Perspective A System of Transforming Growth Factor-S Receptors Patricia R. Segarini Celtrix Laboratories, Palo Alto, California
The initial interaction of polypeptide growth factors with a target cell is the binding to specific receptors present on the plasma membrane. The study, definition, and characterization of these receptors is essential for understanding the mechanism by which growth factors elicit their biologic effects. The identification of receptors that bind members of the transforming growth factor (TGF)-fj family has become a complex investigation involving highly homologous ligands and their binding to several cell surface proteins. Essentially all cells bind TGF-fj, but the parameters of binding vary with each cell type. Dissociation constants range from 1 to 60 pM, and there may be as few as 200 to as many as 100,000 receptors on a cell. It is generally true that cells binding TGF-fj with high affinity have fewer receptors. With many cell types, the half maximal biologic effect is observed at much lower concentrations than the amount required to half-saturate the receptor (1). Thus, TGF-fj appears to mediate metabolic changes at minimal receptor occupancy. The number of receptors available for TGF-fj binding is modulated by a variety of conditions that include cell density, differentiation, treatment with retinoic acid, or the addition of various hormones (2). The dissociation constants appear to remain unchanged with these treatments. Three classes of cell surface TGF-fj binding proteins have been characterized by size and are known as types I, II, and III receptor proteins. The types I and II proteins are glycoproteins of Mr 50,000 and M, 80,000, respectively, and containN-linked sugars. The type III protein (also known as "betaglycan") is heterogeneous in size and has a subunit size of 250,000 M, on reducing gels. It is a proteoglycan containing chains of the glycosaminoglycan (GAG) residues chondroitin sulfate and heparan sulfate. Enzymatic digestion of the GAG chains yields N-glycosylated core proteins that are Mr 120,000 to 140,000. The lGF-fj binding domain is located in the core proteins, leaving the GAG chains free to interact with the extracellular matrix; the GAG chains are not necessary for the binding of TGF-fj to the core protein. On some cells, it is possible to detect binding to proteins of M, 120,000 to 140,000; these proteins are the type III core proteins that have apparently been transported to the cell surface prior to receiving the GAG chains. The type III pro-
(Received for publication March 5, 1991) Address correspondence to: Patricia R. Segarini, Ph.D., Celtrix Laboratories, 2500 Faber Place, Palo Alto, CA 9430l Abbreviations: glycosaminoglycan, GAG; transforming growth factor, TGF. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 395-396, 1991
tein/betaglycan has also been detected in a soluble form (3); it has been extracted from extracellular matrices, culture media of several cell types, and serum. Soluble type III protein/betaglycan binds TGF-fj similarly to the membranebound form. While there is a fair amount of cross reactivity between TGF-fjl and TGF-fj2 for binding to types I, II, and III proteins, there appear to be differential binding affinities for these proteins (4,5). The types I and II proteins have approximately 10-fold higher affinity for TGF-fj) than for TGF-fj2' but there may be a subset of types I and II proteins that have a very high affinity for TGF-fj2' There also appears to be a class of proteins that binds both TGF-fj) and TGF-fj2 with similar affinity (4). The more recently purified recombinant factor TGF-fj3 has been shown to bind to cells in a similar fashion as TGF-fjl (5). Subsets that represent different states of the types I and II binding proteins would account for differential binding by TGF-fjI, TGF-fj2, and TGF-fj3' TGF-fj binding is not inhibited by other polypeptide growth factors (insulin, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors I or II, TGF-a, or nerve growth factor) or the biologically latent form of TGF-fj. Inhibition of TGF-fj binding on rat pituitary tumor cells by two of the less homologous members of the TGF-fj family, activin AB and inhibin B, suggests that these cells have a common receptor for TGF-fjs, activin, and inhibin. This binding protein appears specific to these cells, as activins and inhibins are not able to compete with TGF-fj binding on other cell types. The types I and II proteins are found on all cell types, while