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Whydohemopoieticgrowthfactor receptors interactwith eachother? Hemopoietic growth factors (colony-stimulating factors, CSFs) interact with distinct cellular receptors that recognize only their cognate ligand, Yet, like other growth factor~receptorsystems, the binding of one type of CSF to its receptor can 'downmodulate' the availability of a different type of CSFreceptor. In this art/de Nicos Nicola discusses the distinctive pattern of CSF-receptor modulations and suggests a novel interpretation of such modulations as a means of coupling receptor-derived signals which is consistent with the known biology of the
system. The colony-stimulating factors (CSFs) are a family of glycoprotein growth factors which regulate the survival, proliferation and differentiation of hemopoietic progenitor cells as well as the functional activities of the mature cells1-s. Four distinct CSFscontrolling the production of granulocytes and macrophages in the mouse have been purified and recently molecularly cloned. Three of these CSFs have clear counterparts in the human hemopoietic system (Table 1) but no unequivocal human analogue of murine multi-CSF has yet been identified. The biology, molecular biology and biochemistry of these CSFs have been discussed in detail elsewhere1-s so only those features of their biological action which are particularly relevant to an interpretation of the binding studies will be mentioned here. All four CSFsstimulate the formation of hemopoietic colonies in agar cultures in vitro at very similar concentrations (3-15 pmol/I for half-maximal biological activity). In each case, colony formation is initiated from an undiffer~1
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stimulated by more than one CSF1. These data indicate that at least some progenitor cells should display multiple CSF receptors or that some CSFs can cross-react with other CSF receptors. However, the cross-reactivity hypothesis is made unlikely by the observation of some unique biological properties of the CSFs. For example, although multi-CSF, GM-CSF and G-CSF are equally effective in inducing proliferation and differentiation of neutrophil precursors, G-CSF has a unique capacity to induce terminal differentiation and clonal suppression of myeloid leukemic cells7. The CSFsexhibit distinct concentration dependence in the types of differentiated colonies they induce. At low concentrations G-CSF and M-CSF appear to be essentially lineage-specific, and induce the formation of only neutrophilic granulocyte or macrophage colonies, respectively. However, at higher concentrations they stimulate the formation of additional macrophage or granulocyte colonies, respectively (Fig. 1). Both GM-CSF and multi-CSF induce the formation of predominantly macrophage colonies at low concentrations but also induce the formation of granulocyte colonies at higher concentrations 1. At progressively higher concentrations GM-CSF can also stimulate the formation of eosinophilic, megakaryocytic and erythroid colonies (Fig. 1) (D. Metcalf, unpublished). The binding studies will be discussed in the context of this surprising complexity in the biological crossreactivities of the CSFs.
CI ~ U U I U I I I "
ated fashion, produces differentiated progeny. In general, this process results in the production of a colony containing differentiated cells and very few, if any, proliferative blast cells. The predominant type of differentiated cells in the colonies depends on the type of CSF used to stimulate colony formation and is indicated by the prefix of the CSF (G-CSF produces neutrophilic colonies, M-CSF monocytic colonies, GM-CSF neutrophilic, eosinophilic and monocytic colonies and multi-CSF produces neutrophilic, eosinophilic, monocytic, megakaryocytic, mast cell and erythroid colonies). Since every cell division depends on the continued presence of the appropriate CSF1 and since maturing and mature cells depend on CSF for their continued survival and functional activationS.6 one would expect that appropriate CSF receptors would be expressed on hemopoietic cells throughout the differentiation process. It may be noticed from the foregoing and from Fig. 1 that the CSFs overlap in specificity and are organized hierarchically. Neutrophils and macrophages can be produced by any of the four CSFs but multi-CSF and, to a lesser extent, GM-CSF can also stimulate the production of other cell lineages (Fig. 1). Moreover, clonal analysis has indicated that the same progenitor cell can be 134
