Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites

Betaglycan, also known as the TGF-beta type III receptor, is a membrane- anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.     

Bone morphogenetic proteins signal through the transforming growth factor-beta type III receptor

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-beta (TGF-beta) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-beta type III receptor (TbetaRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-beta and inhibin receptor. Here we demonstrate that TbetaRIII functions as a BMP cell surface receptor. TbetaRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-beta. TbetaRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TbetaRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TbetaRIII function, we demonstrate that BMP-2 specifically stimulates TbetaRIII-mediated epithelial to mesenchymal cell transformation. The ability of TbetaRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-beta signaling suggests a broader role for TbetaRIII in orchestrating TGF-beta superfamily signaling.     

Syndecan-2 regulates transforming growth factor-beta signaling

Transforming growth factor-beta (TGF-beta) has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. We report here that a transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increased in the renal interstitium in diabetes and regulated TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promoted TGF-beta binding and S2 core protein ectodomain directly bound TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increased the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 resulted in a decrease. Syndecan-2 specifically co-immunoprecipitated with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis.     

Membrane binding characteristics of two forms of transforming growth factor-beta

Cartilage-inducing factors A and B (CIF-A and CIF-B) from bovine bone have recently been identified as transforming growth factor-beta (TGF-beta) (Seyedin, S.M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem., 261, 5693-5695) and a unique protein homologous to TGF-beta (Seyedin S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., and Graycar, J. (1987) J. Biol. Chem., 262, 1946-1949), respectively. Although the biological activities of TGF-beta and CIF-B are similar, the divergence of CIF-B from the highly conserved amino acid sequence of TGF-beta prompted an investigation of its receptor binding properties. Three classes of cell surface binding components were identified. Class A has exclusive affinity for TGF-beta; class B has greater affinity for CIF-B; and class C has equal affinity for both proteins. A high molecular weight component, the predominant binding species, was further characterized and shown to consist of two components that are either class B or class C. The differential binding properties of TGF-beta and CIF-B to cell surface components suggest that there are biological activities unique to each of the proteins.     

Ectodomain cleavage and shedding of the type III transforming growth factor-beta receptor in lung membranes effect of temperature, ligand binding and membrane solubilization.

Previous studies from our laboratory [Philip, A. & O'Connor-McCourt, M. D. (1991) J. Biol. Chem. 266, 22290--22296] have shown that the lung exhibited the highest uptake of circulating [125I]-transforming growth factor-beta1 (TGF-beta1) on a per gram basis. This observation, together with the lack of information on TGF-beta receptor expression in the lung, prompted us to attempt to characterize TGF-beta receptors in this tissue. In the present report we show that the type III TGF-beta receptor is the most abundant TGF-beta binding protein in rat lung membranes and that it exhibits a 10-fold higher affinity for TGF-beta2 than for TGF-beta1. We observed that the majority of the type III receptor population in lung membranes is cleaved at a site in the central portion of the ectodomain, the resulting two fragments (95 kDa and 58 kDa) being held together by disulfide bonds. Furthermore, we demonstrate that a soluble form of the ectodomain of the type III receptor is shed from rat lung membranes in an efficient manner, with protease cleavage occurring at a site close to the transmembrane domain. This shedding is controllable by temperature, thus providing a system to study the mechanism of ectodomain release. Using this system, we show that the shedding is inhibited by prior ligand binding and by membrane solubilization. The identification of a membrane preparation which exhibits controllable and quantitative release of the type III receptor ectodomain provides a unique cell-free system for further studies of the mechanism of shedding of the type III TGF-beta receptor ectodomain.     

Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells.

Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.     

Purification of the transforming growth factor-beta (TGF-beta) binding proteoglycan betaglycan.