the type III protein/betaglycan is not detected on primary cells of endothelial or epithelial origin. It is the major TGF-fj binding component found on primary cells of mesenchymal origin (fibroblasts, chondroblasts, and osteoblasts) but may also be found on cultured and transformed cells of epithelial origin. The multiplicity ofTGF-fj binding to different cell surface proteins makes it virtually impossible to define the protein(s) involved in TGF-fj-mediated cell signal transduction; in order to qualify as a true receptor, the protein must not only bind TGF-fj but transmit a signal for metabolic change. Two investigations have been designed to ascertain the function of the individual binding components in order that receptor activity may be assigned to them. In a biologic approach, it has been deduced by the pattern of binding to a wide variety of different cell types that the type III proteoglycan is not involved in transduction of the signal-mediating changes in cell morphology or in the synthesis of DNA, collagen, and
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fibronectin, leaving control of these activities to the types I and II proteins (6). A genetic approach that eliminated the type I protein, similarly suggested that the type I protein is involved in TGF-{j-mediated growth control, fibronectin expression, and cell morphology change (7). More recent evidence suggests that the type II protein may also be involved in mediating these responses, perhaps as a component of a receptor complex that includes the type I protein (8). The mechanism of signal transduction by the TGF-{j receptor system remains ambiguous to date, but it is fairly well accepted that the receptor has no associated tyrosine kinase activity. There is some speculation that a guanine nucleotide binding protein-dependent pathway may be involved in transmitting signals associated with some of the biologic effects mediated by TGF-{j in AKR-2B fibroblasts, but whether this pathway is a direct effect of lGF-{j or a downstream effect remains unclear. Activation of protein kinase C has been observed in brain microvessels and rat brain astrocytes; the response in astrocytes appears coupled to phosphatidylinositol turnover. In summary, it appears that multiple intracellular pathways are involved in the mediation of the biologic response to lGF-{j. The purification, sequencing, and cloning of the types I, II, and III TGF-{j binding proteins will reveal critical domains that will aid in understanding the mechanism of action of the lGF-{j family of proteins. Assignment of biologic activity to individual components that may interact as a recep-
tor complex will be essential for elucidation of the mechanism. Purification of the TGF-{j4 and lGF-{js proteins will enable receptor studies that, coupled with lGF-{ji, lGF-{j2' and lGF-{j3 binding data, will enable the location of critical domains involved in mediation of biologic activity. References 1. Wakefield, L. M., D. M. Smith, T. Masui, C. C. Harris, and M. B. Sporn. 1987. Distribution and modulation of the cellular receptor for transforming growth factor-beta. J. Cell Bioi. 195:965-975. 2. Segarini, P. R. 1991. TGF-,s receptors. In Clinical Applications or ror-s. (Ciba Foundation Symposium 157.) Gregory Bock and Joan Marsh, editors. John Wiley, Chichester. 29-51. 3. Andres, J. L., K. Stanley, S. Cheifetz, and J. Massague. 1989. Membraneanchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-d. J. Cell Bioi. 109:3137-3145. 4. Segarini, P. R., A. B. Roberts, D. M. Rosen, and S. M. Seyedin. 1987. Membrane binding characteristics of two forms of transforming growth factor-d. J. Bioi. Chem. 262:14655-14662. 5. Cheifetz, S., H. Hernandez, M. Laiho, P. ten Dijke, K. K. Iwata, and J. Massague. 1990. Distinct transforming growth factor-S (TGF-(3)subsets as determinants of cellular responsiveness to three TGF-,s isoforms. J. Bioi. Chem. 265:20533-20538. 6. Segarini, P. R., D. M. Rosen, and S. M. Seyedin. 1989. Binding of transforming growth factor-S to cell surface proteins varies with cell type. Mol. Endocrinol. 3:261-272. 7. Boyd, F. T., and J. Massague. 1989. Transforming growth factor-S inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J. Bioi. Chem. 264:2272-2278. 8. Laiho, M., F. M. B. Weis, and J. Massague, 1990. Concomitant loss of transforming growth factor (TGF)-(3 receptor types I and IT in TGF-(3resistant cell mutants implicates both receptor types in signal transduction. J. Bioi. Chem. 265:18518-18524.
