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.     

FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization

Transforming growth factor-beta (TGF-beta) family polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF-beta type I and II receptors. Although interaction screens have shown that the immunophilin FKBP12 interacts with TGF-beta type I receptors, the role of FKBP12 in TGF-beta receptor action is presently unclear. Using a chimeric TGF-beta receptor system, we have shown a specific enhancement of internalization when FKBP12 binding to the type I receptor was prevented with rapamycin. Moreover, although earlier studies demonstrated that type II receptor kinase activity was required for optimal internalization in mesenchymal cells, we found that rapamycin functioned downstream of the type II receptor kinase. Thus, rather than modulating TGF-beta signaling, our data suggest a novel role for FKBP12 as a negative regulator of TGF-beta receptor endocytosis.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

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.     

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.     

In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type I receptor to bind TGF-beta2

Transforming growth factor beta (TGF-beta) ligands exert their biological effects through type II (TbetaRII) and type I receptors (TbetaRI). Unlike TGF-beta1 and -beta3, TGF-beta2 appears to require the co-receptor betaglycan (type III receptor, TbetaRIII) for high affinity binding and signaling. Recently, the TbetaRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-beta2 null mouse, implying the existence of TbetaRIII independent mechanisms for TGF-beta2 signaling. Because a variant of the type II receptor, the type II-B receptor (TbetaRII-B), has been suggested to mediate TGF-beta2 signaling in the absence of TbetaRIII, we directly tested the ability of TbetaRII-B to bind TGF-beta2. Here we show that the soluble extracellular domain of the type II-B receptor (sTbetaRII-B.Fc) bound TGF-beta1 and TGF-beta3 with high affinity (K(d) values = 31.7 +/- 22.8 and 74.6 +/- 15.8 pm, respectively), but TGF-beta2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTbetaRII.Fc). However, sTbetaRII.Fc or sTbetaRII-B.Fc in combination with soluble type I receptor (sTbetaRI.Fc) formed a high affinity complex that bound TGF-beta2, and this complex inhibited TGF-beta2 in a biological inhibition assay. These results show that TGF-beta2 has the potential to signal in the absence of TbetaRIII when sufficient TGF-beta2, TbetaRI, and TbetaRII or TbetaRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-beta receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures.     

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.     

Purification of a new type high molecular weight receptor (type V receptor) of transforming growth factor beta (TGF-beta) from bovine liver. Identification of the type V TGF-beta receptor in cultured cells.

A 400-kDa transforming growth factor beta (TGF-beta) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B affinity chromatography, DEAE-cellulose anion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. This procedure yielded approximately 20 micrograms of the receptor from 1 kg of bovine liver. During purification, the 400-kDa TGF-beta receptor was detected by a cross-linking assay in which the TGF-beta receptor-125I-TGF-beta complex was cross-linked by disuccinimidyl suberate, a bifunctional reagent, and analyzed by 5.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. This novel 400-kDa TGF-beta receptor was also identified on cultured cells including cells reported to lack the type III receptor. The 400-kDa TGF-beta receptor, a nonproteoglycan glycoprotein, appears to be distinct from TGF-beta receptors (types I, II, III, and IV) previously identified on cultured cells and is designated as the type V receptor. The 400-kDa TGF-beta receptor as well as type I, II, and III receptors underwent internalization upon 125I-TGF-beta binding in mink lung epithelial cells.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ursolic acid, an antagonist for transforming growth factor (TGF)-beta1

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which is involved in extracellular matrix modulation, has a major role in the pathogenesis and progression of fibrotic diseases. We now report the effects of ursolic acid on TGF-beta1 receptor binding and TGF-beta1-induced cellular functions in vitro. Ursolic acid inhibited [(125)I]-TGF-beta1 receptor binding to Balb/c 3T3 mouse fibroblasts with an IC(50) value of 6.9+/-0.8 microM. Ursolic acid dose-dependently recovered reduced proliferation of Minc Mv1Lu cells in the presence of 5 nM of TGF-beta1 and attenuated TGF-beta1-induced collagen synthesis and production in human fibroblasts. Molecular dynamics simulations suggest that ursolic acid may interact with the hydrophobic region of the dimeric interface and thereby inhibit the binding of TGF-beta1 to its receptor. All these findings taken together show that ursolic acid functions as an antagonist for TGF-beta1. This is the first report to show that a small molecule can inhibit TGF-beta1 receptor binding and influence functions of TGF-beta1.     

The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors

A new homodimer form of transforming growth factor-beta (TGF-beta), TGF-beta 2, has been identified in porcine blood platelets. TGF-beta 2 is homologous to ordinary TGF-beta (TGF-beta 1), which is also present in platelets. TGF-beta 1.2, a heterodimer containing one TGF-beta 1 chain and one TGF-beta 2 chain, has also been isolated. TGF-beta 1 and TGF-beta 2 interact differently with a family of receptors in target cells. A 280 kd receptor displays high affinity for both TGF-beta 1 and TGF-beta 2. Occupancy of this receptor by TGF-beta 1 or TGF-beta 2 correlates with the ability of these TGF-beta s to inhibit cell proliferation. In contrast, 65 kd and 85 kd receptors have high affinity for TGF-beta 1 but lower affinity for TGF-beta 2. The existence of distinct forms of TGF-beta that interact differently with a family of TGF-beta receptors could provide flexibility to the regulation of tissue growth and differentiation by the TGF-beta system.     

