- Email: [email protected]
A new hematopoietic growth fator receptor superfamily: structural features and implications for signal transduction
A new hematopoietic growth factor receptor superfamily: structural features aid implications for signal transduction A.D. D’Andrea*, *Whitehead
C.D. Fasmant,
H.F. Lodish*
tDepartment of Biochemistry, Brandeis institute, Cambridge, Massachusetts, Waltham, Massachusetts, and li Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA Current
Opinion
in Cell Biology
University,
1990, 2:64&651
Introduction
Extracytoplasmic
KQhin the last year, several new growth factor receptor complementary DNAs (cDNAs) have been isolated, primarily using expression cloning strategies. Analysis of the predicted amino acid sequences of these receptors by sequence and structural-pattern matching techniques has revealed a new growth factor receptor superfamily. The superfamily includes l&and-binding subunits of the receptors for the hematopoietic growth factors erythropoietin (EPO) [I], interleukin (IL)-2 [2], IL-3 [3], IL-4 [4], ~-6 (Yamasaki et al, Science 1988, 241:825-828), IL.-7 [ 51, granulocyte-macrophage colony-stimulating factor (GM-CSF) [6] an d granulocyte colony-stimulating factor (G-CSF) [7], as well as the receptors for growth hormone (Leung et a~!, Nature 1987, 330:537-5431, and prolactin (Boutin et al, Cell 1988, 53~69-77).
Investigators have recently noted other striking similarities among the growth factor receptors [9,10]. All are type 1 membrane-spanning proteins and all are synthesized with a cleaved signal sequence. In the extracytoplas mic domain, members of the receptor superfamily have four cysteines spaced approximately 9, 27, and 15 amino acids apart and also a Trp-Ser-X-Trp-Ser motif positioned just outside the membrane. More recently, other conserved amino acids, including several proline residues, have been observed as part of the extracytoplasmic domains of these receptors [ 51. In contrast, it is possible to identify differences among the receptor types, which may be important for determining the degree of relatedness. The IL-7R lacks two of the conserved cysteines. On the basis of conserved amino acids, the growth hormone receptor and prolactin receptor are more related to each other than to other members of the receptor superfamily. The mature IL6R has a Trp-Ser-X-Trp-Ser motif further from the transmembrane region and, in addition, has an extracellular 86-amino-acid N-terminus that is unrelated to this cytokine receptor family but instead related to the immunoglobulin superfamily. The IL.-3R has an internal duplication of part of the extracytoplasmic domain (Fig. 1). Finally, although the EPO-R and IL-2R P-chains can be aligned on the basis of a second hydrophobic region in the extracytoplasmic region, the other members of the superfamily do not contain this region.
Even prior to their cloning, the hematopoietic growth factor receptors appeared to have several features in common. The receptors are found at low density on the cell surface (generally less than 1000 binding sites per cell) and, in several cases, display multiple subunits, shown by crosslinking analysis, and multiple affinities, shown by equilibrium binding analysis. Although no consistent biochemical mechanism of signal transduction was apparent, occupancy of only a small fraction of the cell surface receptors appeared to initiate a mitogenic signal. Whether the receptor-mediated signal resulted in a proliferative or a differentiative response frequently depended on the concentration of growth factor present or on the characteristics of the particular cell line employed. After publication of the primary amino acid sequences of EPO receptor (R) and IL-2R, we noted gross structural similarities between their P-chains [8]. Both polypeptides are type 1 membrane-spanning proteins, approximately 500 amino acids in length with a single, central transmembrane domain and a cytoplasmic tail that is rich in proline, glutamate, and aspartate. An alignment of these polypeptides revealed approximately 35% amino acid identity in a region of 130 amino acids that included the transmembrane domain [8].
Cytoplasmic
region
region
homology
homology
Analysis of the cytoplasmic domain sequences reveals no clues about signalling mechanisms. The cytoplasmic domains differ greatly in length (Fig. l), and no homologies with protein kinases or with sequences found at phosphorylation acceptor sites for protein tyrosine kinases or protein kinase C are observed. Within the superfamily, there is a subfamily that includes the EPO, K-2, K-3, and IL-4 receptors (Fig. 1); aJ have a high percentage of serine residues and proline residues in their cy toplasmic region, suggesting that they use similar signal
Abbreviations ECF-epidermal CM-CSF-granulocyte-macrophage
648
growth factor colony-stimulating
@ Current
cDNA--complementary factor; IL-lnterleukin;
Biology
DNA; EPO-qhropoietin; R-receptor; SFW-spleen
Ltd ISSN 0955-0674
focus-forming
virus.