NicosA. Nicola
The Walter and Eliza Hall Institute of Medical Research,P.O. Royal MelbourneHospital,3050 Victoria,Australia
Molecularand binding characteristicsof individual CSFreceptors Normal monocyte/macrophage cell populations and macrophage cell lines display a single class of highaffinity cell surface receptor for M-CSF of Mr -- 165 000 (Refs 8 and 9). The purified receptor is a single chain glycoprotein with intrinsic tyrosine kinase activity and can itself be phosphorylated on tyrosines, a property common to several other growth factors lo. The M-CSF receptor may be identical or closely related to the c-fms proto-oncogene since antisera to the v-fms protein immunoprecipitated 12sI M-CSF receptor complexes, and since immunoprecipitates demonstrated M-CSFdependent tyrosine phosphorylation on a protein of Mr 165 000 (Refs 9 and 11). By analogy with v-fms the M-CSF receptor probably has a glycosylated M-CSF binding domain of about 450 amino acids at the Nterminal and an intracellular tyrosine kinase domain of about the same length at the C-terminal. When binding of M-CSF is performed at 2°C no other CSF or growth factor shows competition for M-CSF binding sites12. At this temperature binding is essentially irreversible so that the true dissociation constant (Kd = ko~lkon)is less than 3 x 10-13 mol/I. Nevertheless, a steady state is achieved at low levels of added 12Sl M-CSF because unoccupied receptors are apparently lost from the cell surface by unknown mechanisms13. At 37°C, dissociation of bound 1251 M-CSF is slow but (~ 1987, ElsevierPublications, Cambridge 0167 -4919/87/$02.00
Immunology Today, vol 8, No. 5, 1987 ,
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Table 1. Murine colony-stimulatingfactors and their human equivalentsa
Murine CSF
Othernames
Cellular sources T-cells WEHI-3B
Human equivalent
Multi-CSF
Molecular Subunits weight Interleukin3, BPA, 19 0001 HCGF, MCGF, PSF 28 000 CSF-2(x
GM-CSF
MGI-1GM,CSF-2
M-CSF
MGI-1M, CSF-1
T cells; endothelial cells; macrophages Fibroblasts
CSF-,~,pluripoietin-o= NIF-T M-CSF,CSF-1
23 000
1
Not known?
45 0002 70 000 G-CSF MGI-1G, DF, MGI-2 25 000 1 Macrophages CSF-I~,pluripoietin a MGh macrophageand granulocyteinducer; BPA: burst promoting activity; HCGF: hernopoieticcell growth factor MCGF:mast cell growth factor; PSF:P-cellstimulating factor; NIF-T: neutrophil inhibiting factor, T-cell derived; CSF:colony-stimulatingfactor; G: granulocyte; M: macrophage.For a list of references,see Refs 1-4. measurable and yields a Kd of about 4 x 10 -lo mol/I (Ref. 14). However, following binding of rvi-CSF to its receptor the receptor complex is rapidly internalized (with a half-life of only a few minutes) so that most of the bound M-CSF ends up in the cell. The internalized M-CSF is subsequently degraded either rapidly (peritoneal macrophages) or more slowly (bone marrowderived macrophages) and it has been suggested that the accumulation of intracellular pools of M-CSF receptor may be correlated with the biological response to M-CSF14.~s. Receptor replacement at the cell surface Mulfi-CSF 60
-
occurs fairly rapidly (t,~ 1-3 h) but the relative contributions made from cryptic receptors, receptor recycling and new receptor synthesis are unclear. The G-CSF receptor on normal and leukemic murine myeloid cells has been identified by chemical crosslinking as a single chain protein of Mr "- 150 000 (Ref. 16). No evidence has been obtained for binding site heterogeneity and the receptor is specific for G-CSF, showing no direct cross-reactivity with other CSFs or other growth factors 17. As with M-CSF, binding to normal bone marrow cells at 0°C is essentially irreversi-
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10 102 10:] 10~ 105 Unifs/ml M-CSF
Fig.1. Concentrationdependence of the 3bility of the four different murine CSFsto stimulate the formation of different typesof differentiated hemopoieticcolonies (upper figure) or to 'down-modulate"different typesof CSFreceptorson murine bone marrow cellsat 3"/°C(lower figure). Concentrations of the CSFswere determined by their ability to stimulate colony formation from murine bone marrow cells, 50 units/ml being assigned to that concentration giving half-maximalcolony number. M: macrophage; G: neuzrophil; EO: eosinophil; MEG: megakaryocyte;E: erythroid.
