Transforming growth factors-beta (TGFs-β) are representative of a superfamily whose members were first identified as regulators of morphogenesis and differentiation, and subsequently found to be structurally related. Other members of the family include the activins and inhibins, BMPs, MIS, the DPP-C gene product and Vg-1. When assayed by affinity-labelling techniques, TGFs-β bind to three distinct cell surface proteins which are present on most cells. These proteins are all of relatively low abundance but bind TGFs-β with affinities consistent with the biological potency of the factors. The Type I and Type II binding proteins are glycoproteins with estimated molecular weights of 53 and 73×103Mr, respectively. They both bind TGFS-β significantly better than TGFS-β. The Type I receptor has been identified as the receptor which mediates many of the responses of TGFs-β, based on somatic cell genetic studies of epithelial cell mutants unresponsive to TGFs-β. Betaglycan is the third binding protein present on many, but not all, cell types and is a large proteoglycan (∼280×103Mr) with 100–120 ×103Mr core proteins. A soluble form of this molecule is present in conditioned media of many cell lines and may be derived from the cell surface-associated molecule by cleavage of a small membrane anchor. Betaglycan binds TGFS-β and TGFS-β with similar affinity and this binding is to the core proteins, not the glycosaminoglycan side chains. This molecule may have a function in the localization and delivery or the clearance of activated TGFs-β. The molecular basis of TGFS-β signalling is still largely unknown, but it is possible that one or more of these cell surface molecules signals via a novel mechanism, as the TGFs-β are biologically quite distinct from other factors that act via well-characterized signalling systems.

Transforming growth factors-beta (TGFs-β) are a family of polypeptide hormones which probably act over relatively confined physiological spaces in vivo. They are concentrated in platelets and released in an inactive form upon platelet degranulation (Pircher et al. 1986). Presumably they are activated in a local area by proteases or other specific activating conditions (Lyons et al. 1988). In addition, in situ hybridization and immunohistochemical studies have demonstrated that the factors are synthesized in many in vivo sites (Heine et al. 1987; Lehnert and Adhurst, 1988; Pelton et al. 1989). Our laboratory has attempted to identify and characterize those cell surface proteins with which TGFs-β can interact and which presumably mediate the biological signalling of the factor into the cell.

This is a somewhat complicated task for a number of reasons. As indicated above, TGF-/ÍJ is a family of five closely related factors (TGFS-β to TGFS-β5) within a superfamily of several other factors implicated in essential elements of development. These other factors include the decapentaplegic gene product of Drosophila, the Vg-1 gene product and mesoderm inducing factor in Xenopus, Mullerian Inhibiting Substance, the Bone Morphogenic Proteins, and the activins and inhibins (Massagué, 1990). This suggests that there may be a family of related receptors with differential affinities for different members of the superfamily. Another complicating element is that the known effects of TGFS-β are varied and in some instances diametrically opposed in different cell systems. It is intriguing that TGFS-β has a potent growth inhibitory effect on some epithelial cell lines, but it is also mitogenic in some culture systems, such as osteoblasts (Centrella et al. 1987) and some fibroblast lines (Leof et al. 1986). It is an inhibitor of differentiation in myogenic and adipogénie model systems in vitro (Massagué et al. 1986; Ignotz and Massagué, 1985), but stimulates differentiation of chondroblasts (Seyedin et al. 1985). And finally, experimentally, we have found that there is a relatively low level of specific cell-surface TGFS-β binding and that this activity is distributed among three distinct cell surface binding proteins.

In the course of investigating the activities of TGFS-β over the last six years, our laboratory has characterized the TGFS-β binding patterns of over one hundred different cell lines, primary cells and tissues (Fig. IA) (Massagué, 1990). It is striking that the general pattern is so similar in most cells analyzed. Briefly, the experimental approach is to bind iodinated TGFS-β to cells, crosslink the ligand to cell surface proteins to which it is associated with a bifunctional crosslinking reagent such as disuccinimidyl suberate, solubilize the cell membranes with detergent and separate the labeled proteins on SDS-polyacrylamide gels with subsequent autoradiography. This method commonly identifies three proteins labeled specifically (Fig. IB). The Type I protein is an affinity-labeled species of approximately 65 × 103Mr. The Type II species is approximately 85 × 103Mr, and the Type III species, termed betaglycan, is a broad band typically centered around 280 ×103Mr. All of these apparent molecular weights include an associated monomer of TGF-3 of 12.5 X103Mr, so the presumed size of the binding proteins is correspondingly 12.5 X103Mr smaller than the apparent molecular weight on SDS gels. Each of these proteins have high affinities for TGFs-/?, with Kd values in the range of 5-500 pM. They bind TGFS-β, TGFS-β and TGFS-β3, but not more distantly related members of the TGF-β superfamily. The Type I binding protein is ubiquitous, with every cell type that responds to TGFs-β having the Type I protein. There are several examples of hematopoietic progenitor cell lines which respond to TGFs-βl, TGF-β 1.2 and TGFS-β differentially with an order of potencies that parallels the order of affinities of the factors for the Type I protein, the only TGFS-β binding protein detectable on these cell lines (Ohta et al. 1987; Cheifetz et al. 1988). Human and bovine vascular endothelial cells possess both the Type I and Type II proteins and respond differentially to TGF-β 1 or TGFS-β. In addition, L6E9 myoblasts also have only the Type I and Type II proteins, yet respond equivalently to TGFS-β1 and TGF-β 2. The most common pattern of TGF-β cell surface binding proteins is the presence of the Type I protein, Type II protein and betaglycan together. These data are suggestive, though certainly not definitive, that the Type I protein is sufficient to confer TGFS-β responsiveness to cells. However, the other TGFS-β binding proteins may modulate the availability or activities of the TGFs-/? or mediate particular responses in particular cell types.

