Transforming growth factor beta at 60A
The complete protein-coding region of the Drosophila virilis 60 A gene, a member of the transforming growth factor-beta superfamily, has been isolated and sequenced. The mature domain of the protein-coding region is 99% identical to the Drosophila melanogaster Tgfbeta-60A gene and 73% identical to human bone morphogenetic protein 5. In the pro-domain, a number of large blocks of amino acids are also highly conserved, indicating an important functional role for this portion of the protein as well. In the putative 5' and 3' untranslated regions, several short sequence motifs are conserved between D. virilis and D. melanogaster (Du, 1996).
To improve the understanding of the evolutionary diversification of decapentaplegic the gene was identified in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. A single dpp locus is present in D. melanogaster and S. americana, suggesting that thedpp copy number does not correlate with increasing genome size. Another TGF-beta superfamily member, the D. melanogaster gene Tgfbeta-60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and Tgfbeta-60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. These results for the TGF-beta superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members (Newfeld, 1995).
The ascidian tadpole larva (phylum Urochordata) is thought to be the prototype for the ancestral chordate. Although ascidians show a highly determinate mode of development, recent studies suggest significant roles of cell-cell interaction during embryogenesis. To elucidate the signaling molecules responsible for the cellular interaction, an ascidian homolog of the transforming growth factor beta (TGF-beta) superfamily has been investigated. HrBMPa is an ascidian member
of the 60A subclass of the BMP subfamily. Molecular phylogenetic analysis suggests that HrBMPa branched off prior to further divergence of vertebrate BMPs-5-8. The zygotic expression of HrBMPa is initiated around gastrulation. HrBMPa transcripts are first evident in precursor cells of the spinal cord, notochord, epidermis and nervous system, although signals in the first two regions quickly disappear. In neurulae and early tailbud embryos, transcripts are evident in the adhesive organ, midline of the anterior dorsal neuroectoderm and midline of both ventral and dorsal ectoderm, suggesting that HrBMPa plays a major role in neuroectodermal cell differentiation during embryogenesis. This HrBMPa expression profile resembles that of Xenopus BMP-7, implying a primordial function of BMP-7 among vertebrate BMPs-5-8 (Miya, 1996).
Both Decapentaplegic (Dpp) protein and Tgfbeta-60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, Dpp and Tgfbeta-60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. An examination of the Drosophila proteins Dpp and 60A focussed on whether they have the capacity to induce bone formation in mammals, using the rat subcutaneous bone induction model. Highly purified recombinant Dpp and Tgfbeta-60A induce the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available (Sampath, 1993).
The expression pattern of bone morphogenetic protein-7 (BMP-7) in the hindbrain region of the headfold and early somite stage of the developing mouse embryo suggests a role for BMP-7 in the patterning of this part of the cranial CNS. It is thought that in chick embryos BMP-7 is one of the secreted molecules that mediates the dorsalizing influence of surface ectoderm on the neural tube, and mouse surface ectoderm has been shown to have a similar dorsalizing effect. While it is confirmed that BMP-7 is expressed in the surface ectoderm of mouse embryos at the appropriate time to dorsalize the neural tube, it is also shown that at early stages of hindbrain development BMP-7 transcripts are present in paraxial and ventral tissues, within and surrounding the hindbrain neurectoderm; only later does expression become restricted to a dorsal domain. To determine more directly the effect that BMP-7 may have on the developing hindbrain, COS cells expressing BMP-7 were grafted into the ventrolateral mesoderm abutting the neurectoderm in order to prolong BMP-7 expression in the vicinity of ventral hindbrain. Three distinct actvities of BMP-7 are apparent: (1) as expected from previous work in chick, BMP-7 can promote dorsal characteristics in the neural tube; (2) it can also attenuate the expression of Sonic hedgehog (Shh) in the floorplate without affecting Shh expression in the notochord, and (3) ectopic BMP-7 appears to promote growth of the neurectoderm (Arkell, 1997).