135
Immunology Today, vol. 8, No. 5, 1987
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ble, although dissociation is measurable from the myeloid leukemic cell line WEHI-3B D÷ (koff = 0.003 min-1) at this temperature. Despite this, a steady-state binding rea~ion is achieved at sub-saturating doses of 12Sl G-CSF, suggesting that a similar mechanism to that described for M-CSF binding may exist. Although there is not yet any direct evidence for G-CSFIreceptor internalization at 37°C it is clear that bound G-CSF is degraded slowly at this temperature (t,~ - 4 h) (Nicqla, N.A., unpublished). Cross-linking studies have also been performed on 12Sl GM-CSF bound to normal bone marrow cells or cell lines. Walker and Burgess18 found a receptor Mr of 51 000 while Park et al. 19 identified a receptor of Mr 130 000 and suggested that this receptor might be particularly sensitive to the action of cellular proteases. Walker and Burgess18 demonstrated the existence of both higha[finity (apparent Kd 20-60 pmol/I) and low-affinity (Kd 0.7-1.2 nmol/I) receptors on various cell types while Park et al. 19found only a single class of receptor (apparent Kd 1-3 nmol/I). No direct cross-reactivity with other CSFs or growth factors was seen in the binding of GM-CSF to its receptod 8.19. GM-CSF induced rapid internalization of its receptor on WEHI-3B D+ cells (t,,~ 7 min) and this was followed by slow CSF degradation zo. In contrast to the cross-linking studies with other CSFs, cross-linking of 12Slmulti-CSF bound to various cell lines demonstrated, in some cases, attachment of multi-CSF to two different molecular species16. These were each single-chain proteins, were not covalently attached to each other, and had an apparent Mr of 75 000 and 60 000. 12Sl muiti-CSF became associated with both species with equal affinity and was cross-linked to either species at similar concentrations of cross-linker16. Since most authors have described a single protein of Mr 65 000-70 000 that becomes cross-linked to 12Sl multiCSF21-23 the significance of the two species is at present unclear. There appears to be a single class of multi-CSF receptor which shows no binding competition for other CSFs or growth factors24.2s. The binding interactions of multi-CSF on cell lines are very similar to those of M-CSF. At 0°C the binding is practically irreversible while at 37°C the multi-CSF receptor complex is slowly internalized (t,~ 15-60 rain) and even more slowly degraded (t,~ 2-3 h) (Nicola, N.A., unpublished). For all four CSFsthere are several common features in their receptor binding characteristics. (1) At 0°C the binding interaction is essentially irreversible. How this occurs is unknown but it is not due to covalent bond formation since the binding interaction can be rapidly reversed by decreasing the pH. For M-CSF,
G-CSF and multi-CSF the ligand-bound receptor becomes protease-insensitive. Despite the apparent irreversible binding reaction a sustained steady state is achieved at sub-saturating doses of CSF by unknown mechanisms. (2) At 37°C the CSF receptor complex is internalized and degraded. The rates of internalization and degradation vary considerably with CSF and cell type so that different levels of CSF accumulate in cells w t h time. Stanley has suggested that some types of cells nay serve to deplete CSF by degradation while others may accumulate CSF intracellularly as a mitogenic signal 14.1: (3) Because of the preceding points, equilibri Jm binding constants cannot be determined by zhe Usudl Scatchard analysis at 0°C or 370C since in neither case is a true equilibrium achieved. The same considerations make it difficult to accurately determine kinetic association (kon) and dissociation (kof~)constants. However, Table 2 shows that equilibrium dissociation constants (Kd) determined from the kinetic constants differ considerably from the apparent Kd determined by Scatchard analysis and, in general, are higher than the concentration required for half-maximal biological activity. This suggests that there is no simple relationship between receptor occupancy and biological effect and that, in some cases, the biological response can be mediated at low levels of receptor occupancy. (4) All the CSF receptors are single subunit glycoproteins. The M-CSF and G-CSF receptQrs are large and the former, at least, encodes a tyrosine kinase. The multiCSF, and possibly GM-CSF, receptors are smailer and like the interleukin 2 (IL-2) receptor