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.     

Modulation of TGF-beta type 1 receptor: flow cytometric detection with biotinylated TGF-beta

Transforming growth factor beta type 1 (TGF-beta 1) was reacted with NHS-biotin to yield a derivative of TGF-beta 1 which was biotinylated on lysine residues. The biotinylated form of TGF-beta 1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as active as the starting material. The use of FITC-avidin in conjunction with flow cytometry demonstrated that the binding of biotinylated TGF-beta 1 to its receptor is saturable, competable, and specific. A 100-fold molar excess of underivatized TGF-beta 1 gave 85% inhibition of binding of the biotinylated peptide to the mink lung cell line CCL-64, while TGF-beta 2 showed no inhibition of binding, nor did insulin, calcitonin, or TGF-alpha. Both CCL-64 cells and human umbilical vein endothelial cells showed a density-dependent down-regulation of receptor expression in culture. Several factors were examined that might mediate this effect. The down-regulation was shown not to be due to the secretion of an active form of TGF-beta 1. The extracellular matrix from high-density cells did not decrease expression of the receptor. Fibronectin, collagen, and gelatin were also unable to signal changes in receptor expression, even though in other systems such matrix components can regulate the responsiveness of cells to TGF-beta 1. Lastly, staining simultaneously for DNA content and TGF-beta 1 receptor expression showed that there was no correlation between cell cycle and receptor levels.     

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.     

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.     

Concomitant loss of transforming growth factor (TGF)-beta receptor types I and II in TGF-beta-resistant cell mutants implicates both receptor types in signal transduction

A panel of 71 chemically mutagenized Mv1Lu mink lung epithelial cell clones were selected based on their resistance to the growth inhibitory action of transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2. Characterization of TGF-beta receptors in these mutants indicates that the TGF-beta-binding membrane proteoglycan, betaglycan, is apparently normal in all of them. However, 14 of the mutant clones are defective in TGF-beta receptor type I, and 22 clones are simultaneously defective in receptor types I and II. The clones with type I receptor defects fall into two distinct phenotypes, called R and LR. The R phenotype is characterized by the lack of detectable type I receptors, and has been previously described (Boyd, F. T., and Massagué, J. (1989) J. Biol. Chem. 264, 2272-2278). LR mutants are characterized by expression of low levels of type I receptor and are, like the R mutants, completely resistant to growth inhibition by TGF-beta 1 or -beta 2. Mutant clones that are simultaneously defective in receptor types I and II fall into three distinct phenotypes. These included DRa mutants which are characterized by lack of detectable receptor types I and II, DRb mutants which are characterized by low expression of both receptor types and an anomalously fast electrophoretic mobility of the type II receptor protein. All mutants that have a low level of type II receptor are also defective in type I receptor. In addition to the loss of growth inhibitory response, the receptor-defective mutants described here have lost all other responses to TGF-beta 1 and -beta 2 known to occur in parental Mv1Lu cells. The defects present in these mutant clones are not encountered in clones isolated from nonmutagenized parental Mv1Lu cells or in mutagenized cells that had not been exposed to selection with TGF-beta. The results implicate TGF-beta receptor types I and II in the mediation of a common set of cellular responses to TGF-beta. Furthermore, the high relative frequency of isolation of DR mutants raises the possibility that receptor types I and II interact as part of a common signaling TGF-beta receptor complex.     

Transforming growth factor beta (TGF-beta) type V receptor has a TGF-beta-stimulated serine/threonine-specific autophosphorylation activity.

The transforming growth factor beta (TGF-beta) type V receptor, a newly identified high molecular weight TGF-beta receptor (M(r) approximately 400,000) has been purified from bovine liver plasma membranes (O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 266, 8583-8589). The purified TGF-beta type V receptor underwent autophosphorylation at serine residues when incubated with [gamma-32P]ATP in the presence of 0.1% beta-mercaptoethanol and 2.5 mM MnCl2. This phosphorylation was stimulated by preincubation with TGF-beta. The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. The TGF-beta type V receptor could be affinity-labeled with 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine. Polylysine appeared to stimulate the autophosphorylation of the TGF-beta type receptor in the presence of [gamma-32P]ATP and the incorporation of 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine into the TGF-beta type V receptor. The amino acid sequence analysis of the peptide fragments produced by cyanogen bromide cleavage of the purified TGF-beta type V receptor revealed that a peptide, namely CNBr-19, contained an amino acid sequence which shows homology to the putative ATP binding site of the receptors for activin, the Caenorhabditis elegans daf-1 gene product, and TGF-beta type II receptor (Lin, H. Y., Wang, Y.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785). These results suggest that the TGF-beta type V receptor is a Ser/Thr-specific protein kinase and belongs to the new class of membrane receptors associated with a Ser/Thr-specific protein kinase activity.