The 60A subclass of BMPs contains at least four vertebrate members, BMPs 5-8. Of these four genes, BMP 7 is expressed earliest, in gastrulating embryos. BMP 7 transcripts are present at diverse sites throughout development, in a pattern consistent with a role in a variety of inductive interactions. Recent studies have shown that BMP 2/7 heterodimers have unique activities compared to the corresponding homodimers. For this reason, the patterns of expression of BMP 2 (Drosophila homolog: Decapentaplegic) and BMP 7 were compared using in situ hybridization. These BMPs are coexpressed in a number of tissues that are known to be the source of inductive signals, including the zone of polarizing activity and apical ectodermal ridge of the developing limb and the notochord, raising the possibility that BMP 2/7 heterodimers may mediate aspects of these tissue interactions. BMP 2 transcripts are restricted within the developing gut to dorsal endoderm, whereas Sonic hedgehog has been localized to ventral and medial regions of the developing gut endoderm. These markers provide the first molecular evidence for dorsal/ventral polarity in the developing gut (Lyons, 1995).
A large number of TGF superfamily members, including BMP-2, -4 and -7, are expressed during early embryogenesis in the vertebrate embryo. BMP-7 is shown to have ventralizing activity both in ectodermal explants as well as in whole embryos. As was the case for BMP-2 and BMP-4, BMP-7 is a very poor inducer when provided as a homodimer protein. Because of this weak mesoderm inducing activity, it has been suggested that mesoderm induction by BMPs might represent an artifact of overexpression. Evidence is provided demonstrating that unlike the homodimers of BMP-4 or BMP-7, the purified recombinant heterodimer of Xenopus BMP-4 and BMP-7 (BMP-4/7) has a potent mesoderm inducing activity at physiological concentrations. These results provide the first evidence for an embryonic function of BMP-4/7 heterodimers in the vertebrate embryo (Suzuki, 1997).
The three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution is described. Although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors (Griffith, 1996).
The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, various combinations of BMPs were co-expressed in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, results in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yields heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers are also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results have been obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions (Israel, 1996).
The expression and processing of osteogenic protein-1 (hOP-1), a bone morphogenic protein of the TGF-beta family, has been characterized in Chinese hamster ovary cells. The hOP-1 is initially synthesized as a monomeric 50 kDa pro-protein that is dimerized, glycosylated, and then proteolytically cleaved at the Arg-Xaa-Xaa-Arg maturation site in an acidic cellular compartment before secretion into the medium. Of the four potential N-linked glycosylation sites, two are used, one in the mature domain and one in the pro-domain. Gel permeation chromatography of secreted hOP-1 in physiological buffers yields an apparent molecular weight of 110-120 k, indicating that after proteolytic processing the two pro-domains remain non-covalently associated with the disulfide linked mature dimer in a complex termed soluble hOP-1. Purified soluble hOP-1 is significantly more soluble in physiological buffers than the purified mature OP-1 (Jones, 1994).
Proteins in the TGF-beta superfamily transduce their effects through binding to type I and type II serine/threonine kinase receptors. Osteogenic protein-1 binds activin receptor type I (ActR-I), and BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB) in the presence of activin receptors type II (ActR-II) and type IIB (ActR-IIB). The binding affinity of OP-1 to ActR-II is two- to threefold lower than that of activin A. A transcriptional activation signal is transduced after binding of OP-1 to the complex of ActR-I and ActR-II, or that of BMPR-IB and ActR-II. These results indicate that ActR-II can act as a functional type II receptor for OP-1, as well as for activins. Some of the known biological effects of activin are observed for OP-1, including growth inhibition and erythroid differentiation induction. Compared to activin, OP-1 is a poor inducer of mesoderm in Xenopus embryos. Moreover, follistatin, an inhibitor of activins, inhibits the effects of OP-1, if added at a 10-fold excess. However, certain effects of activin, like induction of follicle stimulating hormone secretion in rat pituitary cells, are not observed for OP-1. OP-1 has overlapping binding specificities with activins, and shares certain but not all of the functional effects of activins. Thus, OP-1 may have broader effects in vivo than hitherto recognized (Yamashita 1995).
Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors [BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB)], and a type II receptor [BMP receptor type II (BMPR-II)]. In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). The binding and signaling properties of GDF-5 through type I and type II receptors has been investigated. GDF-5 induces alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. GDF-5 binds to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. GDF-5 binds to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells are transfected with type II receptor cDNAs, GDF-5 binds to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 binds to different sets of type I receptors, but the binding is most efficient to BMPR-IB, when compared with the other type I receptors. A transcriptional activation signal is efficiently transduced by BMPR-IB in the presence of BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II (Nishitoh, 1996).
Continued: see 60A Evolutionary homologs part 2/3 | part 3/3 |
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