Expression cloning and characterization of the TGF-beta type III receptor.

The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.     

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.     

A kinase subdomain of transforming growth factor-beta (TGF-beta) type I receptor determines the TGF-beta intracellular signaling specificity.

Transforming growth factor-beta (TGF-beta) signals through a heteromeric complex of related type I and type II serine/threonine kinase receptors. In Mv1Lu cells the type I receptor TbetaRI mediates TGF-beta-induced gene expression and growth inhibition, while the closely related type I receptors Tsk7L and TSR1 are inactive in these responses. Using chimeras between TbetaRI and Tsk7L or TSR1, we have defined the structural requirements for TGF-beta signaling by TbetaRI. The extracellular/transmembrane or cytoplasmic domains of TbetaRI and Tsk7L were functionally not equivalent. The juxtamembrane domain, including the GS motif, and most regions in the kinase domain can functionally substitute for each other, but the alphaC-beta4-beta5 region from kinase subdomains III to V conferred a distinct signaling ability. Replacement of this sequence in TbetaRI by the corresponding domain of Tsk7L inactivated TGF-beta signaling, whereas its introduction into Tsk7L conferred TGF-beta signaling. The differential signaling associated with this region was narrowed down to a sequence of eight amino acids, the L45 loop, which is exposed in the three-dimensional kinase structure and diverges highly between TbetaRI and Tsk7L or TSR1. Replacement of the L45 sequence in Tsk7L with that of TbetaRI conferred TGF-beta responsiveness to the Tsk7L cytoplasmic domain in Mv1Lu cells. Thus, the L45 sequence between kinase subdomains IV and V specifies TGF-beta responsiveness of the type I receptor.     

Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC

Transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFbeta-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-beta signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFbeta-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-beta1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-beta signaling.     

Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.     

Transforming growth factor-beta (TGF-beta) binding to the extracellular domain of the type II TGF-beta receptor: receptor capture on a biosensor surface using a new coiled-coil capture system demonstrates that avidity contributes significantly to high affinity binding

Mature TGF-beta isoforms, which are covalent dimers, signal by binding to three types of cell surface receptors, the type I, II and III TGF-beta receptors. A complex composed of the TGF-beta ligand and the type I and II receptors is required for signaling. The type II receptor is responsible for recruiting TGF-beta into the heteromeric ligand/type I receptor/type II receptor complex. The purpose of this study was to test for the extent that avidity contributes to receptor affinity. Using a surface plasmon resonance (SPR)-based biosensor (the BIACORE), we captured the extracellular domain of the type II receptor (TbetaRIIED) at the biosensor surface in an oriented and stable manner by using a de novo designed coiled-coil (E/K coil) heterodimerizing system. We characterized the kinetics of binding of three TGF-beta isoforms to this immobilized TbetaRIIED. The results demonstrate that the stoichiometry of TGF-beta binding to TbetaRIIED was one dimeric ligand to two receptors. All three TGF-beta isoforms had rapid and similar association rates, but different dissociation rates, which resulted in the equilibrium dissociation constants being approximately 5pM for the TGF-beta1 and -beta3 isoforms, and 5nM for the TGF-beta2 isoform. Since these apparent affinities are at least four orders of magnitude higher than those determined when TGF-beta was immobilized, and are close to those determined for TbetaRII at the cell surface, we suggest that avidity contributes significantly to high affinity receptor binding both at the biosensor and cell surfaces. Finally, we demonstrated that the coiled-coil immobilization approach does not require the purification of the captured protein, making it an attractive tool for the rapid study of any protein-protein interaction.     

Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta ) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.     

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.     

GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-gamma1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.     

Expression cloning of TGF-beta receptors.