The initial interaction of polypeptide growth factors with a target cell is the binding to specific receptors present on the plasma membrane. The study, definition, and characterization of these receptors is essential for understanding the mechanism by which growth factors elicit their biologic effects. The identification of receptors that bind members of the transforming growth factor (TGF)-fj family has become a complex investigation involving highly homologous ligands and their binding to several cell surface proteins. Essentially all cells bind TGF-fj, but the parameters of binding vary with each cell type. Dissociation constants range from 1 to 60 pM, and there may be as few as 200 to as many as 100,000 receptors on a cell. It is generally true that cells binding TGF-fj with high affinity have fewer receptors. With many cell types, the half maximal biologic effect is observed at much lower concentrations than the amount required to half-saturate the receptor (1). Thus, TGF-fj appears to mediate metabolic changes at minimal receptor occupancy. The number of receptors available for TGF-fj binding is modulated by a variety of conditions that include cell density, differentiation, treatment with retinoic acid, or the addition of various hormones (2). The dissociation constants appear to remain unchanged with these treatments. Three classes of cell surface TGF-fj binding proteins have been characterized by size and are known as types I, II, and III receptor proteins. The types I and II proteins are glycoproteins of Mr 50,000 and M, 80,000, respectively, and containN-linked sugars. The type III protein (also known as "betaglycan") is heterogeneous in size and has a subunit size of 250,000 M, on reducing gels. It is a proteoglycan containing chains of the glycosaminoglycan (GAG) residues chondroitin sulfate and heparan sulfate. Enzymatic digestion of the GAG chains yields N-glycosylated core proteins that are Mr 120,000 to 140,000. The lGF-fj binding domain is located in the core proteins, leaving the GAG chains free to interact with the extracellular matrix; the GAG chains are not necessary for the binding of TGF-fj to the core protein. On some cells, it is possible to detect binding to proteins of M, 120,000 to 140,000; these proteins are the type III core proteins that have apparently been transported to the cell surface prior to receiving the GAG chains. The type III pro-
(Received for publication March 5, 1991) Address correspondence to: Patricia R. Segarini, Ph.D., Celtrix Laboratories, 2500 Faber Place, Palo Alto, CA 9430l Abbreviations: glycosaminoglycan, GAG; transforming growth factor, TGF. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 395-396, 1991
tein/betaglycan has also been detected in a soluble form (3); it has been extracted from extracellular matrices, culture media of several cell types, and serum. Soluble type III protein/betaglycan binds TGF-fj similarly to the membranebound form. While there is a fair amount of cross reactivity between TGF-fjl and TGF-fj2 for binding to types I, II, and III proteins, there appear to be differential binding affinities for these proteins (4,5). The types I and II proteins have approximately 10-fold higher affinity for TGF-fj) than for TGF-fj2' but there may be a subset of types I and II proteins that have a very high affinity for TGF-fj2' There also appears to be a class of proteins that binds both TGF-fj) and TGF-fj2 with similar affinity (4). The more recently purified recombinant factor TGF-fj3 has been shown to bind to cells in a similar fashion as TGF-fjl (5). Subsets that represent different states of the types I and II binding proteins would account for differential binding by TGF-fjI, TGF-fj2, and TGF-fj3' TGF-fj binding is not inhibited by other polypeptide growth factors (insulin, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors I or II, TGF-a, or nerve growth factor) or the biologically latent form of TGF-fj. Inhibition of TGF-fj binding on rat pituitary tumor cells by two of the less homologous members of the TGF-fj family, activin AB and inhibin B, suggests that these cells have a common receptor for TGF-fjs, activin, and inhibin. This binding protein appears specific to these cells, as activins and inhibins are not able to compete with TGF-fj binding on other cell types. The types I and II proteins are found on all cell types, while the type III protein/betaglycan is not detected on primary cells