The Cytokine Receptor Superfamily

R. S. Kaczmarski, G. J. Mufti S UMMA R Y. The binding of haemopoietic growth factors and cytokines to specific receptors triggers a cascade of intracellular events which results in cell proliferation and differentiation. The knowledge of ligand-receptor-signal pathways is not only important in understanding the pathophysiology of malignant disease but also essential for devising future therapeutic strategies. The advent of recombinant technology has made it possible to test the efllcacy of selective differentiation therapy, and haemopoietic growth factors are undergoing clinical trials for a number of indications. In addition, increasingly the receptors for haemopoietic growth factors and cytokines have come under scientific scrutiny. Recently receptors for IL-2a, JL3B, IL-3, IL-4, IL-5, IL-6, IL7, erythropoietin, GCSF and GM-CSF have been isolated and cloned. It has become apparent that they have structural homology that is shared by receptors for growth hormone and prolactin, and this receptor group makes up the new cytokine receptor superfamily. The finding of sequence homology within these receptors suggests their evolutionary relationship. These receptors are transmembrane proteins 257-856 amino acids and their extracellular ligandbinding domain contains four conserved cysteine residues and a TrpSer-X-Trp-Ser motif. The secondary s&ucture of the extracellular domain is made up of a-helices. High and low at&&y binding forms exist for all these receptors. Binding atlinity may depend on the formation of receptor beterodimers or multimers, association with other membrane proteins or differential glycosylation. Soluble receptor forms have been described for IL-2u, IL-4, IL-5, IL-6 and IL-7. It is not known whether they are actively secreted or represent the degradation products of cell turnover. Their function may be to mop up excess cytokines and thereby confine the cytokine response. There is no sequence homology of the intracytoplasmic domains although several are rich in proline and serine residues, which may be important in mechanisms of signal transduction. No receptor in this superfamily functions as a receptor tyrosine kinase or has intrinsic protein tryosine kinase activity. Detailed study of individual receptors holds clues to the regulation of receptor expression, ligandreceptor interactions and mechanisms involved in signal transduction. Such knowledge might explain the pleotropic effects cytokines may have on different cell types and their overlap in biological functions. Elevated levels of soluble IL& receptor (Tat) are detected in hairy cell leukaemia, lymphomas and adult T-cell leukaemia (IL), and levels reflect tumour burden. Other soluble receptors (eg IL.6 receptor in multiple myeloma) may also prove to be useful in this way. As growth factor therapy is becoming a part of cancer treatment, a knowledge of growth factor receptor distribution and expression by malignant cells may guide as to the appropriate choice of growth factors, avoiding those that may cause proliferation of the malignant clone. Where proliferation of the malignant clone is dependant on autocrine or paracrine growth factor secretion, anti-receptor therapies may be used to block the response. Similarly, soluble receptors, incapable of signal transduction might be used to prevent the action of a cytokine. Blood Reviiws (1991) 5, 193-203 C 1991 Longman Group UK Ltd

194 THE CYTOKINE RECEPTOR SUPERFAMILY

A further understanding of the cytokine-receptor-signal pathway will increase our understanding of the pathogenesis of cancer and manipulation of this axis has prospects for new cancer therapies.

