Identification and functional analysis of transforming growth factor-ß type I receptor (TßR1) from Scylla paramamosain: The first evidence of TßR1 involved in development and innate immunity in crustaceans.

The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24 h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Assembly of TbetaRI:TbetaRII:TGFbeta ternary complex in vitro with receptor extracellular domains is cooperative and isoform-dependent.

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.

Small molecule antagonists of the TGF-beta1/TGF-beta receptor binding interaction.

Excessive and inappropriate action of transforming growth factor (TGF)-beta has been implicated in the pathogenesis of several disease processes, especially cancer and fibrosis. To identify antagonists of the TGF- beta ligand-binding domain that may have therapeutic potential, we screened the National Cancer Institute open access chemical repository for molecules that inhibited binding of TGF-beta to the type II receptor (TbetaRII). About 30,000 molecules were screened resulting in the identification of five structurally related molecules that reduced binding of TGF-beta1 to soluble TbetaRII with an ED50 of approx 10 microM. The chemicals blocked inhibition of Mv1Lu cell growth by TGF-beta, TGF-beta - induced expression of luciferase driven by the TGF-beta response element, and induction of plasminogen inhibitor mRNA detected by Northern blot. In contrast, the chemicals did not block activin-induced inhibition of cell growth. Our results identify a novel chemical group that blocks binding of TGF-beta to its receptor and may result in novel treatment for disease.

Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-ß Superfamily.

The ability to understand the molecular mechanisms by which secreted signaling proteins of the TGF-ß superfamily assemble their cell surface receptors into complexes to initiate downstream signaling is dependent upon the ability to determine atomic-resolution structures of the signaling proteins, the ectodomains of the receptors, and the complexes that they form. The structures determined to date have revealed major differences in the overall architecture of the signaling complexes formed by the TGF-ßs and BMPs, which has provided insights as to how they have evolved to fulfill their distinct functions. Such studies, have however, only been applied to a few members of the TGF-ß superfamily, which is largely due to the difficulty of obtaining milligram-scale quantities of highly homogenous preparations of the disulfide-rich signaling proteins and receptor ectodomains of the superfamily. Here we describe methods used to produce signaling proteins and receptor ectodomains of the TGF-ß superfamily using bacterial and mammalian expression systems and procedures to purify them to homogeneity.

Cellular growth inhibition by IGFBP-3 and TGF-beta1 requires LRP-1.

The type V TGF-beta receptor (TbetaR-V)/IGFBP-3 receptor mediates the IGF-independent growth inhibition induced by IGFBP-3. It also mediates the growth inhibitory response to TGF-beta1 in concert with other TGF-beta receptor types, and its loss may contribute to the malignant phenotype of human carcinoma cells. Here we demonstrate that TbetaR-V is identical to LRP-1/alpha2M receptor as shown by MALDI-TOF analysis of tryptic peptides of TbetaR-V purified from bovine liver. In addition, 125I-IGFBP-3 affinity-labeled TbetaR-V in Mv1Lu cells is immunoprecipitated by antibodies to LRP-1 and TbetaR-V. RAP, an LRP-1 antagonist, inhibits binding of 125I-TGF-beta1 and 125I-IGFBP-3 to TbetaR-V and diminishes IGFBP-3-induced growth inhibition in Mv1Lu cells. Absent or low levels of LRP-1, as with TbetaR-V, have been linked to the malignant phenotype of carcinoma cells. Mutagenized Mv1Lu cells selected for reduced expression of LRP-1 have an attenuated growth inhibitory response to TGF-beta1 and IGFBP-3. LRP-1-deficient mouse embryonic fibroblasts lack a growth inhibitory response to TGF-beta1 and IGFBP-3. On the other hand, stable transfection of H1299 human lung carcinoma cells with LRP-1 cDNA restores the growth inhibitory response. These results suggest that the LRP-1/TbetaR-V/IGFBP-3 receptor is required for the growth inhibitory response to IGFBP-3 and TGF-beta1.

Gene digging. A method for obtaining species-specific sequence based on conserved segments of nucleotides in open reading frames.

A method termed "gene digging" has been developed based on our observation of stretches of highly conserved nucleotide sequence in the coding region of many genes across related species. Rabbit-specific nucleotide sequences corresponding to desired coding segments of 14 different genes were obtained with primers that were designed based on conserved nucleotide stretches. Our success in gene digging could be attributable to the method's inherent ability to reduce the degeneracy of primers by more than two orders of magnitude (sometimes by more than three orders of magnitude) compared to primers designed from conserved amino acids. Our results not only demonstrate the value of the method, but also hint at a thus far unknown functional significance of conserved nucleotide stretches in the coding region of various genes. In our hands the method worked 14 out of 14 times indicating generality of the concept.

An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFbeta type II receptor.