may be too small to encode tyrosine kinases. Cellular distribution of CSF receptors Cell autoradiography has demonstrated the presence of M-CSF receptors on all cells within the monocyte/ macrophage cell lineage and on several cell lines of monocytic or myelomonocytic origin 12.26.The number of receptors per cell increases with cell maturation, adherent macrophages displaying 50 000 or more receptors per cell. Receptors are not present on erythroid, lymphoid, eosinophilic or megakaryocytic cells but some cells with the apparent morphology of neutrophils or their precursors may have small numbers of receptors26.27. Recently, it has been suggested that normal and malignant placental trophoblasts may display M-CSF receptors and the c-fms protein product 9. suggesting a possible role for M-CSF in non-hemopoietic tissue. Although human M-CSF cross-reacts with the murine M-CSF receptor the converse does not occur28.
Table 2. Propertiesof receptorsfor murine colony-stimulatingfactors
Receptorfor
!
i 136
Molecular Subunits kon a weight (m -1 rain-1) (x 1000) Multi-CSF 75 1 2x108 60 1 GM-CSF 51 1 4.5x 108 130 M-CSF 165 1 I>2 x 108 G-CSF 150 1 2 x 109 aMeasuredat 37°C. bFromthe slope of the Scatchardcurve at 37°C.
koffa (rain-1)
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Whydohemopoieticgrowthfactor receptors interactwith eachother? Hemopoietic growth factors (colony-stimulating factors, CSFs) interact with distinct cellular receptors that recognize only their cognate ligand, Yet, like other growth factor~receptorsystems, the binding of one type of CSF to its receptor can 'downmodulate' the availability of a different type of CSFreceptor. In this art/de Nicos Nicola discusses the distinctive pattern of CSF-receptor modulations and suggests a novel interpretation of such modulations as a means of coupling receptor-derived signals which is consistent with the known biology of the
system. The colony-stimulating factors (CSFs) are a family of glycoprotein growth factors which regulate the survival, proliferation and differentiation of hemopoietic progenitor cells as well as the functional activities of the mature cells1-s. Four distinct CSFscontrolling the production of granulocytes and macrophages in the mouse have been purified and recently molecularly cloned. Three of these CSFs have clear counterparts in the human hemopoietic system (Table 1) but no unequivocal human analogue of murine multi-CSF has yet been identified. The biology, molecular biology and biochemistry of these CSFs have been discussed in detail elsewhere1-s so only those features of their biological action which are particularly relevant to an interpretation of the binding studies will be mentioned here. All four CSFsstimulate the formation of hemopoietic colonies in agar cultures in vitro at very similar concentrations (3-15 pmol/I for half-maximal biological activity). In each case, colony formation is initiated from an undiffer~1
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stimulated by more than one CSF1. These data indicate that at least some progenitor cells should display multiple CSF receptors or that some CSFs can cross-react with other CSF receptors. However, the cross-reactivity hypothesis is made unlikely by the observation of some unique biological properties of the CSFs. For example, although multi-CSF, GM-CSF and G-CSF are equally effective in inducing proliferation and differentiation of neutrophil precursors, G-CSF has a unique capacity to induce terminal differentiation and clonal suppression of myeloid leukemic cells7. The CSFsexhibit distinct concentration dependence in the types of differentiated colonies they induce. At low concentrations G-CSF and M-CSF appear to be essentially lineage-specific, and induce the formation of only neutrophilic granulocyte or macrophage colonies, respectively. However, at higher concentrations they stimulate the formation of additional macrophage or granulocyte colonies, respectively (Fig. 1). Both GM-CSF and multi-CSF induce the formation of predominantly macrophage colonies at low concentrations but also induce the formation of granulocyte colonies at higher concentrations 1. At progressively higher concentrations GM-CSF can also stimulate the formation of eosinophilic, megakaryocytic and erythroid colonies (Fig. 1) (D. Metcalf, unpublished). The binding studies will be discussed in the context of this surprising complexity in the biological crossreactivities of the CSFs.