Cytokines, including haemopoietic growth factors and interleukins are polypeptides of diverse structure that exert pleotropic effects on target cells.l*’ These effects require binding of the cytokine to a specific cell surface receptor which triggers signal transduction. The study of cytokine receptors has been hampered by the relative scarcity of these receptors on the cell surface (typically 100-1000 per cell). The establishment of growth factor dependent cell lines, that expresses high numbers of individual cytokine receptors, has recently enabled a large number of cytokine receptors to be isolated, cloned and sequenced. Cytokine receptors can be classified according to their structure or function. A large number of growth factor receptors share structural homology with the immunoglobulin superfamily; these include interleukin-l receptor, platelet derived growth factor (PDGF) receptor and macrophage colony-stimulating factor (M-CSF) receptor, C-FMS. However, as an increasing number of cytokine receptors were identified it became clear that receptors for human IL-2u, IL-2B, IL-4, IL-5, IL-7, erythropoietin, GM-CSF and murine IL-3, were all found to share structural homology3-” and constitute a new receptor superfamily.‘6-‘8 There is however some overlap between the cytokine receptor superfamily and the immunoglobulin superfamily, as illustrated by receptors for IL-6 and G-CSF, both of which have immunoglobulin-like domains but also share structural homology with cytokine receptors (Fig. 1). The finding that a growing number of cytokine receptors have a common structure is evidence of what has long been thought, that the cytokine network has a common evolutionary ancestor, and may explain the interplay between different cytokines. It is interesting to note that the cytokine receptors are not restricted to cells of the haemopoietic system, and are expressed in diverse tissues such as liver, placenta and small-cell lung cancer”V’2*‘3 suggesting a wider role of cytokines outside haemopoiesis and immune regulation. Furthermore receptors for growth hormone and prolactin share the same structure as cytokine receptors16-‘8 and these hormones may play a role in haemopoiesis.” Many growth factors mediate their pleotropic actions through binding to and activating cell surface receptors with intrinsic protein tyrosine kinase activity.20-22 Receptors for M-CSF (C-FMS), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and c-kit (stem cell growth factor receptor 23,24)act as receptor tyrosine kinases to effect signal transduction. 2o--25In contrast receptors of the R. S. Kaczmarski, Wellcome Research Fellow, G. J. Mufti, Senior

Lecturer, Department of Haematological Medicine, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.

cytokine superfamily do not possess intrinsic tyrosine kinase activity and mediate signal transduction through second messangers which remain unidentified. This review highlights the structure, functional relationships and clinical significance of receptor within the cytokine receptor superfamily.

Generic Structure All cytokine receptors are transmembrane glycoproteins made up of an extracellular amino-teminal ligand binding domain, a short hydrophobic transmembrane region and a carboxy-terminal intracellular domain. Amino acid sequencing has shown that this group of receptors share structural homology in a 210 amino acid stretch of the ligand binding domain. Within this region are four conserved cysteine residues and a repeated Tryptophan-Serine motif separated by a random amino acid residue (Trp-Ser-X-Trp-Ser) just proximal to the transmembrane domain (exceptions to this are discussed below). Predictions from the known amino acid sequences, confirmed by spectroscopic data and X-ray crystallography show the ligand-binding domain to have a secondary u-helical structure.17 A comparison of the amino acid sequences of this region shows a significant degree of relatedness which suggests a common ancestral origin of these receptors.‘7g26 There is little amino acid sequence homology in the intracytoplasmic domains of this receptor group all of which are rich in prohne and serine residues. The mechanisms involved in signal transduction are largely unknown. The receptors themselves have no structural homology with, nor function as receptor tyrosine kinases, 20-22v27.28although the activation of intracellular phosphoproteins has been shown to occur following ligand binding.29-33 The nature of second messangers remains to be elucidated. A number of other similarities exist within this receptor superfamily. All receptors identified thus far have been shown to occur as high and low affinity binding forms. The high affinity form is responsible for signal transduction. The role of low affinity binding forms is uncertain but their function may involve regulation of the cytokine response or in an accessory role to the high affinity receptor.34 A number of mechanisms illustrated by different receptors, may account for differences in their binding affinity. For example, the IL-2 receptor is a heterodimer made up of two distinct receptor proteins, IL2a and IL-2B which form the high affinity binding receptor IL-2ccB.5,35Binding affinity may be regulated by the association of receptors with other membrane

BLOOD REVIEWS

195

Cytokine Superfamily

Ligand-binding domain containing conserved cl

sequences cytokine

of superfamily

Immunoglobulin-like domain Fibronectin

Type

III

like domain

l-l M-CSF R PDGF R

IL-1 R

IL-6

R

G-CSF

Fig. 1 The overlap in structure between the immunoglobulin-like

Membrane

ammo

aads

24-27

amino

aclds

13-577

ammo

acids/

GM-CSF R IL-2 R IL-4 R IL-6 R IL-7 R Epo k

mulL-3 R GM-CSFB R

superfamily of receptors and the cytokine receptor superfamily.

as to confine the effect of a cytokine to a localized site. A schematic representation of the receptors and their features and distribution are summarized in Figure 3 and Table 1. A number of characteristics have been identified in individual receptors that may represent models for understanding their function.

