at the NMJ. This deficit can be restored by expression of BMPs in the central nervous system, bypassing retrograde axonal transport as the source of BMPs to the motoneuron cell body. It is tempting to speculate that impaired synaptic homeostasis at the NMJ may play a role in the progression of motoneuron disease associated with impaired retrograde axonal transport (Goold, 2007).

Finally, the data could have relevance to the sustained expression of homeostatic plasticity in regions of the adult nervous system. BMPs and downstream signaling proteins such as the Smads continue to be expressed in the adult nervous system. In particular, BMPs are secreted into the cerebral spinal fluid at concentrations that are relevant for neuronal signaling. It is, therefore, interesting to speculate that circulating levels of BMPs might sustain the competence of neurons to express homeostatic plasticity without driving morphological plasticity in the adult nervous system (Goold, 2007).

Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. This study examined processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which are called the Main and Shadow sites, and one within the prodomain, which is called the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. These data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species (Fritsch, 2012).

While the results of this study support the conventional dogma that proteolytic processing and dissociation of the prodomain are essential steps in BMP activation, this study shows that different BMPs, even if closely related, can have distinct processing requirements. In particular, for the BMP5/6/7/8 proteins, a novel mechanism of prodomain shedding was identified involving Furin-mediated cleavage at a site within the prodomain, and evidence is provided that some BMPs can signal with part or all of the prodomain covalently attached (Fritsch, 2012).

Gbb processing occurs at three sites to give rise to an N-terminal pro-fragment, a C-terminal profragment, and the ligand domain. The processing event that must occur to activate Gbb is the separation of the ligand domain from the N-terminal pro-fragment, which can occur by cleavage at either the Pro site or the Main site. Thus, Gbb can function either as a fully processed ligand or with the C-terminal fragment of the prodomain covalently attached. An inhibitory role for the N-terminal part of the prodomain is not without precedent: functional studies on Myostatin/GDF8 have mapped the inhibitory domain of the protein to amino acids 42 to 115, and the recent crystal structure of TGF-β1 has also implicated this part of the protein in blocking ligand function (Fritsch, 2012).

Scw is processed at three cleavage sites orthologous to those in Gbb (Pro, Main and Shadow), but in this case, cleavage at both the Pro site and the Main site are required to produce a functional ligand. Cleavage at the Pro site is required for dissociation of the prodomain and for stable accumulation of the secreted ligand. Thus, when complexed with the prodomain, Scw is either inefficiently secreted or rapidly internalised following secretion, which is similar to what has been reported for members of the BMP2/4/Dpp group. Cleavage at the Main site is required for dissociation of the ligand domain from the Cterminal prodomain fragment, which, in Scw, inhibits ligand function. The inhibitory effect of the C-terminal prodomain region in Scw is a recent evolutionary adaptation, and could reflect either a newly evolved property of the prodomain or a newly evolved function of the Scw ligand domain with which this prodomain fragment interferes. Notably, a similar rapid functional divergence in the prodomain has also been reported for BMP15, where the prodomains of the mouse and human orthologs confer differential processing efficiencies to the pro-protein that may underlie the distinct functional properties of the ligand in these two species (Fritsch, 2012).

The data support the model that human BMP7 is processed at a single, low-probability Furin cleavage site that lies between the prodomain and ligand domain, and that cleavage at this site is essential for function. Curiously, while the Gbb-BMP7 ligand domain chimera fully rescues gbb mutants, full length BMP7 does not, suggesting that the BMP7 prodomain is incompatible with essential features of Gbb processing and signalling. Thus, while the Gbb and BMP7 ligands are functionally interchangeable, their pro-domains have diverged, presumably to fine-tune their activity to fulfil their endogenous functions (Fritsch, 2012).

Taken together these findings on Scw, Gbb and BMP7 illustrate how evolutionary plasticity in the prodomain sequence serves to modulate the activity of the ligand, which may be subject to stronger evolutionary constraints and thus unable to diverge sufficiently to provide functional diversity. The Furin-mediated cleavage of Gbb and Scw characterized in this study is one of a number of different prodomain processing events that have been shown to modulate the function of TGFβ superfamily ligands. For many ligands, the prodomain has been shown to remain noncovalently associated with the ligand, and a range of different functions have been ascribed to it, including targeting the complex for degradation or to the extracellular matrix. Indeed, the prodomains of BMP-4, -5, -7, -10 and GDF5 have been shown to interact with Fibrillin, and the GDF8 prodomain with Perlecan, and various strategies are deployed to dissociate this complex and release the active ligand. In vitro evidence suggests that for BMP7, the prodomain can be displaced by the Type II receptor. A more general strategy may be proteolytic cleavage of the prodomain. GDF11, GDF8 and BMP10 require proteolytic cleavage by the metalloproteinase Tolloid/BMP-1 to activate signalling in vivo or in tissue culture assays. The data suggest that an additional mechanism for activation is Furin/SPC cleavage of the prodomain (Fritsch, 2012).

The recent crystal structure of the pro-TGF-β1 dimer reveals that the two prodomains form a ring-like shape that wraps around the ligand dimer, altering its structure and shielding it from interaction with receptors. The prodomain ring is subdivided into C-terminal 'arms', an extended loop that encircles the tip of each ligand monomer, and two N-terminal, α-helical forearms that cross one another forming a ‘straitjacket’ around the ligand. In the TGF-β1 model, the N-terminal straitjacket locks the ligand dimer into the prodomain ring, and mechanical force induced by interaction with extracellular matrix proteins unfastens the straitjacket to release the active ligand (Fritsch, 2012).