A new
hematopoletlc
growth
tactor
receptor
supertamlly
U’AncWea,
tasmafi,
Lodlsh-
IL-4R
Fig. 1 Schematic representation of the hematopoietic growth factor receptor superfamily. Cl-C4 denote the four conserved cysteine residues of the extracytoplasmic domain. Note C2 and C4 are absent from the IL-7R. The black box in the extracytoplasmic domain denotes the conserved Trp-Ser-X-Trp-Ser motif, which is absent from the growth hormone receptor (CRH). The cytoplasmic domains of the receptors differ greatly in length. The cytoplasmic domains of the EPO-R, IL-2R P-chain, IL-3R, and IL-4R are related (shaded box) and may function through common signaling mechanisms.
transduction mechanisms. Significantly, Hatakeyama et al. [ll ] have recently shown that the conserved proximal region of the cytoplasmic domain of the IL-2R P-chain is sufficient to signal IL-2-dependent growth of Ba/F3 lymphocytes, and we have similar results with the EPO-R (D’Andrea, unpublished observation). Some of the receptors of the superfamily have multiple representatives differing in the length of the cytoplasmic tail, presumably as a result of alternative splicing. The IL-4R can even exist as a truncated, non-membrane anchored polypeptide [4]. On the basis of the structural similarities among these receptors, several testable hypotheses can be generated. First, the structural homologies of the receptor superfamily may extend to structural homologies among the growth factors themselves. The crystal structure of growth hormone reveals an antiparallel, four-helix bundle core, which might lead us to expect a similar tertiary structure for other growth factors such as EPO or IL-~, although these factors have no amino acid homologies. Second, the conserved Trp-Ser-X-Trp-Ser motif proximal to the membrane may serve some effector function that
is common to all receptor family members, such as the binding of a shared second subunit important for signal transduction. Implications
for signal
transduction
In addition to the shared structural features of the receptor superfamily, the receptors may use a common mechanism of signal transduction. For instance, EPO-R, stably expressed in the IL3-dependent cell line Ba/F3, confers EPO dependence upon these cells [ 12,131. Also, expression of the human IL-4R in the IL-2-dependent cell line CTLL, confers IL-4 dependence. Care must be taken in evaluating these results; for example, epidermal growth factor (EGF) receptor, a receptor with a tyrosine kinase catalytic domain, confers EGF responsiveness on IL-3-dependent cells, even though there is no structural similarity between the cytoplasmic domains of the EGF-R and the IL-3R (Pierce et al, Science 1988, 239:628-631). One way to understand fully the signal transduction by the cytokine receptors will be through an understand-
d b4Y
650
Membranes
ing of the additional receptor subunits. Some cytokine receptor members already have well defined second subunits. For instance, the IL-2R P-chain (~75) forms heterodimeric complexes with the p55 (Tat) subunit, even in the absence of IL-2. The p55 subunit is not required for signalling, but does increase the aifinity of the IL-2R complex for IL-2 (see [l4] >. In contrast, the IL-6R binds to a second subunit, gp130, only in the presence of IL-6 [ 151. That gp130 subunit is the signal transducer is suggested by the observation that a soluble, extracytoplasmic truncated form of the IL-GR, rebound to IL-~, will promote growth when bound to the gp130 subunit. The association of cytokine receptors with critical signal transducing polypeptides, such as gp130, fundamentally distinguishes them from the EGF family of receptors which contain an intramolecular tyrosine kinase in their cytoplasmic domain. Further, the expression of the additional cytokine receptor subunits may be developmentally regulated. For instance, expression of Tut is induced by IL-2 binding to the p75 subunit of the IL-2 receptor, thus increasing the sensitivity of the cells to low concentrations of IL-2 [ 14,181. This could be an important cellular mechanism to control the particular stage of differentiation at which a cell is responsive to a growth factor. Several lines of evidence suggest that tyrosine phosphotylation is a component of the receptor superfamily signalling mechanism. The IL2R, bound to IL-2, activates cellular tyrosine kinase activity and the IL-2R p-chain (~75) is itself phosphorylated in response to IL-2 binding (Mills et aA, J Biol cbem 1330, 265:3561-3567). It is possible that the cytokine receptors are non-covalently associated with a second tyrosine kinase subunit in a similar marner to the association of CD4 with the tyrosine kinase, 1cK [16]. Also, IL-3 stimulates the tyrosine phosphorylation of a 140 kD protein that may be a subunit of the IL-3 receptor [ 171. While a role for phosphorylation is supported by these experimental data, other second messengers for signal transduction have apparently been ruled out by studies with IL-2R [ 181. According to several independent investigations (reviewed ln [14,18]), the IL-2. mediated mitogenie signal does not involve cyclic AMP or phosphatidylnositol hydrolysis or elevations of Ca2+. Given the structural similarities of these growth factor receptors, any molecular insight into one particular receptor member, such as its subunit structure, the means of generation of multiple affiities, or the method of signalling, should be generalizable to the other family members. Such an insight is provided by the recent observation that the glycoprotein, gp55, encoded by the Friend spleen focus-forming virus (SFFV) binds to and stimulates the EPO-R. It is well known that a class of viruses, the mink cell focus-forming viruses, promote cellspecific proliferation; the gp55, encoded by SFFV, can itself induce EPO-independent proliferation of erythroblasts and its expression accounts for the early stage of Friend erythroleukemia - that is, the Friend SFFV transforms erythroblasts to a growth-factor-independent state by producing an envelope protein capable of activating the EPO receptor directly We have recently shown a physical and functional interaction between the EPO-R