Cytoplasmic Domain

206-603

R

IL-2 Receptor (IL-2 R) /

Intracellular Domain COO”

Fig. 2 The generic structure of cytokine receptors.

proteins; for example the association of IL-6 receptor with a membrane glycoprotein gpl30 is known to be involved in signal transduction, but may in addition confer a high binding affinity.j6s3’ Some evidence exists for receptors forming dimers (eg IL-7 and which may represent the high G-CSF receptors ‘OV3*), affinity binding forms of the receptor. Differential receptor glycosylation may also influence binding affinity as in the case of Epo receptor.3g Soluble receptors have been found for IL-2cq5 IL4,40 IL-5,15 IL-636 and IL-71°: they consist of the extracellular domain and are capable of ligand-binding. Whether they are actively secreted or represent the degradation product of the receptor or cell turnover is uncertain. The function of soluble receptors might be to mop up excess circulating cytokines so

IL-2 R was one of the first to be identified and its function, expression and regulation have been studied in detail. The high affinity IL-2 receptor is made up of two distinct receptor proteins, 01 (~55) and j3 (~70-75).‘~~*~‘~~~IL-201 R binds and dissociates from ligand rapidly, whereas IL-2b R binds ligand with a slow rate of dissociation (Table 2).42 The IL-2aB R heterodimer combines these features to form a receptor that binds IL-2 rapidly with a slow rate of dissociation which ensures an optimal response. IL2B R is expressed constitutively on resting T-cells, NK-cells and some B- and T-cell lines.3s*43 IL-2u R is highly inducible and mechanisms controlling its expression have been studied in detail. The IL-2aR gene is localized to chromosome 10 (p15-~14). A number of regulatory sites within the 5’ promotor region of this gene bind several nuclear transcription factors.44 One of these is a KB-like binding site45 which has been shown to be the site of a complex of DNA-protein and protein-protein interactions. Two DNA binding factors (86kDa and 5lkDa proteins) that bind specifically to the IL-2ct R KB site have

196 THE CYTOKINE RECEPTOR SUPERFAMILY Table 1

Receptor (species)

Chromosomal localization

Amino acids Binding Receptor domain

IL-2a Human IL-28 Human

lO(pl5-p14)

257

219

-

52s

214

IL-3 Murine IL-4 Human

-

856

417

-

785

208

IL-5 Murine

-

398

322

IL-6 Human

-

468

340

IL-7 Human Epo Human GM-CSF~L Human

-

439

219

19P

508

250

X-Y pseudoautosomal region

378

297

GM-CSFB Human

-

G-CSF Human

-

7591 852

603

Receptor distribution

Other features

Not expressed Highly inducible T-cells, B-cells NK-cells T-, B-cell lines

Low affinity binding No signal transduction Pro/Ser cytoplasmic domain/ Intermediate affinity binding Forms al3 heterodimers Pro/Ser cytoplasmic domain Duplicated conserved cysteines Pro/Ser cytoplasmic domain Soluble form

Haemopoietic progenitors Leukaemic cells and cell lines All haemopoietic cells Fibroblasts, epithelial cells, hepatocytes, tumour cell lines B-cells, T-cells Eosinophils, cell lines T-cells, activated B-cells, myeloma cells and cell lines Hepatoma cell lines Lymphoid and myeloid cells and cell lines BFU-E, CFU-E, placenta Murine receptor on Ba/F3 cell lines Monocytes neutrophils Eosinophils + precursors Leukaemic cells and lines Small Cell lung cancer

