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A dual receptor system is required for basic fibroblast growth factor activity
Cell, Vol. 67, 229-231, October18, 1991,Copyright© 1991 by Cell Press
Minireview
A Dual Receptor System Is Required For Basic Fibroblast Growth Factor Activity Michael Klagsbrun* and Andrew Bairdt *Departments of Surgery, Biological Chemistry, and Molecular Pharmacology Harvard Medical School and Children's Hospital Boston, Massachusetts 02115 tDepartment of Molecular and Cellular Growth Biology Whittier Institute at Scripps Memorial Hospitals La Jolla, California 92037
The notion that interaction with a single high affinity receptor is the most important step that mediates growth factor binding to the cell surface and its initiation of a mitogenic cascade is now regarded by many as simplistic. The complex interactions of fibroblast growth factors (FGFs) with high and low affinity receptors provide a good case for amending this view. To date four human genes have been identified that each encode a distinct high affinity receptor (Kd of -10 -11 M)(Houssaint et al., 1990; Johnson et al., 1990; Keegan et al., 1991; Partanen et al., 1991). Each of these genes (shown in Figure 1 as A-D) encodes multiple proteins derived from alternative mRNA splicing. Despite their structural similarities, these high affinity receptors may differ in their ability to bind various members of the FGF family, for example, acidic FGF and basic FGF (Partanen et al., 1991). Basic FG F also binds to cell surface heparan sulfate proteoglycans (HSPGs) that have been determined to be low affinity receptors (K~ of -2 x 10-9 M) (Moscatelli, 1987). Low affinity HSPG-binding sites for basic and acidic FGF exist in the extracellular matrix of cells as well. The existence of at least seven genes encoding FGF family members, at least four genes encoding high affinity receptors, and heterogeneous populations of HSPG low affinity receptors on cell surfaces and in the extracellular matrix suggests novel mechanisms for regulating FGF activity and determining responsiveness of target cells to FGF. The binding of basic FGF to the extracellular domain of a high affinity receptor and to heparan sulfate moieties on HSPG is shown schematically in Figure 1. Basic FGF exported into the extracellular matrix by the cell (step 1) is sequestered as a complex with HSPG but is made available (through unkown processes) to cell surface HSPG' low affinity receptors (step 2). This cell-associated HSPG can deliver the growth factor to one or several high affinity receptors (step 3) that internalize basic FGF and initiate the cellular response (step 4). The law of mass action predicts that the relative concentration of low and high affinity sites on the cell surface determines which type of binding site is available to a ligand. In the absence of high affinity receptors, all ligands will be inactive; in the absence of low affinity binding, insufficient ligand accumulates at the cell surface. There presumably exists a low affinity receptor number that may concentrate the ligand at the cell surface and optimally mediate its transfer to the high affinity site. It is of paramount importance to study the characteristics
of the low affinity binding sites, establish whether cell surface HSPG is different from extracellular matrix-binding HSPG, and determine how HSPG interacts with high affinity receptors. Is binding to cell surface HSPG important for growth factor activity, and if so, does it participate in signal transduction and cellular responsiveness? Recent studies have demonstrated that, in the absence of cell surface HSPG, basic FGF does not bind to its high affinity receptor and is not active. Mutagenized Chinese hamster ovary (CHO) cells that lack cell surface HSPGs have no low affinity binding sites, even when the structurally related glycosaminoglycan chondroitin sulfate is overexpressed (Yayon et al., 1991). These results demonstrate that HSPG is indeed a low affinity receptor for basic FGF and that low affinity binding is specific for heparin-related molecules (as opposed to other glycosaminoglycans). These mutant CHO cells, unlike wild-type CHO cells, do not bind basic FGF, even when the high affinity FGF receptor FGFR-1 is expressed. This suggests that binding to cell surface HSPG is a prerequisite for high affinity binding. A similar conclusion was reached in two types of studies in which HSPGs are removed from cell surfaces by heparatinase treatment or in which cells are incubated with chlorate to block the sulfation of heparan sulfate (Rapraeger et al., 1991). Either treatment substantially reduced binding of basic FGF to high and low cell surface receptors, blocked its mitogenic activity for Swiss 3T3 fibroblasts, and blocked its ability to repress the terminal differentiation of MM14 skeletal muscle cells. Taken together, it appears that cell surface heparan sulfate modulates basic FGF activity by facilitating binding to high affinity receptors and that the low affinity sites are directly involved in basic FGF cell signaling. One mechanism proposed to explain these observations is that interaction with cell surface