More direct evidence supporting specific roles of these proteins in TGFS-β signalling has come from studies of somatic cell mutants in the MvlLu epithelial cell line which are non-responsive to TGFs-β The parental line is exquisitely sensitive to TGFS-β and is virtually completely growth inhibited by 5pM TGFs-β1; it also responds to TGFS-β with increased expression of extracellular matrix components such as fibronectin and plasminogen activator inhibitor (PAI-1). In addition, the parental line has all three types of putative TGFS-β receptor proteins. Ethyl methane sulfonate (EMS)-mutagenized MvlLu cells were selected which were able to grow in the presence of 100 pM TGFS-β. The mutant clones were completely resistant to the growth inhibitory effects of TGF-β1 and TGFS-β2 and have also lost all other responses to TGFs-β assayed for. In addition, several of the clones were defective in expression of the Type I binding protein (Fig. 2). Clones of MvlLu cells obtained from a non-mutagenized population and analyzed for TGFS-β binding proteins always have all three binding proteins present. On this basis, we suggest that the Type I protein is the receptor which mediates epithelial cell responsiveness to TGFS-β (Boyd and Massagué, 1989). In addition to these receptor defective mutants, termed R mutants, mutants were isolated which have normal binding protein patterns, yet are deficient in all TGFS-β responses. These are called signalling, or S, mutants. When complementation analysis was performed, none of the mutant hybrids were complementary, suggesting that all the R and S mutants isolated are mutants in the same gene, presumably the TGFS-β receptor gene. It is also apparent from this analysis that all the mutants isolated were recessive mutations. All mutant-parental fusions were fully responsive to TGFS-β with normal receptor profiles.

Fig. 1.

Distribution of TGF-β receptors and binding proteoglycans in various cell types. A. Summary of TGFS-β receptors in various cell types. All cell lines were screened for the presence of TGFS-β receptors (I, II, III) using the affinity-labelling protocol outlined in the text. The presence of a receptor type in any cell line is signified with an open circle (O). The majority of cell lines screened express all three protein species, some lines express only the Type II and Type I receptors, a few lines express only the Type I receptor. No cell line which responds to TGFS-β with established assays lacks the Type I receptor. Cell lines which lack any receptor type are signified with a closed circle (•) under the column relating to that receptor type. B. Receptor profiles from representative cell lines. Mouse BALB/c-3T3 cells, 3T3-L1 cells, rat NRK cells, chick embryo fibroblasts (CEF) and mink lung epithelial cells (MvlLu) were screened by affinity labelling with 50 pM 125I-TGFs-β in the presence of no (a), 50 (b), 100 (c), 200 Cd), 700 (e), or 3000 (f) pM native TGFS-β These experiments show the affinity of these proteins for TGFS-β and the general nature of the affinity labeled species (Cheifetz et al. 1986).

Fig. 1.

Distribution of TGF-β receptors and binding proteoglycans in various cell types. A. Summary of TGFS-β receptors in various cell types. All cell lines were screened for the presence of TGFS-β receptors (I, II, III) using the affinity-labelling protocol outlined in the text. The presence of a receptor type in any cell line is signified with an open circle (O). The majority of cell lines screened express all three protein species, some lines express only the Type II and Type I receptors, a few lines express only the Type I receptor. No cell line which responds to TGFS-β with established assays lacks the Type I receptor. Cell lines which lack any receptor type are signified with a closed circle (•) under the column relating to that receptor type. B. Receptor profiles from representative cell lines. Mouse BALB/c-3T3 cells, 3T3-L1 cells, rat NRK cells, chick embryo fibroblasts (CEF) and mink lung epithelial cells (MvlLu) were screened by affinity labelling with 50 pM 125I-TGFs-β in the presence of no (a), 50 (b), 100 (c), 200 Cd), 700 (e), or 3000 (f) pM native TGFS-β These experiments show the affinity of these proteins for TGFS-β and the general nature of the affinity labeled species (Cheifetz et al. 1986).