Neutrophils + precursors Leukaemic cells and lines. Endothelial cells, placenta

been identified and three additional proteins (63SkDa, 70kDa and 71kDa) also take part in this interaction (Fig. 4). The importance of the role of the KB site in the regulation of IL-2a R expression is illustrated by experiments where deletion of the KB site from the IL-2a R promotor results in abrogation of induction of IL-2a R by mitogens eg PMA (phorbol 1Zmyristate 13-acetate) and cytokines eg TNFu (tumor necrosis factor).42 The genetic mechanisms involved in the regulation of IL-2u R expression have provided insights into the pathogenesis of the HTLV1 associated adult T-cell leukaemia/lymphoma (ATLL).45-47 HTLV-1 encodes a 40kDa trans-activating regulatory protein Tax, that up-regulates viral replication through binding to a NF-KB site within the viral long term repeat (LTR) sequences.48 The viral NF-KB site is homologous to the KB sites in the promotor regions of IL-2aR and IL-2. The binding of Tax protein to these sites4’ sets up an autocrine loop enabling both IL-2 secretion as well as IL-2clR expression with resulting T-cell proliferation.50*51 Signal transduction by IL-2 R involves serine/threonine and protein tyrosine kinases,27,28*30-32 although in contrast to other growth factor receptors such as M-CSF R, EGF R and PDGF R the intracellular domain of IL-2R does not possess a kinase that becomes activated on ligand binding.22 Tyrosine phosphorylation therefore involves indirect mechanisms which are unknown at present, although it has been suggested that IL-2R may be associated with a

Low affinity receptor cloned Soluble form Pro-rich cytoplasmic domain Ig-like amino-terminal gpl30 role in signal transduction Soluble form Pro/Ser cytoplasmic domain, Soluble form Point mutation in mmine receptor results in autonomous cell growth Low affinity receptor ?shares affinity for IL-3

Protein product of KH97 cDNA, has homology with IL-3R. Does not bind ligand directly. Confers high affinity binding to GM-CSF R. Ig-like amino terminal Fibronectin Type III domain Pro/Ser-rich cytoplasmic domain

gamma chain 41 that might possess intrinsic protein kinase activity. Elevated levels of soluble IL-2 receptor (Tat) occur in a number of pathological states, including lymphomas, 52*53hairy-cell leukaemias4 and ATLLsos51 and serve as a useful marker of disease, reflecting tumor load and response to treatment.54 IL-3 Receptor (IL-3 R) Murine IL-3 R has been cloned.7 It differs from other members in this group, as the large extracellular ligand binding domain has a duplicate set of conserved cysteine residues (fig. 3). This receptor binds IL-3 with only low affinity therefore high affinity binding may depend on receptor-receptor or receptoror protein interactions, receptor oligomerization differential glycosylation as discussed above. In view of the considerable degree of overlap in target cell specificity and funcitonal effects of IL-3 and GM-CSF, it has been proposed that they either share a common receptor or can bind to one-anothers receptors. 55 However competitive binding studies have shown no ligand-receptor promiscuity.7v” The observation that IL-3 and GM-CSF can cross compete for binding to receptors in the KG-l cell lines5 and some human leukaemic cells raises the possibility that there may exist an as yet unidentified receptor capable of binding both IL-3 and GM-CSF or that their receptors may interact to form a common ligand binding site.

BLOOD REVIEWS Murine

IL-3

GM-CSF

197

B

Extracellular Domain G-CSF

IL-20

IL-4

IL-2B

I I

IL-7

IL-5

GM-CSFo

EPO

--

J

-

I

I-

-

-

-

-.

-

Trp-Ser-X-Trp-Ser

-

Conserved

Cysteine

Residues

Immunoglobulln-like Intracellular Domain

domain

Fibronectin

Type

Ill-like

Membrane

glycoprotein

domoin

0 gp130

Fig. 3 The structural domains of receptors in the cytokine receptor superfamily.

Table 2 Properties of the IL-2 Receptors IL-2 receptor Molecular weight Binding affinity Expression Signal transduction IL-2 binding Kd(pM) t+association t+dissociation

P 70-75K Intermediate Constitutive Yes 500-1000 45min 5h

&K Low Induced No loo00 SS

7s

7OkDa

++ High Induced Yes 10-50 30s 5h

7lkDa

Neither IL-3 nor GM-CSF receptors have intrinsic tyrosine kinase activity, however it has been shown that a common set of cytoplasmic phosphoproteins pp93 and pp70 become phosphorylated after binding of IL-3 and GM-CSF to their respective receptors.33 This may explain some of the overlap in function of these two cytokines. IL-4 Receptor (IL-4 R) Three forms of IL-4 R have been identified; a complete transmembrane protein, a protein comprising

63.5kDa

000 .@Q. 1

KB domain

Downstream Elements (DE)

. 1 Upstream

2 Elements

(UE)

Fig. 4 The DNA binding proteins at the KB-like site in the promotor region of IL-2a receptor, and the flanking upstream and downstream elements (UE, DE).

