The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor

The transforming growth factor-beta (TGF-beta) receptor type III is a low abundance cell surface component that binds TGF-beta 1 and TGF-beta 2 with high affinity and specificity, and is present in many mammalian and avian cell types. Type III TGF-beta receptors affinity-labeled with 125I-TGF-beta migrate in sodium dodecyl sulfate-polyacrylamide electrophoresis gels as diffuse species of 250-350 kDa. Here we show that type III receptors deglycosylated by the action of trifluoromethanesulfonic acid yield affinity-labeled receptor cores of 110-130 kDa. This marked decrease in molecular weight is also achieved by combined treatment of type III receptors with heparitinase and chondroitinase ABC. Digestion of receptor-linked glycosaminoglycans by treatment of intact cell monolayers with heparitinase and chondroitinase does not prevent TGF-beta binding to the type III receptor core polypeptide and does not release the receptor polypeptide from the membrane. The type III TGF-beta receptor binds tightly to DEAE-Sephacel and coelutes with cellular proteoglycans at a characteristically high salt concentration. Thus, the type III TGF-beta receptor has the properties of a membrane proteoglycan that carries heparan and chondroitin sulfate glycosaminoglycan chains. The binding site for TGF-beta appears to reside in the 100-120-kDa core polypeptide of this receptor. The type III receptor is highly sensitive to cleavage by trypsin. Trypsin action releases the glycosaminoglycan-containing domain of the receptor leaving a 60-kDa membrane-associated domain that contains the cross-linked ligand. A model for the domain structure of the TGF-beta receptor type III is proposed based on these results.     

Possible involvement of transforming growth factor-beta 1 and transforming growth factor-beta receptor type II during luteinization in the marmoset ovary

The expression of transforming growth factor-beta 1 (TGF-beta 1), and transforming growth factor-beta receptor type II (T beta R-II), were evaluated in periovulatory marmoset ovaries. Histochemical methods were used, in particular double-labelling techniques, in order to correlate growth factor/receptor expression with proliferation (Ki 67), apoptosis (TUNEL method) and luteinization (3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)). The latter was used as a luteinization marker. Periovulatory ovaries are especially suited for studying all aspects since they typically consist of small non-luteinized follicles, large luteinizing follicles and corpora lutea accessoria (Clas), which have developed from large luteinizing follicles. TGF-beta 1 and T beta R-II expression was found in luteinizing theca cells of large periovulatory follicles and in all luteal cells of Clas. Non-luteinized theca cells, including those of small follicles were always devoid of any immunostaining. Granulosa cells of small follicles were immunopositive for T beta R-II. Large follicles with granulosa cell immunoreactivity of both antibodies coexisted with non-reactive follicles of comparable size. The highest activity of the luteal marker enzyme 3 beta-HSD was co-localized in the same cells that expressed TGF-beta 1 and T beta R-II. The double-labelling experiments revealed that TGF-beta 1 and T beta R-II expression is not correlated with proliferation or apoptosis of follicular cells. Our results indicate that TGF-beta 1 and T beta R-II participate in differentiation processes, i.e. luteinization, rather than proliferation. In particular, the dynamics of T beta R-II expression appear highly related to the process of luteinization.     

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.     

Selection of RNA aptamers against recombinant transforming growth factor-beta type III receptor displayed on cell surface

In most cases, anti-protein aptamers are selected by systematic evolution of ligands by exponential enrichment (SELEX) using purified recombinant protein targets. Cell surface proteins, however, are not easy targets for SELEX due to the difficulties associated with their purification. Here, we developed a novel SELEX procedure (referred to as TECS-SELEX) in which cell-surface displayed recombinant protein is directly used as the selection target. Using this method, we isolated RNA aptamers against transforming growth factor-beta type III receptor expressed on Chinese hamster ovary (CHO) cells. One of the RNA aptamers has a dissociation constant in the 1 nM range and competed with transforming growth factor-beta to bind to the cell surface receptor in vitro.     

Endocytosis of the type III transforming growth factor-beta (TGF-beta) receptor through the clathrin-independent/lipid raft pathway regulates TGF-beta signaling and receptor down-regulation