CI ~ U U I U I I I "
ated fashion, produces differentiated progeny. In general, this process results in the production of a colony containing differentiated cells and very few, if any, proliferative blast cells. The predominant type of differentiated cells in the colonies depends on the type of CSF used to stimulate colony formation and is indicated by the prefix of the CSF (G-CSF produces neutrophilic colonies, M-CSF monocytic colonies, GM-CSF neutrophilic, eosinophilic and monocytic colonies and multi-CSF produces neutrophilic, eosinophilic, monocytic, megakaryocytic, mast cell and erythroid colonies). Since every cell division depends on the continued presence of the appropriate CSF1 and since maturing and mature cells depend on CSF for their continued survival and functional activationS.6 one would expect that appropriate CSF receptors would be expressed on hemopoietic cells throughout the differentiation process. It may be noticed from the foregoing and from Fig. 1 that the CSFs overlap in specificity and are organized hierarchically. Neutrophils and macrophages can be produced by any of the four CSFs but multi-CSF and, to a lesser extent, GM-CSF can also stimulate the production of other cell lineages (Fig. 1). Moreover, clonal analysis has indicated that the same progenitor cell can be 134
NicosA. Nicola
The Walter and Eliza Hall Institute of Medical Research,P.O. Royal MelbourneHospital,3050 Victoria,Australia
Molecularand binding characteristicsof individual CSFreceptors Normal monocyte/macrophage cell populations and macrophage cell lines display a single class of highaffinity cell surface receptor for M-CSF of Mr -- 165 000 (Refs 8 and 9). The purified receptor is a single chain glycoprotein with intrinsic tyrosine kinase activity and can itself be phosphorylated on tyrosines, a property common to several other growth factors lo. The M-CSF receptor may be identical or closely related to the c-fms proto-oncogene since antisera to the v-fms protein immunoprecipitated 12sI M-CSF receptor complexes, and since immunoprecipitates demonstrated M-CSFdependent tyrosine phosphorylation on a protein of Mr 165 000 (Refs 9 and 11). By analogy with v-fms the M-CSF receptor probably has a glycosylated M-CSF binding domain of about 450 amino acids at the Nterminal and an intracellular tyrosine kinase domain of about the same length at the C-terminal. When binding of M-CSF is performed at 2°C no other CSF or growth factor shows competition for M-CSF binding sites12. At this temperature binding is essentially irreversible so that the true dissociation constant (Kd = ko~lkon)is less than 3 x 10-13 mol/I. Nevertheless, a steady state is achieved at low levels of added 12Sl M-CSF because unoccupied receptors are apparently lost from the cell surface by unknown mechanisms13. At 37°C, dissociation of bound 1251 M-CSF is slow but (~ 1987, ElsevierPublications, Cambridge 0167 -4919/87/$02.00
Immunology Today, vol 8, No. 5, 1987 ,
i
,H
Ll
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Table 1. Murine colony-stimulatingfactors and their human equivalentsa
Murine CSF
Othernames
Cellular sources T-cells WEHI-3B
Human equivalent
Multi-CSF
Molecular Subunits weight Interleukin3, BPA, 19 0001 HCGF, MCGF, PSF 28 000 CSF-2(x
GM-CSF
MGI-1GM,CSF-2
M-CSF
MGI-1M, CSF-1
T cells; endothelial cells; macrophages Fibroblasts
CSF-,~,pluripoietin-o= NIF-T M-CSF,CSF-1
23 000
1
Not known?