We report the purification of betaglycan, a low-abundance membrane proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). Betaglycan solubilized from rat embryo membrane preparations was purified to near-homogeneity by sequential chromatography through DEAE-Trisacryl, wheat germ lectin-Sepharose, and TGF-beta 1-agarose. Purified betaglycan has properties similar to betaglycan affinity-labeled in intact cells: it binds TGF-beta 1 and TGF-beta 2 with KD approximately 0.2 nM, contains heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains and N-linked glycans attached to a 110-kDa core protein, and can spontaneously associate with phosphatidylcholine liposomes. The betaglycan core obtained by enzymatic removal of the GAG chains has high affinity for TGF-beta and associates with artificial liposomes, indicating that the core protein binds TGF-beta and anchors to membranes independently of the GAG chains present on the native protein or of any ancillary protein.     

Transforming growth factor-beta (TGF-beta) receptor proteoglycan. Cell surface expression and ligand binding in the absence of glycosaminoglycan chains

The type III transforming growth factor-beta (TGF-beta) receptor is a cell surface chondroitin/heparan sulfate proteoglycan that binds various forms of TGF-beta with high affinity and specificity. Here, we have used a genetic approach to determine the requirement for glycosaminoglycan (GAG) chains for normal TGF-beta receptor expression and the role that the receptor proteoglycan core and GAG chains play in TGF-beta binding. Chinese hamster ovary (CHO) cells defective in GAG synthesis express on their surface 110-130-kDa type III receptor proteoglycan cores that can bind normal levels of TGF-beta compared to wild type CHO cells. The affinity of the receptor core for TGF-beta 1 and TGF-beta 2 in CHO cell mutants is similar to that of the TGF-beta receptor proteoglycan forms present in wild type CHO cells or in CHO cell mutants that have been allowed to bypass their metabolic defect and express the wild type proteoglycan phenotype. The binding properties of TGF-beta receptor types I and II in CHO cells and the growth-inhibitory response of CHO cell mutants to TGF-beta are not impaired by the absence of GAG chains in the type III receptor. These results show that the GAG chains are dispensable for type III receptor expression on the cell surface, binding of TGF-beta to the receptor core, and growth inhibitory response of the cells to TGF-beta. The evidence also suggests that the type III receptor may act as a multifunctional proteoglycan able to bind TGF-beta via the receptor core while performing another as yet unidentified function(s) via the GAG chains.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.     

Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.

Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta 1 and TGF-beta 2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta 1 and TGF-beta 2. The Kd values obtained from Scatchard analyses were approximately 65 pM for 125I-TGF-beta 1 and approximately 40 pM for 125I-TGF-beta 2, with 70,000 and 85,000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta 1 and TGF-beta 2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 125I-TGF-beta 1 and 125I-TGF-beta 2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta 1 than for -beta 2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta 2 than for -beta 1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta 2 than for -beta 1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.     

Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells.

Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.     

Identification of distinct inhibin and transforming growth factor beta-binding sites on betaglycan: functional separation of betaglycan co-receptor actions

Betaglycan is a co-receptor that mediates signaling by transforming growth factor beta (TGFbeta) superfamily members, including the distinct and often opposed actions of TGFbetas and inhibins. Loss of betaglycan expression, or abrogation of betaglycan function, is implicated in several human and animal diseases, although both betaglycan actions and the ligands involved in these disease states remain unclear. Here we identify a domain spanning amino acids 591-700 of the betaglycan extracellular domain as the only inhibin-binding region in betaglycan. This binding site is within the betaglycan ZP domain, but inhibin binding is not integral to the ZP motif of other proteins. We show that the inhibin and TGFbeta-binding residues of this domain overlap and identify individual amino acids essential for binding of each ligand. Mutation of Val614 to Tyr abolishes both inhibin and TGFbeta binding to this domain. Full-length betaglycan V614Y, and other mutations, retain TGFbeta binding activity via a distinct site, but are unable to bind inhibin-A. These betaglycan mutants fail to mediate inhibin antagonism of activin signaling but can present TGFbeta to TbetaRII. Separating the co-receptor actions of betaglycan toward inhibin and TGFbeta will allow the clarification of the role of betaglycan in disease states such as renal cell carcinoma and endometrial adenocarcinoma.     