Transforming growth factor-beta (TGF-beta) signals through three highly conserved cell surface receptors, the type III TGF-beta receptor (T beta RIII), the type II TGF-beta receptor (T beta RII), and the type I TGF-beta receptor (T beta RI) to regulate diverse cellular processes including cell proliferation, differentiation, migration, and apoptosis. Although T beta RI and T beta RII undergo ligand-independent endocytosis by both clathrin-mediated endocytosis, resulting in enhanced signaling, and clathrin-independent endocytosis, resulting in receptor degradation, the mechanism and function of T beta RIII endocytosis is poorly understood. T beta RIII is a heparan sulfate proteoglycan with a short cytoplasmic tail that functions as a TGF-beta superfamily co-receptor, contributing to TGF-beta signaling through mechanisms yet to be fully defined. We have reported previously that T beta RIII endocytosis, mediated by a novel interaction with beta arrestin-2, results in decreased TGF-beta signaling. Here we demonstrate that T beta RIII undergoes endocytosis in a ligand and glycosaminoglycan modification-independent and cytoplasmic domain-dependent manner, with the interaction of Thr-841 in the cytoplasmic domain of T beta RIII with beta-arrestin2 enhancing T beta RIII endocytosis. T beta RIII undergoes both clathrin-mediated and clathrin-independent endocytosis. Importantly, inhibition of the clathrin-independent, lipid raft pathway, but not of the clathrin-dependent pathway, results in decreased TGF-beta1 induced Smad2 and p38 phosphorylation, supporting a specific role for clathrin-independent endocytosis of T beta RIII in regulating both Smad-dependent and Smad-independent TGF-beta signaling.     

Oncogenic Ki-ras confers a more aggressive colon cancer phenotype through modification of transforming growth factor-beta receptor III

Transforming growth factor-beta1 (TGF-beta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras(G12V) transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivo than controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928-30936). We now show that stable expression of oncogenic Ki-ras(G12V) converts the HD6-4 colon cancer cell line from insensitive to TGF-beta1 to growth-promoted by TGF-beta1. Each of three Ki-ras(G12V) transfectants responded to TGF-beta1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-beta1. The wild-type Ki-ras transfectants lack functional TGF-beta receptors, whereas all three Ki-ras(G12V) transfectants expressed functional TGF-beta receptors that bound (125)I-TGF-beta1. The previous studies showed that in cells with wild-type Ki-ras, TGF-beta receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-beta receptor III (TbetaRIII) was incomplete. We now show that the betaglycan form of TbetaRIII is highly modified following translation when transiently expressed in Ki-ras(G12V) cells, whereas no such post-translational modification of TbetaRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TbetaRIII post-translational modification in Ki-ras(G12V) cells and TGF-beta1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TbetaRIII post-translational modification.     

Positive and negative modulation of vitamin D receptor function by transforming growth factor-beta signaling through smad proteins

Several lines of experiments demonstrated the interplay between the transforming growth factor-beta (TGF-beta) and vitamin D signaling pathways. Recently, we found that Smad3, a downstream component of the TGF-beta signaling pathway, potentiates ligand-induced transactivation of vitamin D receptor (VDR) as a coactivator of VDR (Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science 283, 1317-1321). Here, we investigated the roles of inhibitory Smads, Smad6 and Smad7, which are negative regulators of the TGF-beta/bone morphogenetic protein signaling pathway, on the Smad3-mediated potentiation of VDR function. We found that Smad7, but not Smad6, abrogates the Smad3-mediated VDR potentiation. Interaction studies in vivo and in vitro showed that Smad7 inhibited the formation of the VDR-Smad3 complex, whereas Smad6 had no effect. Taken together, our results strongly suggest that the interplay between the TGF-beta and vitamin D signaling pathways is, at least in part, mediated by the two classes of Smad proteins, which modulate VDR transactivation function both positively and negatively.     

Control of type II transforming growth factor-beta receptor expression by integrin ligation

Ectopic expression of the alpha5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of alpha5 integrin is due to induction of autocrine negative transforming growth factor-beta (TGF-beta) activity. MCF-7 breast cancer cells do not express either alpha5 integrin or type II TGF-beta receptor and hence are unable to generate TGF-beta signal transduction. Ectopic expression of alpha5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-beta receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in alpha5 transfectants by growth on fibronectin-coated plates. Induction of type II TGF-beta receptor expression also resulted in the generation of autocrine negative TGF-beta activity because colony formation was increased after TGF-beta neutralizing antibody treatment. Transient transfection with a TGF-beta promoter response element in tandem with a luciferase cDNA into cells stably transfected with alpha5 integrin resulted in basal promoter activities 5-10-fold higher than those of control cells. Moreover, when alpha5 transfectants were treated with a neutralizing antibody to either TGF-beta or integrin alpha5, this increased basal promoter activity was blocked. Autocrine TGF-beta activity also induced 3-fold higher endogenous fibronectin expression in alpha5 transfectants relative to that of control cells. Re-expression of type II receptor by alpha5 transfection also restored the ability of the cells to respond to exogenous TGF-beta and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-beta type II receptor expression can be controlled by alpha5beta1 ligation and integrin signal transduction. Moreover, TGF-beta and integrin signal transduction appear to cooperate in their tumor-suppressive functions.     