45 0002 70 000 G-CSF MGI-1G, DF, MGI-2 25 000 1 Macrophages CSF-I~,pluripoietin a MGh macrophageand granulocyteinducer; BPA: burst promoting activity; HCGF: hernopoieticcell growth factor MCGF:mast cell growth factor; PSF:P-cellstimulating factor; NIF-T: neutrophil inhibiting factor, T-cell derived; CSF:colony-stimulatingfactor; G: granulocyte; M: macrophage.For a list of references,see Refs 1-4. measurable and yields a Kd of about 4 x 10 -lo mol/I (Ref. 14). However, following binding of rvi-CSF to its receptor the receptor complex is rapidly internalized (with a half-life of only a few minutes) so that most of the bound M-CSF ends up in the cell. The internalized M-CSF is subsequently degraded either rapidly (peritoneal macrophages) or more slowly (bone marrowderived macrophages) and it has been suggested that the accumulation of intracellular pools of M-CSF receptor may be correlated with the biological response to M-CSF14.~s. Receptor replacement at the cell surface Mulfi-CSF 60
-
occurs fairly rapidly (t,~ 1-3 h) but the relative contributions made from cryptic receptors, receptor recycling and new receptor synthesis are unclear. The G-CSF receptor on normal and leukemic murine myeloid cells has been identified by chemical crosslinking as a single chain protein of Mr "- 150 000 (Ref. 16). No evidence has been obtained for binding site heterogeneity and the receptor is specific for G-CSF, showing no direct cross-reactivity with other CSFs or other growth factors 17. As with M-CSF, binding to normal bone marrow cells at 0°C is essentially irreversi-
6M-CSF
M-CSF
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10 102 10:] 10~ 105 Unifs/ml M-CSF
Fig.1. Concentrationdependence of the 3bility of the four different murine CSFsto stimulate the formation of different typesof differentiated hemopoieticcolonies (upper figure) or to 'down-modulate"different typesof CSFreceptorson murine bone marrow cellsat 3"/°C(lower figure). Concentrations of the CSFswere determined by their ability to stimulate colony formation from murine bone marrow cells, 50 units/ml being assigned to that concentration giving half-maximalcolony number. M: macrophage; G: neuzrophil; EO: eosinophil; MEG: megakaryocyte;E: erythroid.
135
Immunology Today, vol. 8, No. 5, 1987
ros i
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m ................
ble, although dissociation is measurable from the myeloid leukemic cell line WEHI-3B D÷ (koff = 0.003 min-1) at this temperature. Despite this, a steady-state binding rea~ion is achieved at sub-saturating doses of 12Sl G-CSF, suggesting that a similar mechanism to that described for M-CSF binding may exist. Although there is not yet any direct evidence for G-CSFIreceptor internalization at 37°C it is clear that bound G-CSF is degraded slowly at this temperature (t,~ - 4 h) (Nicqla, N.A., unpublished). Cross-linking studies have also been performed on 12Sl GM-CSF bound to normal bone marrow cells or cell lines. Walker and Burgess18 found a receptor Mr of 51 000 while Park et al. 19 identified a receptor of Mr 130 000 and suggested that this receptor might be particularly sensitive to the action of cellular proteases. Walker and Burgess18 demonstrated the existence of both higha[finity (apparent Kd 20-60 pmol/I) and low-affinity (Kd 0.7-1.2 nmol/I) receptors on various cell types while Park et al. 19found only a single class of receptor (apparent Kd 1-3 nmol/I). No direct cross-reactivity with other CSFs or growth factors was seen in the binding of GM-CSF to its receptod 8.19. GM-CSF induced rapid internalization of its receptor on WEHI-3B D+ cells (t,,~ 7 min) and this was followed by slow CSF degradation zo. In contrast to the cross-linking studies with other CSFs, cross-linking of 12Slmulti-CSF bound to various cell lines demonstrated, in some cases, attachment of multi-CSF to two different molecular species16. These were each single-chain proteins, were not covalently attached to each other, and had an apparent Mr of 75 000 and 60 000. 