Structural determinants of binding and specificity in transforming growth factor-receptor interactions.

Transforming growth factor (TGF-beta) protein families are cytokines that occur as a large number of homologous proteins. Three major subgroups of these proteins with marked specificities for their receptors have been found-TGF-beta, activin/inhibin, and bone morphogenic protein. Although structural information is available for some members of the TGF-beta family of ligands and receptors, very little is known about the way these growth factors interact with the extracellular domains of their cell surface receptors, especially the type II receptor. In addition, the elements that are the determinants of binding and specificity of the ligands are poorly understood. The structure of the extracellular domain of the receptor is a three-finger fold similar to some toxin structures. Amino acid exchanges between multiply aligned homologous sequences of type II receptors point to a residue at the surface, specifically finger 1, as the determinant of ligand specificity and complex formation. The "knuckle" epitope of ligands was predicted to be the surface that interacts with the type II receptor. The residues on strands beta2, beta3, beta7, beta8 and the loop region joining beta2 and beta3 and joining beta7 and beta8 of the ligands were identified as determinants of binding and specificity. These results are supported by studies on the docking of the type II receptor to the ligand dimer-type I receptor complex.     

Growth factor-binding sequence in human alpha2-macroglobulin targets the receptor-binding site in transforming growth factor-beta

alpha(2)-Macroglobulin (alpha(2)M) binds transforming growth factor-beta1 (TGF-beta1) and TGF-beta2, forcing these growth factors into a state of latency. The mechanism by which this occurs remains unclear. In this paper, we demonstrate that peptides, derived from the structure of human alpha(2)M (amino acids 714-729), bind directly to TGF-beta1 and block the binding of TGF-beta1 to the type I and II TGF-beta receptors. The alpha(2)M-derived peptides are notable for hydrophobic tripeptide sequences (WIW or VVV) and acidic residues (Glu(714) and Asp(719) in the mature alpha(2)M subunit), which may function analogously to the structural elements that mediate TGF-beta-binding in the type II receptor. Mutating Glu(714) and Asp(719) in the alpha(2)M-peptide-GST fusion protein, FP3, which contains the putative growth factor-binding site, significantly decreased the binding affinity of FP3 for TGF-beta1. The alpha(2)M-derived peptides, which bind TGF-beta1, inhibited the interaction of TGF-beta1 with its receptors in fetal bovine heart endothelial cells. The same peptides also inhibited the activity of TGF-beta1 in endothelial cell proliferation assays. These results demonstrate that alpha(2)M-derived peptides target the receptor-binding sequence in TGF-beta.     

Distribution and modulation of the cellular receptor for transforming growth factor-beta

Scatchard analyses of the binding of transforming growth factor-beta (TGF-beta) to a wide variety of different cell types in culture revealed the universal presence of high affinity (Kd = 1-60 pM) receptors for TGF-beta on every cell type assayed, indicating a wide potential target range for TGF-beta action. There was a strong (r = +0.85) inverse relationship between the receptor affinity and the number of receptors expressed per cell, such that at low TGF-beta concentrations, essentially all cells bound a similar number of TGF-beta molecules per cell. The binding of TGF-beta to various cell types was not altered by many agents that affect the cellular response to TGF-beta, suggesting that modulation of TGF-beta binding to its receptor may not be a primary control mechanism in TGF-beta action. Similarly, in vitro transformation resulted in only relatively small changes in the cellular binding of TGF-beta, and for those cell types that exhibited ligand-induced down-regulation of the receptor, down-regulation was not extensive. Thus the strong conservation of binding observed between cell types is also seen within a given cell type under a variety of conditions, and receptor expression appears to be essentially constitutive. Finally, the biologically inactive form of TGF-beta, which constitutes greater than 98% of autocrine TGF-beta secreted by all of the twelve different cell types assayed, was shown to be unable to bind to the receptor without prior activation in vitro. It is proposed that this may prevent premature interaction of autocrine ligand and receptor in the Golgi apparatus.     