1%

THE CYTOKINE RECEPTOR SUPERFAMILY

only the extracellular and transmembrane domains and a soluble form.8v40 The intracellular domain is essential for signal transduction, as the receptor lacking this region is only capable of ligand binding without signal transduction. Soluble IL4R neutralizes the effects of IL-4, a function shared by the receptor lacking the intracellular domain.

IL-5 Receptor (IL-5 R) Murine IL-5 R is the most recent of the cytokine receptor superfamily to have been cloned.15 This receptor binds ligand with low affinity, and high affinity binding is proposed to depend on the association of the receptor with other membrane proteins.56*57 The ligand binding domain has sequence homology with growth hormone and prolactin receptors; the short intracytoplasmic domain is rich in proline residues. IL-5 R does not appear to have any protein tyrosine kinase activity, and mechanisms of signal transduction have not yet been studied. A soluble receptor has been identified but its functions are unknown.

IL-6 Receptor (IL-6 R) IL-6 R differs from other members in this receptor group (Fig. 1). The first 90 amino-terminal amino acids have sequence homology with the immunoglobulin superfamily, 58 however by virtue of the presence of the four conserved cysteines and the Trp-Ser-XTrp-Ser motif IL-6 R is included in the cytokine receptor superfamily.g IL-6 R is present in membrane bound and soluble forms. Mutants of IL-6 R in which the 65 amino acids of the intracytoplasmic domain are deleted are still capable of signal transduction which suggests that this region is not essential for full receptor function.36 IL-6 R has been shown to be associated with another membrane glycoprotein, gp130 which does not bind ligand but is essential for signal transduction. This glycoprotein co-precipitates with IL-6 R only after IL-6 stimulation, indicating that IL-6 R and gp130 interact only after ligand binding. Soluble IL6 R can also associate with gp130 in the presence of IL-6 to induce signal transduction.36 The association of a cytokine receptor with other membrane proteins may serve as a model for other receptors to explain their binding affinity as well as the mechanisms involved in signal transduction. IL-6 has pleotropic effects on a wide range of cells including B-cells, T-cells, plasmacytomas, myeloid stem cells and megakaryocytes. 37*5gWhether ligand-receptor binding results in immunoglobulin synthesis, cell proliferation or differentiation may depend on the presence of gpl30-variants in different cell types.

IL-7 Receptor (IL-7 R) IL-7 R has only two of the conserved cysteine residues at the amino terminal domain (Fig. 3) however, the Trp-Ser-X-Trp-Ser motif is conserved. IL-7 R shares sequence homology with the intracytoplasmic domains of IL-2B and growth hormone receptors. Like other receptors in this group a soluble form of IL-7 R has been found.” High and low affinity binding forms exist and evidence from dissociation kinetics and cross-linking studies suggest that the murine IL7 R may form non-covalently bound dimers in the cell membrane.60 This may be analagous to the growth factor tyrosine kinase receptors EGF-R, PDGF-R and insulin-R where it has been shown that high affinity ligand binding receptors exist as oligomers of the low affinity receptor.20V22