®
©
basicFGF ~ ~
Glycosamlrloglycan / : FGFR-1 Highaffinityreceptors (2and3IgG-Ilkedomains)~ " FGFR-2 FGFR-3 FGFR-4
\:
Figure 1. High and Low Affinity Receptorsfor Basic FGF
Cell 230
(a) Quiescence
% basicFGF ~
r~ept°rbindingd°maln
"~pGAG
~
Ac~binding~,te
bindingd~main
~ ~ ~
Heparansulfateproteoglycan Highaffinityreceptors
(2 and $ IgG-Ilkadomains)
~
Transmembrane domain
[]
"l~/roslneklnsse
Figure2. A Complexof BasicFGF, HPSG,and High AffinityReceptorResultsin MitogenicActivation
HSPG changes the conformation of basic FGF so that it can interact with the high affinity receptor. Alternatively, it is possible that cell surface HSPG modulates the structure of the high affinity receptor, allowing it to bind basic FGF. In the absence of cell surface HSPG, exogenous heparin facilitates basic FGF binding to its high affinity receptor. Basic FGF binds to (Yayon et al., 1991) and is mitogenic for (Bernard et al., 1991) cells lacking cell surface HSPG and expressing FGFR-1, but only in the presence of exogenous heparin. One must be somewhat cautious, however, in assessing the role of exogenous heparin in modulating basic FGF activity. Kiefer et al. (1991) have expressed the extracellulardomain of one form of FGFR-1 and have shown that, while high affinity receptor binding of basic FGF is significantly increased by heparin, this interaction is not obligatory in vitro. Taken together, all these studies point to the same conclusion-the binding and mitogenic activity of basic FGF are dependent to some degree on cell surface heparin-like molecules or, in their absence, exogenously added heparin-like molecules. Whether the dual requirement of growth factor binding to protein receptors and glycosaminoglycan-type receptors is a general phenomenon has yet to be demonstrated. There are a number of other heparin-binding growth factors, including PDGF, acidic FGF, vascular endothelial cell growth factor, heparin-binding EGF, Schwannoma-derived growth factor, GM-CSF, hepatocyte growth factor, and pleiotropin. Binding of growth factors to columns of immobilized heparin need not be necessarily synonymous with binding to cell-associated glycosaminoglycan. Nevertheless, the biological activities of other heparin-binding growth factors might also be modulated by their ability to
bind to cell surface HSPG. To this end, parathyroid cells possess a 150 kd high affinity receptor for acidic FGF that appears to contain heparan sulfate side chains (Sakaguchi et al., 1991). Removal of the heparan sulfate with heparatinase completely eliminates high affinity acidic FGF binding. TGFI3 also binds to proteoglycan-type receptors (K~ of 10-11M; Ruoslahti and Yamaguchi, 1991). However, unlike basic FGF, TGFI~ binds to the proteoglycan betaglycan via the core protein rather than the glycosaminoglycan. In its simplest form, the take-home message is that basic FGF utilizes a dual receptor system composed of a classical protein-type receptor and a lower affinity glycosaminoglycan-type receptor. Thus, the cellular responses to basic EGF are mediated by a receptor complex rather than by a single protein. If this is the case, then a combination of binding sites, rather than any single component, may mediate ligand specificity. A general model for how basic FGF is activated by binding to each of the two receptor types is shown in Figure 2. Basic FGF has both heparin-binding and high affinity receptor-binding domains. One possibility is that basic FGF straddles the two receptor types, with the heparinbinding domain(s) acting to concentrate basic FGF and present it to high capacity HSPG-binding sites. Alternatively, basic FGF might bind to cell surface HSPG first and, via an activation mechanism, be transferred to the high affinity receptors. If this is the case, then mechanisms presumably exist to facilitate this transfer. One intriguing possibility is the role proposed for the phosphorylation of basic FGF by a target cell-associated ectokinase (Vilgrain and Baird, 1991). Given that syndecan, a low affinity receptor, has a cytoplasmic actin-binding site (Saunders et al., 1989; Kiefer et al., 1990), delivery of basic FGF to its high