TGFbeta signaling is initiated by binding of growth factor ligand to two related single-pass transmembrane receptor serine/threonine kinases, known as the TGFbeta type I (TbetaRI) and type II (TbetaRII-ED) receptors. TbetaRII-ED is essential for all TGFbeta-induced signals. The DNA sequence encoding the extracellular domain of human TbetaRII-ED (TbetaRII-ED, residues 4-136) was synthesized from 20 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. High level expression ( approximately 1 gL(-1)) of thioredoxin/TbetaRII-ED fusion was achieved in Escherichia coli BL21(DE3) strain mainly in soluble form. The soluble thioredoxin/TbetaRII-ED fusion has been purified and refolded on Ni-NTA agarose. After cleavage of purified thioredoxin/TbetaRII-ED fusion by recombinant human enteropeptidase light chain (L-HEP) the target protein of TbetaRII-ED was separated from thioredoxin on Ni-NTA agarose. Fourteen milligrams of highly purified TbetaRII-ED without N- or C-terminal tags was yielded from 100mL cell culture. The purified preparation of TbetaRII-ED was highly homogenous, as shown by SDS-PAGE with silver staining, HPLC and mass spectroscopy analysis. The binding of TbetaRII-ED purified from E. coli to TGFbeta1 was shown to be comparable to commercial material purified from NSO cells. Recombinant TbetaRII-ED could be employed as an antagonist of TGFbeta1 and TGFbeta3 in vitro and in vivo as well as for therapy of fibrotic disorders and some types of cancer.

The transforming growth factor beta receptors types I, II, and III form hetero-oligomeric complexes in the presence of ligand.

Transforming growth factors beta (TGF-beta s) are disulfide-linked dimers. In Rat-1 cells both radioiodinated TGF-beta 1 and -beta 2 bind to and can be chemically cross-linked to type I and II receptors (which are thought to mediate effects of cell growth suppression and gene activation), to type III proteoglycan receptors, and to a novel approximately 50-kDa protein. After detergent solubilization of cells that were cross-linked with radioiodinated TGF-beta, antibodies specific for the type II receptor precipitated labeled receptor types I and III as well as type II. In these cells, the type III receptor is the predominant TGF-beta-binding protein, and antibodies specific for it precipitate mainly this cross-linked receptor. Thus, in the presence of TGF-beta ligand, receptor types II and III and types II and I form heteromeric complexes. The majority of the type III receptor does not associate with receptor types I and II, probably reflecting the relative amounts of the three receptors on the surface of Rat-1 cells. Since TGF-beta 1 but not TGF-beta 2 binds to the exoplasmic domain of the type II receptor in the absence of the type III receptor, and since both TGF-beta 1 and -beta 2 bind with high affinity to the type III receptor, we suggest that TGF-beta 2, and possibly TGF-beta 1, bind initially to the type III receptor. The TGF-beta 2-type III receptor complex would then interact with a type II receptor, thus modulating the affinity of the type II receptor for TGF-beta 2.

Molecular cloning of a novel type I receptor serine/threonine kinase for the TGF beta superfamily from rat brain.

Growth factors belonging to the TGF beta superfamily bind to and signal through a receptor complex comprising two transmembrane serine/threonine kinases, called type I and type II. Each receptor is responsible for the signaling of the individual TGF beta superfamily members. So far, five type II and six type I receptors have been cloned from mammalian sources. We report here the molecular cloning of a novel type I receptor serine/threonine kinase, ALK7 (activin receptor-like kinase 7), from rat brain. ALK7 shows a significant sequence similarity with TGF beta RI and ActRIB in the intracellular kinase domain and is quite distinct from other type I receptors in the extracellular domain. ALK7 mRNA is expressed in embryonic and in adult rat brain, where it was localized in superficial layers of the forebrain, the CA3 pyramidal subfield of hippocampus, the basal ganglia, the thalamus, and the cerebellar cortex. The functionality of the receptor was demonstrated by the identification of a constitutively active point mutant of ALK7 that activates the TGF beta/activin-responsive reporter without any ligand stimulation. Although the endogenous ligand for ALK7 has yet to be identified, its extensive anatomic distribution in brain, gut, spleen, and lung suggests important roles for this orphan receptor.

Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues.