Using a powerful expression cloning method in COS cells, we have cloned the TGF-beta types II and III receptors. The type III TGF-beta receptor is a membrane-bound proteoglycan with a core protein of about 110 kDa. Stable expression of the type III receptor in L6 myoblasts leads to an apparent increase in the ability of the type II receptor to bind iodinated TGF-beta 1. The cloned type II receptor has a predicted protein core of about 60 kDa with a cysteine-rich extracellular domain, a single transmembrane domain, and a functional serine/threonine kinase domain that is homologous to the activin receptor and to the C. elegans protein daf-1. These results implicate serine/threonine phosphorylation as an important mechanism of TGF-beta action.     

Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.     

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.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

A PDZ protein regulates the distribution of the transmembrane semaphorin, M-SemF.

M-SemF is a membrane-associated, neurally enriched member of the semaphorin family of axon guidance signals. We considered whether the cytoplasmic domain of M-SemF might possess a signaling function and/or might control the distribution of M-SemF on the cell surface. We identify a PDZ-containing neural protein as an M-SemF cytoplasmic domain-associated protein (SEMCAP-1). SEMCAP-2 is a closely related nonneuronal protein. SEMCAP-1 has recently also been identified as GIPC, by virtue of its interaction with the RGS protein GAIP in vitro (De Vries, L., Lou, X., Zhao, G., Zheng, B., and Farquhar, M. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12340-12345). Expression studies support the notion that SEMCAP-1(GIPC) interacts with M-SemF, but not GAIP, in brain. Lung SEMCAP-2 and SEMCAP-1(GIPC) are potential partners for both GAIP and M-SemF. The protein interaction requires the single PDZ domain of SEMCAP-1(GIPC) and the carboxyl-terminal four residues of M-SemF, ESSV. While SEMCAP-1(GIPC) also interacts with SemC, it does not interact with other proteins containing a class I PDZ binding motif, nor does M-SemF interact with other class I PDZ proteins. Co-expression of SEMCAP-1(GIPC) induces the redistribution of dispersed M-SemF into detergent-resistant aggregates in HEK293 cells. Thus, SEMCAP-1(GIPC) appears to regulate the subcellular distribution of M-SemF in brain, and SEMCAPs could link M-SemF to G protein signal transduction pathways.     

Isolation and characterization of a transforming growth factor-beta Type II receptor cDNA from Xenopus laevis

Transforming Growth Factor-beta (TGF-beta) and their receptors have been characterized from many organisms. Two TGF-beta signaling receptors called Type I and II have been described for various ligands of the superfamily from organisms ranging from Drosophila to humans. In Xenopus laevis, TGF-beta2 and 5 have been reported and presumably, play important roles during early development. Several Type I and type II receptors for many ligands of the TGF-beta superfamily except TGF-beta type II receptor (TbetaIIR), have been characterized in Xenopus laevis. A chemical cross linking experiment using iodinated TGF-beta1 and -beta5, revealed four specific binding proteins on XTC cells. In order to understand the TGF-beta involvement during Xenopus development, a TGF-beta type II receptor (XTbetaIIR) has been isolated from a XTC cDNA library. XTbetaIIR was a partial cDNA lacking a portion of the signal peptide. The sequence analysis and homology comparison with the human TbetaIIR revealed 67% amino acid similarity in the extra cellular domain, 60% similarity in the transmembrane domain and 87% similarity in the cytoplasmic kinase domain, suggesting that XTbetaIIR is a putative TGF-beta type II receptor. In addition, the consensus amino acid motif for serine threonine receptor kinases was also present. Further, a dominant negative expression construct lacking the cytoplasmic kinase domain (engineered with the signal peptide from human TGF-beta type II receptor), was able to abolish TGF-beta mediated induction of a luciferase reporter plasmid, in a transient cell transfection assay. This substantiates the notion that XTbetaIIR cDNA can act as a receptor for TGF-beta. RT-PCR analysis using RNA isolated from various developmental stages of Xenopus laevis revealed expression of this gene in all the early stages of development and in the adult organs, except in stages 46/48.     

Two novel patterns of transforming growth factor beta (TGF-beta) binding to cell surface proteins are dependent upon the binding of TGF-beta 1 and indicate a mechanism of positive cooperativity.

Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF-beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex.     

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.     

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.