Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice

Tilak, Anup; Nelsen, Sylvia; Kim, Hyung Seok; Donley, Nathan; McKnite, Autumn; Lee, Hyunjung; Christian, Jan L.

doi:10.1242/dev.110130

Cited by 18 publications

(24 citation statements)

References 52 publications

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“…Specifically, we have shown that mice carrying a knock-in mutation that causes mature BMP4 to be released prematurely from the prodomain die during embryogenesis due to destabilization of the cleaved ligand (53). Our findings support a model in which the transient prodomain/mature ligand complex that forms during sequential cleavage targets mature BMP4 to binding partners in the ECM that protect the ligand from degradation and thus promote signaling (Fig.…”

Section: Discussionsupporting

confidence: 77%

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The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

Neugebauer¹,

Kwon

Kim³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.B one morphogenetic proteins (BMPs) are members of the TGFβ superfamily that were originally isolated as boneinducing morphogens and were subsequently found to play central roles during embryogenesis and in adult homeostasis (1). BMPs are clinically important therapeutic agents that are used to reverse bone loss caused by trauma, disease, and tumor resection (2). Their use as regenerative agents is limited, however, by their short half-life and low specific activity when implanted in vivo. Understanding how BMP activity is regulated is important for the development of more effective therapeutic agents for the treatment of bone injury and disease.BMPs bind to and activate a receptor complex consisting of type I and type II transmembrane serine/threonine kinases. Following ligand binding, activated receptors propagate their signal by phosphorylating one of the SMADs that is specific for the BMP pathway (SMAD1, -5, or -8). The phosphorylated Smads then form heterooligomers with the common Smad, Smad4, and this complex translocates into the nucleus where it binds to BMP response elements and activates transcription of target genes (1).BMPs are classified into subfamilies based on sequence homology. They signal as either homodimers, or as heterodimers from different subfamilies. For example, class I BMPs, which consist of BMP2 and BMP4, can heterodimerize with class II BMPs, consisting of BMP5-8 (3). Heterodimers composed of distinct BMP family members show a higher specific activity than do homodimers of either subunit. Homodimers of BMP2, -4, or -7, for example, can all induce bone formation, but heterodimers of BMP2 plus BMP7, or BMP4 plus BMP7 are significantly more potent (5-to 20-fold) than any of the homodimers in osteogenic differentiation assays (4-6). BMP2/7 and BMP4/7 heterodimers also sho...

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Section: Discussionsupporting

confidence: 77%

“…6A). Specifically, we have shown that BMP4 is sequentially cleaved at the S1 and S2 sites in a post-trans-Golgi network (TGN) vesicle and that cleavage is closely coupled to secretion (53) (Fig. 6A).…”

Section: Discussionmentioning

confidence: 99%

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

Neugebauer¹,

Kwon

Kim³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…It is interesting to note that both HEK and undifferentiated SW3 cells secrete the vast majority of FGF23 cleavage fragments they synthesize, suggesting that a convertase-mediated cleavage event occurs either just prior to or during secretion in a poorly understood coupled proteolysis/secretion process. Similar cleavage timing occurs for other constitutively-trafficked precursors in other cell systems, for example pro-atrial natriuretic factor [35], proaugurin [36], and bone morphogenetic protein 4 (BMP4) [37]. …”

Section: Discussionmentioning

confidence: 93%

Posttranslational processing of FGF23 in osteocytes during the osteoblast to osteocyte transition

Yamamoto

Ramos‐Molina

Lick

et al. 2016

Bone

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FGF23 is an O-glycosylated circulating peptide hormone with a critical role in phosphate homeostasis; it is inactivated by cellular proprotein convertases in a pre-release degradative pathway. We have here examined the metabolism of FGF23 in a model bone cell line, IDG-SW3, prior to and following differentiation, as well as in regulated secretory cells. Labeling experiments showed that the majority of 35S-labeled FGF23 was cleaved to smaller fragments which were constitutively secreted by all cell types. Intact FGF23 was much more efficiently stored in differentiated than in undifferentiated IDG-SW3 cells. The prohormone convertase PC2 has recently been implicated in FGF23 degradation; however, FGF23 was not targeted to forskolin-stimulatable secretory vesicles in a regulated cell line, suggesting that it lacks a targeting signal to PC2-containing compartments. In vitro, PC1/3 and PC2, but not furin, efficiently cleaved glycosylated FGF23; surprisingly, PC5/6 accomplished a small amount of conversion. FGF23 has recently been shown to be phosphorylated by the kinase FAM20C, a process which was shown to reduce FGF23 glycosylation and promote its cleavage; our in vitro data, however, show that phosphorylation does not directly impact cleavage, as both PC5/6 and furin were able to efficiently cleave unglycosylated, phosphorylated FGF23. Using qPCR, we found that the expression of FGF23 and PC5/6, but not PC2 or furin, increased substantially following osteoblast to osteocyte differentiation. Western blotting confirmed the large increase in PC5/6 expression upon differentiation. FGF23 has been linked to a variety of bone disorders ranging from autosomal dominant hypophosphatemic rickets to chronic kidney disease. A better understanding of the biosynthetic pathway of this hormone may lead to new treatments for these diseases.

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“…Often, multiple PC cleavage motifs are present (Fig. 1) and are used (Israel et al 1992; Akiyama et al 2012; Tilak et al 2014). …”

Section: Structures and Functions Of Tgf-β Family Procomplexesmentioning

confidence: 99%

Structural Biology and Evolution of the TGF-β Family

Hinck

Mueller

Springer

2016

Cold Spring Harb Perspect Biol

317

360

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We review the evolution and structure of members of the transforming growth factor β (TGF-β) family, antagonistic or agonistic modulators, and receptors that regulate TGF-β signaling in extracellular environments. The growth factor (GF) domain common to all family members and many of their antagonists evolved from a common cystine knot growth factor (CKGF) domain. The CKGF superfamily comprises six distinct families in primitive metazoans, including the TGF-β and Dan families. Compared with Wnt/Frizzled and Notch/Delta families that also specify body axes, cell fate, tissues, and other families that contain CKGF domains that evolved in parallel, the TGF-β family was the most fruitful in evolution. Complexes between the prodomains and GFs of the TGF-β family suggest a new paradigm for regulating GF release by conversion from closed- to open-arm procomplex conformations. Ternary complexes of the final step in extracellular signaling show how TGF-β GF dimers bind type I and type II receptors on the cell surface, and enable understanding of much of the specificity and promiscuity in extracellular signaling. However, structures suggest that when GFs bind repulsive guidance molecule (RGM) family coreceptors, type I receptors do not bind until reaching an intracellular, membrane-enveloped compartment, blurring the line between extra- and intracellular signaling. Modulator protein structures show how structurally diverse antagonists including follistatins, noggin, and members of the chordin family bind GFs to regulate signaling; complexes with the Dan family remain elusive. Much work is needed to understand how these molecular components assemble to form signaling hubs in extracellular environments in vivo.

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Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice

Cited by 18 publications

References 52 publications

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

Posttranslational processing of FGF23 in osteocytes during the osteoblast to osteocyte transition

Structural Biology and Evolution of the TGF-β Family

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