of endothelial or epithelial origin. It is the major TGF-fj binding component found on primary cells of mesenchymal origin (fibroblasts, chondroblasts, and osteoblasts) but may also be found on cultured and transformed cells of epithelial origin. The multiplicity ofTGF-fj binding to different cell surface proteins makes it virtually impossible to define the protein(s) involved in TGF-fj-mediated cell signal transduction; in order to qualify as a true receptor, the protein must not only bind TGF-fj but transmit a signal for metabolic change. Two investigations have been designed to ascertain the function of the individual binding components in order that receptor activity may be assigned to them. In a biologic approach, it has been deduced by the pattern of binding to a wide variety of different cell types that the type III proteoglycan is not involved in transduction of the signal-mediating changes in cell morphology or in the synthesis of DNA, collagen, and
396
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fibronectin, leaving control of these activities to the types I and II proteins (6). A genetic approach that eliminated the type I protein, similarly suggested that the type I protein is involved in TGF-{j-mediated growth control, fibronectin expression, and cell morphology change (7). More recent evidence suggests that the type II protein may also be involved in mediating these responses, perhaps as a component of a receptor complex that includes the type I protein (8). The mechanism of signal transduction by the TGF-{j receptor system remains ambiguous to date, but it is fairly well accepted that the receptor has no associated tyrosine kinase activity. There is some speculation that a guanine nucleotide binding protein-dependent pathway may be involved in transmitting signals associated with some of the biologic effects mediated by TGF-{j in AKR-2B fibroblasts, but whether this pathway is a direct effect of lGF-{j or a downstream effect remains unclear. Activation of protein kinase C has been observed in brain microvessels and rat brain astrocytes; the response in astrocytes appears coupled to phosphatidylinositol turnover. In summary, it appears that multiple intracellular pathways are involved in the mediation of the biologic response to lGF-{j. The purification, sequencing, and cloning of the types I, II, and III TGF-{j binding proteins will reveal critical domains that will aid in understanding the mechanism of action of the lGF-{j family of proteins. Assignment of biologic activity to individual components that may interact as a recep-
tor complex will be essential for elucidation of the mechanism. Purification of the TGF-{j4 and lGF-{js proteins will enable receptor studies that, coupled with lGF-{ji, lGF-{j2' and lGF-{j3 binding data, will enable the location of critical domains involved in mediation of biologic activity. References 1. Wakefield, L. M., D. M. Smith, T. Masui, C. C. Harris, and M. B. Sporn. 1987. Distribution and modulation of the cellular receptor for transforming growth factor-beta. J. Cell Bioi. 195:965-975. 2. Segarini, P. R. 1991. TGF-,s receptors. In Clinical Applications or ror-s. (Ciba Foundation Symposium 157.) Gregory Bock and Joan Marsh, editors. John Wiley, Chichester. 29-51. 3. Andres, J. L., K. Stanley, S. Cheifetz, and J. Massague. 1989. Membraneanchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-d. J. Cell Bioi. 109:3137-3145. 4. Segarini, P. R., A. B. Roberts, D. M. Rosen, and S. M. Seyedin. 1987. Membrane binding characteristics of two forms of transforming growth factor-d. J. Bioi. Chem. 262:14655-14662. 5. Cheifetz, S., H. Hernandez, M. Laiho, P. ten Dijke, K. K. Iwata, and J. Massague. 1990. Distinct transforming growth factor-S (TGF-(3)subsets as determinants of cellular responsiveness to three TGF-,s isoforms. J. Bioi. Chem. 265:20533-20538. 6. Segarini, P. R., D. M. Rosen, and S. M. Seyedin. 1989. Binding of transforming growth factor-S to cell surface proteins varies with cell type. Mol. Endocrinol. 3:261-272. 7. Boyd, F. T., and J. Massague. 1989. Transforming growth factor-S inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J. Bioi. Chem. 264:2272-2278. 8. Laiho, M., F. M. B. Weis, and J. Massague, 1990. Concomitant loss of transforming growth factor (TGF)-(3 receptor types I and IT in TGF-(3resistant cell mutants implicates both receptor types in signal transduction. J. Bioi. Chem. 265:18518-18524.