Interactions of high affinity insulin-like growth factor-binding proteins with the type V transforming growth factor-beta receptor in mink lung epithelial cells

High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-beta receptor (TbetaR-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572-20576). To characterize the interactions of high affinity IGFBPs with TbetaR-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. 125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to TbetaR-V as demonstrated by the detection of the approximately 400-kDa 125I-IGFBP.TbetaR-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of 125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for TbetaR-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of 125I-IGFBP-3 preferentially bound to and was cross-linked to TbetaR-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-beta type I/type II receptor (TbetaR-I.TbetaR-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for TbetaR-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the TbetaR-I and TbetaR-II heterocomplex.     

Binding of transforming growth factor-beta to cell surface proteins varies with cell type

Transforming growth factor-beta (TGF beta 1 and TGF beta 2) bind to several different cell surface proteins, including a high Mr proteoglycan. We found that on primary and early passage cultures of fibroblasts, chondroblasts, and osteoblasts TGF beta 1 binds to both the high Mr proteoglycan and to lower Mr components, whereas on epithelial, endothelial, and lymphoid-derived cells TGF beta 1 only binds to the lower Mr species. With cell lines, this distinction is lost. Further analysis indicated that binding to the high Mr proteoglycan is not necessary for TGF beta 1 induced regulation of DNA, collagen and fibronectin synthesis, change in cell morphology, or reorganization of the actin cytoskeleton. We propose that the lower Mr components are the active receptors mediating these events.     

Identification of soluble transforming growth factor-beta receptor III (sTbetaIII) in rat milk

Transforming growth factor-beta (TGF-beta) is present at high concentrations in maternal milk. In milk TGF-beta2 is the predominant isoform. For function TGF-beta2 requires TbetaRIII to facilitate efficient binding to the TGF-beta receptor types I and II signalling complex. We have shown that TGF-beta receptor types I (TbetaRI), II (TbetaRII) and III (TbetaRIII) are coexpressed in the suckling rat intestine. Immunostaining for TbetaRIII was also observed in the intestinal lumen prior to weaning. TbetaRIII (or betaglycan) has been reported in serum, cell culture medium and extracellular matrix. To determine whether a soluble form of TbetaRIII is present in milk, the rat milk aqueous phase was analysed by slot-blot and Western blot. Soluble TbetaRIII was detected in milk throughout lactation. Western blot analysis of rat milk revealed a high molecular weight band of glycosylated protein of >200 kDa, with a core protein of approximately 110-120 kDa that comigrated with recombinant TbetaRIII. Immunoabsorption of soluble TbetaRIII (sTbetaRIII) from milk resulted in partial depletion of active TGF-beta from milk, suggesting that the receptor may interact with ligand in milk. In addition rat pups suckled on mother's milk demonstrated an enhanced labelling of TbetaRIII in the gut, as compared with pups fed on a rat milk substitute (RMS). These findings suggest that milk sTbetaRIII is functional, and may modulate milk-derived TGF-beta function in the developing intestine.     

Hyaluronan regulates transforming growth factor-beta1 receptor compartmentalization

Transforming growth factor-beta1 (TGF-beta1) is a key cytokine involved in the pathogenesis of fibrosis in many organs. We previously demonstrated in renal proximal tubular cells that the engagement of the extracellular polysaccharide hyaluronan with its receptor CD44 attenuated TGF-beta1 signaling. In the current study we examined the potential mechanism by which the interaction between hyaluronan (HA) and CD44 regulates TGF-beta receptor function. Affinity labeling of TGF-beta receptors demonstrated that in the unstimulated cells the majority of the receptor partitioned into EEA-1-associated non-lipid raft-associated membrane pools. In the presence of exogenous HA, the majority of the receptors partitioned into caveolin-1 lipid raft-associated pools. TGF-beta1 increased the association of activated/phosphorylated Smad proteins with EEA-1, consistent with activation of TGF-beta1 signaling following endosomal internalization. Following addition of HA, caveolin-1 associated with the inhibitory Smad protein Smad7, consistent with the raft pools mediating receptor turnover, which was facilitated by HA. Antagonism of TGF-beta1-dependent Smad signaling and the effect of HA on TGF-beta receptor associations were inhibited by depletion of membrane cholesterol using nystatin and augmented by inhibition of endocytosis. The effect of HA on TGF-beta receptor trafficking was inhibited by inhibition of HA-CD44 interactions, using blocking antibody to CD44 or inhibition of MAP kinase activation. In conclusion, we have proposed a model by which HA engagement of CD44 leads to MAP kinase-dependent increased trafficking of TGF-beta receptors to lipid raft-associated pools, which facilitates increased receptor turnover and attenuation of TGF-beta1-dependent alteration in proximal tubular cell function.