Erythropoietin Receptor (Epo R) The human Epo R has been cloned, sequenced and localized to the short arm of chromosome 19.12*13 Human Epo-R has over 80% amino acid sequence homology with the murine receptor.3g*61 High and low affinity binding receptors have been identified. Mouse erythroleukaemia line cells (MEL) express only the low affinity binding receptor and are unresponsive to Epo, however mouse splenic erythroblasts respond to Epo and express both high and low affinity receptors.39 These observations suggest that the physiological effects of erythropoietin are realized through the high affinity binding receptor.62*63 Since muEpo R and human IL-2B R are highly homologous it raises the possibility that the Epo R may be associated with a Tat-like protein analagous to IL-2 R which may explain differential binding affinities.61 The envelope glycoprotein of the Friend spleen focusforming virus, gp55, binds to muEpo R and triggers growth activation in the absence of E~o.~~ Recently two mutations of muEpo R have been described which result in Epo-independent cell activation and tumorgenicity.65 muEpo R was expressed in the IL3 dependant cell line Ba/F3. A single point mutation in the ligand binding domain at codon 129 (Arg to Cys) resulted in autonomous cell growth. This point mutation has been shown to prolong the half life of the mutant receptor by delaying intracellular receptor transport and degradation. This is also seen in the action of gp55 on muEpo R.66 It was also shown that deletion of the 42 carboxy-terminal amino acids leads to a tenfold increase in sensitivity of the cells to E~o?~ The activation of Epo R by a point mutation makes Epo R a likely candidate for an oncogene analogous to the receptor tyrosine kinase M-CSF (CFMS) in which altered receptor function by point mutations in the ligand binding domain can activate the tyrosine kinase that results in the transformation of the ce11.25

BLOOD REVIEWS

GCSF Receptor (G-CSF R) Two high affinity binding receptors for human G-CSF have recently been cloned. I4 Like IL-6 R these receptors have an amino terminal immunoglobulin domain, and differ further by having three Fibronectin Type III domains between the region containing the conserved cysteines and the transmembrane domain (Fig. 3). These Type III domains have also been described in growth hormone and prolactin receptors.67 The two receptors are made up of 759 and 812 amino acids respectively. The extracellular, membrane-spanning domains and first 96 amino acids of the intracellular domains are identical; the receptors differ at the carboxy-terminal 34 and 87 amino acid residues respectively. The two receptors are probably derived from alternatively processed transcripts of the same gene. Differential splicing may result in tissue-specific forms of receptor as has been demonstrated for two other cell-surface proteins, the rat Prolactin receptor6* and chicken neural cell adhesion molecules.69 Tissue specificity of the G-CSF R isoforms has yet to be demonstrated, but this would provide yet another model to explain the pleotorpic effects of a cytokine in different cells. Alternatively the two receptor forms may differ in signal transduction pathways to account for the diverse effects of G-CSF. The 812 amino acid receptor has close homology to the murine G-CSF receptor which has also recently been cloned.” The function of the structural domains in the G-CSF receptors is unclear. Fibronectin has been implicated in cell to ce1171and heparin7’ binding. The Fibronectin Type III domain of the murine G-CSF R has marked similarity with a chicken neuronal surface glycoprotein contactin, involved in cellular communication. 73 It is therefore possible that the fibronectin Type III region of G-CSF R is involved in the binding and interactions of meyloid progenitor cells with marrow stromal elements and heparan sulphate-bound growth factors.74 Like IL-7 R, there is evidence that the high affinity binding of murine G-CSF R results from the formation of oligomers of the low affinity binding receptor. 38 Neither receptor isoform of the human G-CSF R contains sequences indicative of tyrosine kinase activity, although the intracellular domain of the 812 amino acid G-CSF R has a potential protein kinase C phosphorylation site. l4 Both human G-CSF receptors have intracytoplasmic domains containing a high proportion of proline and serine residues. The murine G-CSF R shares sequence homology with the intracytoplasmic domain of IL-4 R,70 and it is possible that these receptors may share some common pathways of signal transduction. GM-CSF Receptor (GM-CSF R) The low affinity human GM-CSF R has been cloned and sequenced, l1 and localized to the pseudoautoso-