The effects of Bmp-4 on interdigital cell death were investigated in the mouse. Affi-Gel beads, loaded with recombinant Bmp-4 protein, were transplanted into the interdigital tissues of day 12.5 hindlimb, ex utero. It was established that Bmp-4 could induce precocious interdigital cell death. Using in situ hybridization, the expression patterns of bmp-4 and alk-6 receptor were established. Both genes were found coexpressed in the interdigital region of 12.5- and 13. 5-day hindlimbs. This suggests that Bmp-4 may act in an autocrine fashion. We have also studied the effects of Bmp-4 on 12.5-day interdigital tissue cultures. In all specimens examined, the interdigital tissues produced cartilage instead of participating in cell death. The addition of exogenous Bmp-4 to the interdigital cultures did not induce apoptosis but instead enhanced chondrogenesis. The discrepancy between the effects of Bmp-4 in vitro and ex utero was attributed to the presence of digits. When a flanking digit was left attached to the interdigital tissues, in vitro, Bmp-4 promoted apoptosis instead of chondrogenesis. In sum, the results suggest that Bmp-4 is a multifunctional protein and its effect on the interdigital tissues is dependent on the modulating influence of the digits.

Interaction of the transforming growth factor-beta type I receptor with farnesyl-protein transferase-alpha.

Transforming growth factor-beta 1 (TGF-beta 1) is the prototype of a large family of molecules that regulate a variety of biological processes. The type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta 1 are transmembrane serine/threonine kinases, forming a heteromeric signaling complex. Recent studies have shown that T beta R-II is a constitutively active kinase and phosphorylates T beta R-I upon ligand binding, suggesting that T beta R-I is the effector subunit of the receptor complex, which transduces signals to intracellular targets. This model has been further confirmed by the identification of constitutively active T beta R-I that mediates TGF-beta 1-specific cellular responses in the absence of ligand and T beta R-II. To investigate signaling by TGF-beta 1, we have sought to isolate proteins that interact with the cytoplasmic region of T beta R-I. One of the proteins identified was the alpha subunit of farnesyl-protein transferase (FT alpha) that modifies a series of peptides including Ras. T beta R-I specifically interacts with FT alpha in the yeast two-hybrid system. Glutathione S-transferase-T beta R-I fusion proteins bind FT alpha translated in vitro. T beta R-I also phosphorylates FT alpha. We further show that the constitutively active T beta R-I interacted with FT alpha very strongly whereas an inactive form of T beta R-I did not. These results suggest that FT alpha may be one of the substrates of the activated T beta R-I kinase.

Cell surface proteoglycan expression by human periodontal cells.

Cell surface proteoglycans are known to be involved in many functions including interactions with components of the extracellular microenvironment and serve to influence cell shape, adhesion, proliferation, and differentiation. They also can act as co-receptors, to help bind and modify the action of various growth factors and cytokines. Despite their strategic location and relevance to cell function, few studies have considered the nature of the cell surface proteoglycans associated with cells of the periodontium. Due to the structural complexity and multiplicity of cell types in the periodontium, we have selected three different cell lines (gingival connective tissue fibroblast, periodontal ligament fibroblast, and osteoblast) which each represent the unique functions within the periodontium to study the expression of cell surface proteoglycans. We hypothesized that a number of cell surface proteoglycans will be expressed by human periodontal cells and these may be related to the source and function of the cell. Western blotting and RT-PCR methods were used to study the expression of five cell surface proteoglycans (syndecan-1, -2, -4, glypican and betaglycan) in three cell lines of human periodontal cells in vitro. Our results demonstrated the expression of protein cores for syndecan-1 (43 kDa), syndecan-2 (48 kDa), syndecan-4 (35 kDa), glypican (64 kDa), and betaglycan (100-110 kDa). RT-PCR results confirmed that all of these cells produced mRNA for the cell surface proteoglycans under study, of which syndecan-2 showed a significant difference in expression between the periodontal ligament fibroblasts, gingival fibroblasts and osteoblasts. We conclude that the presence of specific cell surface proteoglycans on periodontal cells implies a likely role for these molecules in cell-cell, cell-matrix interactions involved in periodontal disease and/or regeneration of the periodontium, of which they may have distinctive functions related to the source and function of these cells.

Induction of transforming growth factor beta 1 and its receptor expression during myeloid leukemia cell differentiation.

The human myeloid leukemia cell lines HL-60, U-937, and THP-1 were used to analyze the alterations of transforming growth factor beta (TGF-beta) during hematopoietic cell growth and differentiation. Differentiation of these cell lines was induced by the phorbol ester phorbol 12-myristate 13-acetate, tumor necrosis factor alpha or by retinoic acid. Northern hybridization analyses indicated that the basal levels of TGF-beta 1, latent TGF-beta binding protein, and type II TGF-beta receptor (T beta IIR) mRNAs were low in untreated cells. Major increases of these mRNAs were observed in cells treated with phorbol 12-myristate 13-acetate, with maximal induction after 12-72 h of stimulation. Retinoic acid and tumor necrosis factor alpha elevated significantly only the expression of T beta IIR mRNA. TGF-beta 1 induced its receptor mRNA in HL-60 and U937-1SF cells but not in THP-1 cells. These changes in gene expression were related to the differentiation of myeloid leukemia cells. Affinity labeling with 125I-TGF-beta 1 indicated that type I TGF-beta receptor was coregulated with T beta IIR. Types I and II receptors were coprecipitated by T beta IIR-specific antibodies. Differentiation of myeloid cells induced secretion of latent TGF-beta 1 protein, as shown by immunoblotting, but significant changes in the levels of active TGF-beta were not observed. These results indicate that the genes involved in TGF-beta signal transduction are coordinately up-regulated during myeloid differentiation.