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.     

Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms.

Characterization of the three mammalian transforming growth factor-beta (TGF-beta) isoforms, TGF-beta 1, -beta 2, and -beta 3, indicates that TGF-beta 3 is somewhat more potent (ED50 = 0.5 pM versus 2 pM) than TGF-beta 1 and TGF-beta 2 as a growth inhibitor of the Mv1Lu mink lung epithelial cell line. In the fetal bovine heart endothelial (FBHE) cell line, however, TGF-beta 1 and -beta 3 are at least 50-fold more potent than TGF-beta 2 which is a very weak growth inhibitor (ED50 greater than or equal to 0.5 nM). Thus, as growth inhibitors, TGF-beta 1 and -beta 3 resemble each other more than TGF-beta 2. The presence of serum alpha 2-macroglobulin in the FBHE cell assays decreases the biological potency of TGF-beta s, in particular TGF-beta 2. This effect of alpha 2-macroglobulin, however, is not sufficient to explain the low responsiveness of FBHE cells to TGF-beta 2. Evaluation of the role of TGF-beta receptors as determinants of cell-specific responsiveness to TGF-beta isoforms indicates that TGF-beta 1, -beta 2, and -beta 3 have similar affinity for the membrane proteoglycan, betaglycan. They differ, however, in their ability to bind to receptor types I and II which are implicated in TGF-beta signal transduction. TGF-beta 1 is similar, albeit not identical, to TGF-beta 3 and much more potent than TGF-beta 2 as a competitor for binding to the overall population of receptors I and II in all cell lines tested. A subset of receptors I and II has been identified in Mv1Lu cells which has high affinity for TGF-beta 2 (KD approximately 10 pM) and binds this factor at concentrations that are biologically active in Mv1Lu cells. This receptor subset could not be detected in FBHE cells, suggesting that cell-specific differences in the level of high affinity of TGF-beta 2 receptors may lead to cell-specific differences in responsiveness to this isoform. Thus, despite their structural and biological similarities, TGF-beta 1, -beta 2, and -beta 3 diverge in their ability to bind to receptors in a manner that correlates with their potency as growth inhibitors.     

Assignment of transforming growth factor beta1 and beta3 and a third new ligand to the type I receptor ALK-1

Germ line mutations in one of two distinct genes, endoglin or ALK-1, cause hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disorder of localized angiodysplasia. Both genes encode endothelial cell receptors for the transforming growth factor beta (TGF-beta) ligand superfamily. Endoglin has homology to the type III receptor, betaglycan, although its exact role in TGF-beta signaling is unclear. Activin receptor-like kinase 1 (ALK-1) has homology to the type I receptor family, but its ligand and corresponding type II receptor are unknown. In order to identify the ligand and type II receptor for ALK-1 and to investigate the role of endoglin in ALK-1 signaling, we devised a chimeric receptor signaling assay by exchanging the kinase domain of ALK-1 with either the TGF-beta type I receptor or the activin type IB receptor, both of which can activate an inducible PAI-1 promoter. We show that TGF-beta1 and TGF-beta3, as well as a third unknown ligand present in serum, can activate chimeric ALK-1. HHT-associated missense mutations in the ALK-1 extracellular domain abrogate signaling. The ALK-1/ligand interaction is mediated by the type II TGF-beta receptor for TGF-beta and most likely through the activin type II or type IIB receptors for the serum ligand. Endoglin is a bifunctional receptor partner since it can bind to ALK-1 as well as to type I TGF-beta receptor. These data suggest that HHT pathogenesis involves disruption of a complex network of positive and negative angiogenic factors, involving TGF-beta, a new unknown ligand, and their corresponding receptors.