199

ma1 region of the sex chromosomes.‘1,75,76 A high affinity binding receptor is known to exist, and recently a second component to the human GM-CSF R has been identified that confers high affinity binding to GM-CSF R analagous to IL-2b.77,78 Using the mouse IL-3 R cDNA as a probe, a cDNA designated KH97 was obtained from the human erythroleukaemia cell line TF-I. KH97 encodes a 120kDa protein that has 56% amino acid identity with the low affinity murine IL-3 R, and has features in common with proteins of the cytokine receptor superfamily. KH97 was transfected to COS-7 cells and this system used to investigate the binding of cytokines to the KH97 product. Neither IL-3, IL-2, IL-4, IL-5, GM-CSF nor Epo showed any specific binding. To investigate the possibility that KH97 protein formed a component of another receptor, KH97 was co-transfected with the low affinity GM-CSF R cDNA. This resulted in the expression of both high and low affinity binding sites for GM-CSF. Further studies showed that KH97 protein is cross-linked to the low affinity GM-CSF R and that formation of the high affinity receptor does not require the cytoplasmic domain of KH97 protein. Dissociation kinetics show that high affinity binding is due to a slow dissociation of GM-CSF from the high affinity receptor. In view of the analagous situation with the IL-2 R the low affinity GM-CSF R has been designated the a-chain and the KH97 protein the B-chain7’ Neither the c1- nor B-chains of GM-CSF R have intrinsic tyrosine kinase activity, yet as discussed above, GM-CSF and IL-3 induce phosphorylation of a similar set of cytoplasmic phosphoproteins.33 Despite its structural homology with IL-3 R, the KH97 protein does not bind IL-3, however it is possible that in the same way as it confers high ffinity binding to GM-CSF R, it may be involved in forming the high affinity IL-3 R. A common component to the IL-3 and GM-CSF receptors might explain the overlap in biological activities of these cytokines. GM-CSF R has been demonstrated in immature haemopoietic progenitors, mature peripheral blood cells, leukaemic cells and leaukaemia cell lines, and it has been shown that the greatest expression of receptors appears on the most differentiated cells,79-81 reflecting the fact that GM-CSF not only acts as a growth and differentiating factor, but also enhances the biological properties of differentiated cells. Binding of GM-CSF to its receptor results in rapid ligand-receptor internalization.80 GM-CSF can significantly downregulate its own receptor and this may represent an important regulatory mechanism to limit the proliferative effects of the ligand.79 GM-CSF has been shown to be a growth factor for leukaemic blasts and many cell lines in vitro.82-85 Autocrine secretion of GM-CSF has been postulated to account for this, but equally over expression of GM-CSF R, mutations of GM-CSF R or failure of receptor down

200

THE CYTOKINE RECEPTOR SUPERFAMILY

regulation may be important leukaemias.

in the pathogenesis of

Applications of Growth Factor Receptors and Future Prospects The binding of haemopoietic growth factors to specific cell surface receptors, triggers a cascade of intracellular events resulting in cell proliferation and differentiation. Knowledge of ligand-receptor-signal pathways is important in understanding the regulation of haemopoiesis and the immune response, and the pathophysiology of malignant disease. There is considerable evidence that implicates the autocrine and paracrine secretion of growth factors in the pathogenesis of haematological malignancies.85-87 For example it has been demonstrated that in acute myeloid leukaemias, clonogenic blast cells (CFU-Bl) may proliferate in response to a number of haemopoietic growth factors such as GM-CSF, G-CSF, IL-l, and IL-258~82~85-gowhich are secreted by the leukaemic cells in an autocrine manner. Whether autocrine growth is stimulated by a growth factor or depends on receptor expression or intracellular binding of the growth factor is unclear at present, but is of importance in devising future therapeutic strategies. It is likely however that mechanisms involved may vary with respect to tumour type. For example, using antibodies to growth factors present in cell culture supernatants of CFU-AML, it has been shown that the proliferation of these cells can be markedly inhibited. *’ This argues in favour of the classical autocrine loop hypothesis. By contrast, evidence exists for autocrine growth mechanisms acting through the intracellular action of growth factors. Experiments where the genes coding for murine IL3 and the PDGR analogue p28 (v-sis) have been mutated in order to prevent secretion of these growth factors from cells normally expressing their receptors, have resulted in autonomous cell growth without detectable levels of secreted growth factors.g1’g2 Autocrine secretion of IL-6 has been implicated in the pathogenesis of multiple myeloma and plasma cell dyscrasias.” Myeloma cells constitutively secrete IL6 and express IL-6 R. IL-6 can augment the proliferation of plasma cells in vitro, which can be blocked by anti-IL-6. Furthermore bone marrow stromal cells have been shown to produce IL-6 in multiple myeloma, implying a paracrine mechanism for plasma cell growth. However in concordance with the multistep evolution of the clonal proliferation of plasma cells, other factors such as rearrangements of C-MYC oncogene and mis-sense mutations of the RAS genes, have been shown to have a pathogenic role. However the precise sequence of events remains unclear and the role of the autocrine loop in this sequence requires elucidation. There are prospects therefore for novel cancer therapies including antibodies or drugs directed at growth factors of growth factor receptors which might be effective in blocking an autocrine or