Minireview 231
affinity receptor m a y be linked to m e c h a n o c h e m i c a l changes that are associated with injury, cell growth, and differentiation (Ingber and Folkman, 1989). In the end, it is clear that it is important to identify the mechanisms that regulate growth factor responsiveness of target cells in order to understand the c o n s e q u e n c e s of their pleiotropic activities. As the c o m p l e x i t y of this process is better understood, it m a y then be possible to understand how growth factors achieve target cell specificity in vivo and to d e v e l o p a c o m p r e h e n s i v e rationale for the design of selective antagonists and agonists. References Bernard, O., Li, M., and Reid, H. H. (1991). Proc. Natl. Acad. Sci. USA 88, 7625-7629. Houssaint, E., Blanquet, P. R., Champion-Arnaud, P., Gesnel, M. C., Torriglia, A., Courtois, Y., and Breathnach, R. (1990). Proc. Natl. Acad. Sci. USA 87, 8180-8184. Johnson, D. E., Lee, P. L., Lu, J., and Williams, L. T. (1990). Mol. Cell. Biol. 10, 4728-4736. Ingber, D. E., and Folkman, J. (1989). J. Cell Biol. 109, 317-330. Keegan, K., Johnson, D. E., Williams, L. T., and Hayman, M. J. (1991). Proc. Natl. Acad. Sci. USA 88, 1095-1099. Kiefer, M. C., Stephans, J. C., Crawford, K., Okino, K., and Barr, P. J. (1990). Proc. Natl. Acad. Sci. USA 87, 6985-6989. Kiefer, M. C., Baird, A., Nguyen, T., George-Nascimento, C., Mason, O. B., Boley, L. J., Valenzuela, P., and Barr, P. J. (1991). Growth Factors, in press. Moscatelli, D. (1987). J. Cell. Physiol. 131, 123-130. Partanen, J., M~kel~, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson-Welsh, L., and Alitalo, K. (1991). EMBO J. 10, 1347-1354. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991). Science 252, 1705-1708. Ruoslahti, E., and Yamaguchi, Y. (1991). Cell 64, 867-869. Sakaguchi, K., Yanagashita, M., Takeuchi, Y., and Aurbach, G. D. (1991). J. Biol. Chem. 266, 7270-7278. Saunders, S., Jalkenen, M., O'Farrell, S., and Barnfield, M. (1989). J. Cell Biol. 108, 1547-1556. Vilgrain, I., and Baird, A. (1991). Mol. Endocrinol. 5, 1003-1012. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991). Cell 64, 841-848.
Minireview
A Dual Receptor System Is Required For Basic Fibroblast Growth Factor Activity Michael Klagsbrun* and Andrew Bairdt *Departments of Surgery, Biological Chemistry, and Molecular Pharmacology Harvard Medical School and Children's Hospital Boston, Massachusetts 02115 tDepartment of Molecular and Cellular Growth Biology Whittier Institute at Scripps Memorial Hospitals La Jolla, California 92037
The notion that interaction with a single high affinity receptor is the most important step that mediates growth factor binding to the cell surface and its initiation of a mitogenic cascade is now regarded by many as simplistic. The complex interactions of fibroblast growth factors (FGFs) with high and low affinity receptors provide a good case for amending this view. To date four human genes have been identified that each encode a distinct high affinity receptor (Kd of -10 -11 M)(Houssaint et al., 1990; Johnson et al., 1990; Keegan et al., 1991; Partanen et al., 1991). Each of these genes (shown in Figure 1 as A-D) encodes multiple proteins derived from alternative mRNA splicing. Despite their structural similarities, these high affinity receptors may differ in their ability to bind various members of the FGF family, for example, acidic FGF and basic FGF (Partanen et al., 1991). Basic FG F also binds to cell surface heparan sulfate proteoglycans (HSPGs) that have been determined to be low affinity receptors (K~ of -2 x 10-9 M) (Moscatelli, 1987). Low affinity HSPG-binding sites for basic and acidic FGF exist in the extracellular matrix of cells as well. The existence of at least seven genes encoding FGF family members, at least four genes encoding high affinity receptors, and heterogeneous populations of HSPG low affinity receptors on cell surfaces and in the extracellular matrix suggests novel mechanisms for regulating FGF activity and determining responsiveness of target cells to FGF. The binding of basic FGF to the extracellular domain of a high affinity receptor and to heparan sulfate moieties on HSPG is shown schematically in Figure 1. Basic FGF exported into the extracellular matrix by the cell (step 1) is sequestered as a complex with HSPG