Signaling activity of homologous and heterologous transforming growth factor-beta receptor kinase complexes.

Transforming growth factor-beta (TGF-beta) signaling in Mv1Lu lung epithelial cells requires coexpression of TGF-beta receptors I (T beta R-I) and II (T beta R-II), two distantly related transmembrane serine/threonine kinases that form a heteromeric complex upon ligand binding. Here, we examine the formation of TGF-beta receptor homo-oligomers and their possible contribution to signaling. T beta R-I can contact ligand bound to T beta R-II, but not ligand free in the medium, and thus cannot form ligand-induced homo-oligomers. T beta R-II, which binds ligand on its own, formed oligomeric complexes when overexpressed in transfected COS cells. However, these complexes were largely ligand-independent and involved immature receptor protein. Since ligand-induced homo-oligomers could not be obtained with the wild-type TGF-beta receptors, we studied receptor cytoplasmic domain homo-oligomerization by using receptor chimeras. The extracellular domain of T beta R-II was fused to the transmembrane and cytoplasmic domains of T beta R-I, yielding T beta R-II/I, and the extracellular domain of T beta R-I was fused to the transmembrane and cytoplasmic domains of T beta R-II, yielding T beta R-I/II. When contransfected with wild-type receptors and exposed to ligand, T beta R-II/I formed a complex with T beta R-I, and T beta R-I/II formed a complex with T beta R-II, thus yielding complexes with homologous cytoplasmic domains. T beta R-II/I transfected alone or with T beta R-I did not restore TGF-beta responsiveness in T beta R-II-defective cell mutants. Furthermore, T beta R-II/I acted in a dominant negative fashion, inhibiting restoration of TGF-beta responsiveness by a cotransfected T beta R-II in T beta R-II-defective cells and by a cotransfected T beta R-I in T beta R-I-defective cells. Similarly, T beta R-I/II transfected alone or with T beta R-II did not restore TGF-beta responsiveness and acted in a dominant negative fashion against T beta R-I. Together with previous genetic and biochemical evidence, these results suggest that TGF-beta mediates transcriptional and antiproliferative responses through the heteromeric T beta R-I.T beta R-II complex and not through homo-oligomeric T beta R-I or T beta R-II complexes.

A mouse TGF-beta type I receptor that requires type II receptor for ligand binding.

A cDNA for a serine/threonine kinase receptor was isolated from a mouse brain cDNA library. The receptor transiently expressed on COS cells bound TGF-beta 1 not by itself but only when TGF-beta type II receptor was coexpressed. The molecular mass of the ligand-receptor complex was estimated to be 75 kDa. The type II receptor-dependent binding and the molecular mass of the complex suggest that the receptor is a TGF-beta type I receptor.

The type II transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.

The type I and type II receptors for transforming growth factor-beta (TGF-beta) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.

TGF-beta receptor regulation mediates the response to exogenous ligand but is independent of the degree of cellular differentiation in human oral keratinocytes.

This study examined the expression of TGF-beta cell-surface receptors, the response to exogenous TGF-beta 1 and the autocrine production of TGF-beta in normal and squamous cell carcinoma-derived human oral keratinocytes with variable degrees of cellular differentiation. TGF-beta receptor expression, the response to exogenous ligand and the autocrine production of TGF-beta appeared unrelated to cellular differentiation. Cells expressed variable proportions of type-I, -II and -III TGF-beta receptors. The expression of type-III receptors correlated inversely with the expression of type-I receptors, but there was no relationship between type-II and either type-I or type-III TGF-beta receptors. Normal cells and the majority (7 of 8) of tumour-derived keratinocytes were inhibited by exogenous TGF-beta 1 and the degree of inhibition correlated with the expression of type-I, but not type-II or type-III, TGF-beta receptors. One tumour-derived cell line was refractory to exogenous TGF-beta 1 although it expressed all 3 receptor types. Endogenous TGF-beta was produced by both normal and tumour-derived keratinocytes and correlated inversely to the expression of type-I, but not type-II, TGF-beta receptors. Further, cells that produced more autocrine TGF-beta had a diminished response to exogenous TGF-beta 1. The data indicate a complex interaction between the expression of TGF-beta cell-surface receptors, endogenous ligand production and the cellular response to exogenous TGF-beta 1.