paracrine loop. Indeed a trial of anti-IL-6 is underway in multiple myeloma. In the same way, soluble receptors, capable of ligand binding but not signal transduction, might be used to block the action of a cytokine in an autocrine loop. Mutations within a growth factor receptor may confer oncogenic potential. Recent in vitro work has shown that point mutations within the receptor for M-CSF coded by FMS have transforming activity.” Furthermore point mutations within the M-CSF receptor (c-fms) at codons 301 or 969 analogous to those found in v-fms have been found in vivo and may result in a constitutively active or upregulated receptor with resultant cell transformation.g3 Similarly mutations within the stem cell growth factor receptor gene (c-kit) result in autonomous cell growth,22,24 and the recent demonstration of a point mutation in the murine Epo R resulting in autonomous cell growth,‘j5 suggests that human Epo R may be a potential proto oncogene. Other oncogenes may confer growth autonomy by affecting signal transduction pathways either in the cytoplasm (e.g. ras) or the nucleus (e.g. myc, myb,fos). Haemopoietic growth factors and cytokines are playing an increasing part in cancer therapy; they hold potential for accellerating recovery following myelosuppressive therapy, they are being investigated for their potential to eliminate the leukaemic clone in myelodysplastic syndromes through differentiating clonogenic cells. g4 However, growth factor receptors are present on many leukaemic ce11s,58~88-go*g5~g6 and increasingly they are being found outside the haemopoietic system, e.g. GM-CSF R in small cell lung cancer I1 G-CSF R and Epo R in the liver and placen;a.‘3,14,63 A knowledge of growth factor receptor distribution and expression particularly on malignant cells, may guide as to the appropriate choice of growth factors in therapy, avoiding those that might proliferate a malignant clone. As well as furthering our understanding of the pathophysiology of disease, cytokine receptors have potential diagnostic uses. Soluble receptors have been found for IL-2a, IL-4, IL-5, IL-6, and IL-7, and it is likely that soluble forms of the other receptors in this superfamily exist. Elevated levels of soluble IL2cr R occur in hairy cell leukaemia, Hodgkin’s disease, non-Hodgkin’s lymphomas and ATLL. Serum levels of sIL-201 R have been shown to be a reliable marker of disease bulk in HCL, and can be used to monitor response to treatment. Multiple myeloma is a patchy disease, and can be difficult to stage and monitor therapy. Soluble IL-6 R may prove to be a useful marker of disease burden and response to treatment in this condition and studies are underway to evaluate its use. Potential benefits from cytokine receptor studies have not been confined to cancer treatments. Monoclonal antibodies to (Tat), have been used in the treatment of graft versus host disease following bone marrow transplantationg7 and to treat rejection in

BLOOD REVIEWS

organ transplantation, ‘* by preventing IL-Zdependent T-cell proliferation. In years to come this list is likely to be considerably longer. Concluding Remarks The identification of a novel group of receptor proteins having sequence homology, common cl-helix structure and other similarities supports the view that the cytokine network appears to be evolutionarily related. The inclusion of receptors for growth hormone and prolactin in this superfamily is not surprising as we find that cytokines and their receptors are increasingly identified in roles outside the haemopoietic system. The study of cytokine receptors and mechanisms of signal transduction has a long way to go, but is already furthering our understanding of regulation of haemopoiesis and the immune response and pathophysiology of diseases. Manipulation of the ligandreceptor-signal axis holds promise for diagnostic and therapeutic advances. References 1. Nicola N A 1989 Haemopoietic growth factors and their

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