Fig. 2.

TGFS-β receptor profiles of TGFS-β resistant mutants of mink lung epithelial cells. Mutants of MvlLu cells which are not responsive to TGF-β with respect to growth inhibition were generated as described in the text. Representative clones of several mutant phenotypes were affinity labeled with 100 pM 125I-TGFS-β. Three distinct types of mutants have been isolated as characterized by TGFS-β binding profiles. When compared with the parental MvlLu cells, it is apparent that two of the mutant types have distinct deficits in TGF-β binding. The R-mutants are lacking the Type I receptor. In addition, the DR-mutants are lacking both the Type I and the Type II receptors. In contrast, the S-mutants have an apparently normal receptor profile.

Fig. 2.

TGFS-β receptor profiles of TGFS-β resistant mutants of mink lung epithelial cells. Mutants of MvlLu cells which are not responsive to TGF-β with respect to growth inhibition were generated as described in the text. Representative clones of several mutant phenotypes were affinity labeled with 100 pM 125I-TGFS-β. Three distinct types of mutants have been isolated as characterized by TGFS-β binding profiles. When compared with the parental MvlLu cells, it is apparent that two of the mutant types have distinct deficits in TGF-β binding. The R-mutants are lacking the Type I receptor. In addition, the DR-mutants are lacking both the Type I and the Type II receptors. In contrast, the S-mutants have an apparently normal receptor profile.

These studies have been extended and selection of MvlLu mutants has been performed with lower doses (25 pM) of TGFS-β. In addition to the R and S mutant classes, a third class of mutants defective in the expression of both the Type I and Type II proteins has been isolated (DR mutants) (M-. Laiho and others, unpublished observations). Complementation analysis of these mutants is not complete, so the genetic basis of these mutations is still unknown. Several explanations for this phenotype are possible. DR mutants may be the result of mutations of two distinct loci encoding for the Type I and Type II proteins, although the frequency of isolation of these mutants argues strongly against this. Another possibility is that the two genes are linked and a single large deletion can knock out expression of both genes. A third possibility is that the two proteins are associated intracellularly and some mutations in one or the other of the proteins prevent the expression of both. These possibilities are the subject of active investigation in the laboratory. We are also attempting to complement these mutants by cDNA and genomic transfection to clone the genes responsible for these mutations.

While mutant analysis has revealed the functional significance of the Type I and Type II proteins, the fact remains that in the majority of cells we have screened, the major component of cell surface TGFS-β binding activity is associated with a large proteoglycan species with apparent molecular weight of 200–400 ×103Mr. This molecule is a complex mixed chondroitin/heparan sulfate proteoglycan (Segarini and Seyedin, 1988; Cheifetz et al. 1988) with multiple deglycosylated core proteins of 100–120×103Mr. Unlike other growth factors which associate with proteoglycans via relatively non-specific binding to the glycosaminoglycan chains, TGFs-β bind to betaglycan via the core proteins. The core proteins are expressed and bind TGFs-β in metabolic mutants which do not synthesize glycosaminoglycan side chains (Cheifetz and Massagué, 1989). There is also a soluble form of betaglycan, found in the media of tissue culture cells which express betaglycan, which is capable of binding TGFs-β (Andres et al. 1989). This form is slightly smaller than the membrane-associated form and is incapable of being incorporated into phospholipid vesicles. The soluble form of betaglycan also associates with the extracellular matrix. It is intriguing that TGF-β, which is a well-characterized modulator of the extracellular matrix, binds specifically to a TGFS-β binding proteoglycan which associates with the extracellular matrix and is the major species of TGFS-β binding activity associated with cells. This may be a mechanism which can be modulated by TGFs-β, by which TGFs-β are sequestered in the intercellular space.

The definitive identification of the TGFS-β receptor awaits cloning of the gene and reconstitution of a TGFS-β-responsive phenotype to TGFS-β-receptor mutants. However, the features of TGFS-β-binding outlined above suggests the following model. The identification of cell lines which possess only the Type I protein and the common defect in Type I binding activity in TGFS-β-response mutants suggests that the Type I protein is the signalling receptor for TGFS-β. The identification of TGFS-β-response mutants defective in both the Type I and Type II proteins suggests that the Type II protein may be part of a higher order complex with the Type I protein that associates and is a functional entity in some cell types. The dimeric structures of TGFs-β are reminiscent of the structure of other dimeric growth factors which bind to dimeric receptors. Finally, betaglycan may be an extracellular storage site or mechanism of inactivation of the ligand. The physiology of the TGFs-β suggests that specificity of action may come about as a result of acute regulation of availability of the active ligand. The fact that betaglycan is a proteoglycan and is present in membrane associated form as well as soluble and matrix associated forms suggests the possibility of acute regulation of the molecule, which might make it well suited to a role in clearance or storage of the ligands.

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