This model for the prodomain fold provides insight into the molecular basis for results on the processing requirements for Gbb, Scw and BMP7, and also raises intriguing questions. The Pro site in Gbb and Scw lies within the extended loop between the arm and straitjacket, and thus is in a key position to influence ligand accessibility and function. In the case of Gbb, cleavage at this site effectively opens the straitjacket and reveals the ligand dimer irrespective of cleavage at the Main/Shadow sites. Scw, on the other hand, cannot function even with the straitjacket removed in this way. This distinction suggests there is a difference between the arm domains of Gbb and Scw, the former of which is neutral to ligand function and the latter of which is inhibitory. Moreover, Scw requires cleavage at the Pro site to shed the prodomain and activate the ligand, while Gbb functions without this cleavage. Thus, Scw follows the TGF-β1 paradigm of requiring more than just cleavage at the Main site to shed the prodomain, while Gbb presents a distinct situation where cleavage at the Main site is sufficient for prodomain shedding (Fritsch, 2012).

The results with BMP7 also depart from the TGF-β1 paradigm where the prodomain locks the ligand into an inactive form. While cleavage-resistant BMP7 only retains a low level of signalling activity, the data show that it can activate the signalling pathway despite having the prodomain covalently attached. While the ability for a BMP pro-protein to bind to its receptor has been previously shown for BMP2 and BMP7 in vitro, the data provide the first evidence that a BMP pro-protein can signal in vivo without displacement of the prodomain (Fritsch, 2012).

The expression of cleavage-resistant TGFβ superfamily proteins has been shown to generate dominant negative effects by blocking secretion of the wild type protein. In these studies, it was assumed that the mutant proteins formed heterodimers with the wild type monomers, thus promoting their degradation within the cell. The results with cleavage resistant Gbb do not support this type of model for two reasons. First, cleavage resistant Gbb is secreted at normal levels, and thus is not degraded within the cell. Second, it was found that cleavage-resistant Gbb knocks down the function of endogenous Gbb, but not Dpp, with which it forms heterodimers. Thus, the dominant negative effect is exclusive to the homotypic ligand. This specificity has been reported previously, but it is not clear how it might arise for a protein that is known to function by forming heterodimers with other ligands. This suggests that either the relationship between dimerization and processing is different than currently thought, or that the mechanism underlying the dominant negative behaviour is not exclusively due to heterodimerization (Fritsch, 2012).

Phylogenetic analysis of the BMP5/6/7/8 proteins has shown that the processing sites are embedded in blocks of sequence that are poorly conserved even between closely related species. This feature is also a characteristic of the cleavage sites in the BMP2/4/Dpp proteins, and thus the domains that include the cleavage sites in the BMPs appear to be in constant flux, with the core and flanking sequences changing from one species to the next. Indeed, in both subgroups there is evidence that the arrangement of cleavage sites can change, presumably influencing the processing mechanism. For example, in the vertebrate BMP2/4 proteins, the S1 site is a high probability site and is cleaved first, while the S2 site is a low probability site and is only cleaved after processing at the S1 site. In Arthropods, these cleavage probabilities are reversed (and an additional cleavage site is added) and the order of cleavage is correspondingly inverted. Similarly, in the Gbb proteins, the Main site is typically a high probability site, and the Shadow site a low probability site, but in Glossina, Anopheles, and Tribolium, the cleavage probabilities are reversed, indicating that, in Gbb, it is the presence, and not the position of the site that is important. In this light, the Furin cleavage sites appear to evolve like transcription factor binding sites in a promoter where the key feature is maintaining the function of the element irrespective of the position, number, or affinity of the binding sites that comprise it. Given this, the plasticity in processing requirements the observations for the BMP5/6/7/8 proteins may well apply to other BMPs and be a general mechanism whereby the ligands are fine-tuned for their particular functions (Fritsch, 2012).


GENE STRUCTURE

cDNA length - 1667

Bases in 5' UTR - 171

Exons - 1

Bases in 3' UTR - 130


PROTEIN STRUCTURE

Amino Acids - 455

Structural Domains

The Tgfbeta-60A gene, a member of the transforming growth factor beta superfamily of signaling proteins, has been identified in Drosophila. From its inferred protein sequence it is predicted that the precursor is secreted and processed to release a growth factor-like molecule. The putative precursor contains an amino-terminal signal sequence and four N-linked glycosylation sites, consistent with the proposed secreted nature of this protein. The TGF-beta homologous sequences are located in 100 amino acids of the C-terminal region. The multibasic cleavage sites, RSKR and RKRKK (positions 322 and 331, respectively), found upstream of the TGF-beta homologous region are candidate sites for the proteolytic cleavage of the precursor molecule. As would be expected based on the primers used to isolate this gene, the C-terminal sequence of the Tgfbeta-60A gene exhibits expecially high levels of sequence identity in amino acid residues that are conserved within the Vg-1/dpp subgroup. The putative Tgfbeta-60A protein shows greater sequence similarity to three vertebrate family members (human bone morphogenetic proteins 5, 6, and 7) than to its only Drosophila relative, the protein product of the dpp gene. This observation suggests that the duplication event that gave rise to the two transforming growth factor beta-like proteins in Drosophila predates the divergence of chordates and arthropods (Wharton, 1991).

Using PCR methods, a second Drosophila gene in the TGF-beta family has been identified. It encodes a protein product that is more similar to the TGF-beta-related human bone morphogenetic proteins (BMPs) 5, 6, and 7 than it is to the Drosophila dpp gene product. All seven cysteines are precisely conserved between Tgfbeta-60A and other family members Because of its localization on the polytene chromosome map, this gene is termed Tgfbeta-60A. Expression of a Tgfbeta-60A cDNA in Drosophila S2 cells was used to determine that Tgfbeta-60A encodes a preproprotein that is processed to yield secreted amino- and carboxy-terminal polypeptides (Doctor, 1992).


glass bottom boat/Transforming growth factor beta at 60A: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 June 98

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