12Sl muiti-CSF became associated with both species with equal affinity and was cross-linked to either species at similar concentrations of cross-linker16. Since most authors have described a single protein of Mr 65 000-70 000 that becomes cross-linked to 12Sl multiCSF21-23 the significance of the two species is at present unclear. There appears to be a single class of multi-CSF receptor which shows no binding competition for other CSFs or growth factors24.2s. The binding interactions of multi-CSF on cell lines are very similar to those of M-CSF. At 0°C the binding is practically irreversible while at 37°C the multi-CSF receptor complex is slowly internalized (t,~ 15-60 rain) and even more slowly degraded (t,~ 2-3 h) (Nicola, N.A., unpublished). For all four CSFsthere are several common features in their receptor binding characteristics. (1) At 0°C the binding interaction is essentially irreversible. How this occurs is unknown but it is not due to covalent bond formation since the binding interaction can be rapidly reversed by decreasing the pH. For M-CSF,
G-CSF and multi-CSF the ligand-bound receptor becomes protease-insensitive. Despite the apparent irreversible binding reaction a sustained steady state is achieved at sub-saturating doses of CSF by unknown mechanisms. (2) At 37°C the CSF receptor complex is internalized and degraded. The rates of internalization and degradation vary considerably with CSF and cell type so that different levels of CSF accumulate in cells w t h time. Stanley has suggested that some types of cells nay serve to deplete CSF by degradation while others may accumulate CSF intracellularly as a mitogenic signal 14.1: (3) Because of the preceding points, equilibri Jm binding constants cannot be determined by zhe Usudl Scatchard analysis at 0°C or 370C since in neither case is a true equilibrium achieved. The same considerations make it difficult to accurately determine kinetic association (kon) and dissociation (kof~)constants. However, Table 2 shows that equilibrium dissociation constants (Kd) determined from the kinetic constants differ considerably from the apparent Kd determined by Scatchard analysis and, in general, are higher than the concentration required for half-maximal biological activity. This suggests that there is no simple relationship between receptor occupancy and biological effect and that, in some cases, the biological response can be mediated at low levels of receptor occupancy. (4) All the CSF receptors are single subunit glycoproteins. The M-CSF and G-CSF receptQrs are large and the former, at least, encodes a tyrosine kinase. The multiCSF, and possibly GM-CSF, receptors are smailer and like the interleukin 2 (IL-2) receptor may be too small to encode tyrosine kinases. Cellular distribution of CSF receptors Cell autoradiography has demonstrated the presence of M-CSF receptors on all cells within the monocyte/ macrophage cell lineage and on several cell lines of monocytic or myelomonocytic origin 12.26.The number of receptors per cell increases with cell maturation, adherent macrophages displaying 50 000 or more receptors per cell. Receptors are not present on erythroid, lymphoid, eosinophilic or megakaryocytic cells but some cells with the apparent morphology of neutrophils or their precursors may have small numbers of receptors26.27. Recently, it has been suggested that normal and malignant placental trophoblasts may display M-CSF receptors and the c-fms protein product 9. suggesting a possible role for M-CSF in non-hemopoietic tissue. Although human M-CSF cross-reacts with the murine M-CSF receptor the converse does not occur28.
Table 2. Propertiesof receptorsfor murine colony-stimulatingfactors
Receptorfor
!
i 136
Molecular Subunits kon a weight (m -1 rain-1) (x 1000) Multi-CSF 75 1 2x108 60 1 GM-CSF 51 1 4.5x 108 130 M-CSF 165 1 I>2 x 108 G-CSF 150 1 2 x 109 aMeasuredat 37°C. bFromthe slope of the Scatchardcurve at 37°C.
koffa (rain-1)
koffikon
(pM)
Kdb (app) (pM)
~