but is made available (through unkown processes) to cell surface HSPG' low affinity receptors (step 2). This cell-associated HSPG can deliver the growth factor to one or several high affinity receptors (step 3) that internalize basic FGF and initiate the cellular response (step 4). The law of mass action predicts that the relative concentration of low and high affinity sites on the cell surface determines which type of binding site is available to a ligand. In the absence of high affinity receptors, all ligands will be inactive; in the absence of low affinity binding, insufficient ligand accumulates at the cell surface. There presumably exists a low affinity receptor number that may concentrate the ligand at the cell surface and optimally mediate its transfer to the high affinity site. It is of paramount importance to study the characteristics
of the low affinity binding sites, establish whether cell surface HSPG is different from extracellular matrix-binding HSPG, and determine how HSPG interacts with high affinity receptors. Is binding to cell surface HSPG important for growth factor activity, and if so, does it participate in signal transduction and cellular responsiveness? Recent studies have demonstrated that, in the absence of cell surface HSPG, basic FGF does not bind to its high affinity receptor and is not active. Mutagenized Chinese hamster ovary (CHO) cells that lack cell surface HSPGs have no low affinity binding sites, even when the structurally related glycosaminoglycan chondroitin sulfate is overexpressed (Yayon et al., 1991). These results demonstrate that HSPG is indeed a low affinity receptor for basic FGF and that low affinity binding is specific for heparin-related molecules (as opposed to other glycosaminoglycans). These mutant CHO cells, unlike wild-type CHO cells, do not bind basic FGF, even when the high affinity FGF receptor FGFR-1 is expressed. This suggests that binding to cell surface HSPG is a prerequisite for high affinity binding. A similar conclusion was reached in two types of studies in which HSPGs are removed from cell surfaces by heparatinase treatment or in which cells are incubated with chlorate to block the sulfation of heparan sulfate (Rapraeger et al., 1991). Either treatment substantially reduced binding of basic FGF to high and low cell surface receptors, blocked its mitogenic activity for Swiss 3T3 fibroblasts, and blocked its ability to repress the terminal differentiation of MM14 skeletal muscle cells. Taken together, it appears that cell surface heparan sulfate modulates basic FGF activity by facilitating binding to high affinity receptors and that the low affinity sites are directly involved in basic FGF cell signaling. One mechanism proposed to explain these observations is that interaction with cell surface
®
©
basicFGF ~ ~
Glycosamlrloglycan / : FGFR-1 Highaffinityreceptors (2and3IgG-Ilkedomains)~ " FGFR-2 FGFR-3 FGFR-4
\:
Figure 1. High and Low Affinity Receptorsfor Basic FGF
Cell 230
(a) Quiescence
% basicFGF ~
r~ept°rbindingd°maln
"~pGAG
~
Ac~binding~,te
bindingd~main
~ ~ ~
Heparansulfateproteoglycan Highaffinityreceptors
(2 and $ IgG-Ilkadomains)
~
Transmembrane domain
[]
"l~/roslneklnsse
Figure2. A Complexof BasicFGF, HPSG,and High AffinityReceptorResultsin MitogenicActivation
HSPG changes the conformation of basic FGF so that it can interact with the high affinity receptor. Alternatively, it is possible that cell surface HSPG modulates the structure of the high affinity receptor, allowing it to bind basic FGF. In the absence of cell surface HSPG, exogenous heparin facilitates basic FGF binding to its high affinity receptor. Basic FGF binds to (Yayon et al., 1991) and is mitogenic for (Bernard et al., 1991) cells lacking cell surface HSPG and expressing FGFR-1, but only in the presence of exogenous heparin. One must be somewhat cautious, however, in assessing the role of exogenous heparin in modulating basic FGF activity. Kiefer et al. (1991) have expressed the extracellulardomain of one form of FGFR-1 and have shown that, while high affinity receptor binding of basic FGF is significantly increased by heparin, this interaction is not obligatory in vitro. Taken together, all these studies point to the same conclusion-the binding and mitogenic activity of basic FGF are dependent to some degree on cell surface heparin-like molecules or, in their absence, exogenously added heparin-like molecules. Whether the dual requirement of growth factor binding to protein receptors and glycosaminoglycan-type receptors is a general phenomenon has yet to be demonstrated. There are a number of other heparin-binding growth factors, including PDGF, acidic FGF, vascular endothelial cell growth factor, heparin-binding EGF, Schwannoma-derived growth factor, GM-CSF, hepatocyte growth factor, and pleiotropin. Binding of growth factors to columns of immobilized heparin need not be necessarily synonymous with binding to cell-associated glycosaminoglycan. Nevertheless, the biological activities of other heparin-binding growth factors might also be modulated by their ability to
bind to cell surface HSPG. To this end, parathyroid cells possess a 150 kd high affinity receptor for acidic FGF that appears to contain heparan sulfate side chains (Sakaguchi et al., 1991). Removal of the heparan sulfate with heparatinase completely eliminates high affinity acidic FGF binding. TGFI3 also binds to proteoglycan-type receptors (K~ of 10-11M; Ruoslahti and Yamaguchi, 1991). However, unlike basic FGF, TGFI~ binds to the proteoglycan betaglycan via the core protein rather than the glycosaminoglycan. In its simplest form, the take-home message is that basic FGF utilizes a dual receptor system composed of a classical protein-type receptor and a lower affinity glycosaminoglycan-type receptor. Thus, the cellular responses to basic EGF are mediated by a receptor complex rather than by a single protein. If this is the case, then a combination of binding sites, rather than any single component, may mediate ligand specificity. A general model for how basic FGF is activated by binding to each of the two receptor types is shown in Figure 2. Basic FGF has both heparin-binding and high affinity receptor-binding domains. One possibility is that basic FGF straddles the two receptor types, with the heparinbinding domain(s) acting to concentrate basic FGF and present it to high capacity HSPG-binding sites. Alternatively, basic FGF might bind to cell surface HSPG first and, via an activation mechanism, be transferred to the high affinity receptors. If this is the case, then mechanisms presumably exist to facilitate this transfer. One intriguing possibility is the role proposed for the phosphorylation of basic FGF by a target cell-associated ectokinase (Vilgrain and Baird, 1991). Given that syndecan, a low affinity receptor, has a cytoplasmic actin-binding site (Saunders et al., 1989; Kiefer et al., 1990), delivery of basic FGF to its high
Minireview 231
affinity receptor m a y be linked to m e c h a n o c h e m i c a l changes that are associated with injury, cell growth, and differentiation (Ingber and Folkman, 1989). In the end, it is clear that it is important to identify the mechanisms that regulate growth factor responsiveness of target cells in order to understand the c o n s e q u e n c e s of their pleiotropic activities. As the c o m p l e x i t y of this process is better understood, it m a y then be possible to understand how growth factors achieve target cell specificity in vivo and to d e v e l o p a c o m p r e h e n s i v e rationale for the design of selective antagonists and agonists. References Bernard, O., Li, M., and Reid, H. H. (1991). Proc. Natl. Acad. Sci. USA 88, 7625-7629. Houssaint, E., Blanquet, P. R., Champion-Arnaud, P., Gesnel, M. C., Torriglia, A., Courtois, Y., and Breathnach, R. (1990). Proc. Natl. Acad. Sci. USA 87, 8180-8184. Johnson, D. E., Lee, P. L., Lu, J., and Williams, L. T. (1990). Mol. Cell. Biol. 10, 4728-4736. Ingber, D. E., and Folkman, J. (1989). J. Cell Biol. 109, 317-330. Keegan, K., Johnson, D. E., Williams, L. T., and Hayman, M. J. (1991). Proc. Natl. Acad. Sci. USA 88, 1095-1099. Kiefer, M. C., Stephans, J. C., Crawford, K., Okino, K., and Barr, P. J. (1990). Proc. Natl. Acad. Sci. USA 87, 6985-6989. Kiefer, M. C., Baird, A., Nguyen, T., George-Nascimento, C., Mason, O. B., Boley, L. J., Valenzuela, P., and Barr, P. J. (1991). Growth Factors, in press. Moscatelli, D. (1987). J. Cell. Physiol. 131, 123-130. Partanen, J., M~kel~, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson-Welsh, L., and Alitalo, K. (1991). EMBO J. 10, 1347-1354. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991). Science 252, 1705-1708. Ruoslahti, E., and Yamaguchi, Y. (1991). Cell 64, 867-869. Sakaguchi, K., Yanagashita, M., Takeuchi, Y., and Aurbach, G. D. (1991). J. Biol. Chem. 266, 7270-7278. Saunders, S., Jalkenen, M., O'Farrell, S., and Barnfield, M. (1989). J. Cell Biol. 108, 1547-1556. Vilgrain, I., and Baird, A. (1991). Mol. Endocrinol. 5, 1003-1012. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991). Cell 64, 841-848.