and the gp55 envelope protein [ 121. It is therefore possible that analogous retroviruses could transform other cell types using, for example, the IL-2R or the IL-3R. An unusual aspect of the EPO system concerns receptor metabolism. The EPO-R is synthesized as a 64kD species with one N-linked high mannose sugar. Within 30-60mi1-1 of manufacture, much of this protein has passed through the Golgi apparatus, and emerges as a mature 66kD species containing complex oligosaccharides. Surprisingly, most of the fully processed 66kD EPO-R is not found on the cell surface; presumably, it is en route either from the trunsGolgi to the cell surface or from the cell surface to the lysosome. The 66kD receptor has a half-life of only 60 min. We predict that other hematopoetic growth factor receptors will have a similar cell surface distribution and metabolism (Yoshimura et al, Proc Nati Acud Sci USA 1!990,87:413H143). Importantly, when the SFFV gp55 is co-expressed with EPO-R, very little mature EPO-R is generated; most newly made EPO-R remains in the endoplasmic reticulum, bound to gp55, and has a half-life of approximately 2 h. This raises the intriguing possibility that a growth-promoting signal from the gp55/EPO-R complex is generated from within the cell, and not from the plasma membrane. Conclusion
A new growth factor receptor superfamily has been identified on the basis of various extracytoplasmic and cytoplasmic amino acid sequence homologies. There is currently little data available on specific second messengers which may be involved in the signal transduction by the receptors in this superfamily. Several members of the superfamily, including the EPO-R, the IL-2R and the IL-3R, have multiple receptor ailinities and multiple subunits. We have recently demonstrated that a retroviral envelope protein, the gp55 of the Friend SFFV, directly activates the EPO-R This raises the intriguing possibility that other members of the receptor superfamily are activated by an analogous mechanism. Annotated reading l
.* 1.
references
Of interest Of outstanding
and recommended
interest
D’ANDREA AD, LODL~H HP, WONG GG: Expression the murine erythropoietirt receptor. Cell 1989, &s paper describes the cloning of the cDNA encoding the mouse erythroleukemia cells. The cDNA, expressed in COS the expression of high (2OpM) and low (2OOpM) atfinity EPO crosslinked to its receptors generates two crosslinked
cloning of 57:177-285. EPO-R from cells, directs receptors. bands.
2. 0
HATAKEY~ M, Tsuw M, MJNMOTO S, KONO T, DOI T, MNATA T, MNA.C&A M, TANIGUCHI T: Interleulcin-2 receptor p-chain gene: generation of three receptor forms by cloned human a and g-chain cDNAs. Science 1989, 244:551-556. Using a monoclonal antibody directed against the lL2R g-chain, these investigators isolated the cDNA encoding the b-chain (~75) by an expression cloning strategy in COS cells. The p75 polypeptide, coexpressed with Tat (p55), generates high-afTinky receptors for IL-2. 3. l
ITOH N, YOHEHARA S, !%HREURS J, CORMAN DM, MARtNAhtANA K, ISHU A Y,wtAkt~ I, ARAI K-l MNAJ~MA A: Cloning of an
A new hematopoietic interleukin-3 receptor gene: a member of a distinct receptor gene family. Science 1990, 247:32C327. Fibroblasts &ansfected~ with IL-3R cDNA bound IL-3 with a low Alanity. No consensus sequence for a tyrosine kinase was present in the cytoplasmic domain. 4. 0
MOS~EY B, BECKMANN MP, MARCH CJ, IDZERDA RI+ GUMPEL SD, VAND~NBOS T, F~UEND D, ALPERT A, ANDERSON D, JACKSON J: The murine interleukin-4 receptor: molecular cloning characterization of secreted and membrane bound forms. Cell 1989, 59:33%348. Fluorescence-acthated celI sorting of a subclone of CTLL which expresses more than one million receptors per cell surface was accomplished. Then these cells were used as a source of messenger RNA for preparation of an expression cDNA Library and isolation of the IL-4R clone from COS cells. 5. 0
GOODY RG, FRIEND D, ZIEGLER SF, JERZY R. FAIX BA, GWPEL S, COWAN D, DOWER SK, MARCH CJ, NAMEN AE, PARK Is: Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily. Ceil 1990, 60:941-951. The binding properties and molecular size of the recombinant IL-7R were comparable to those of the native receptor. In addition, several cDNA clones were isolated that encode a secreted form of the IL-7R capable of binding IL-7 in solution. 6. 0
GEARING DP, KING JA, GOUGH NM, NICOLA NA: Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J 1989, 8~3667-3676. A cDNA clone encoding a receptor for human GM-CSF was isolated by expression screening of a library made from human placental mes senger RNA Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodlnated GM-CSF using a sensitive microscopic autoradiographic approach. 7. 0
F~KLINAYA R, ISHIX-XIKEDA E. Sflo Y, NACATA S: Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell 1990, 61:341-350. Two cDNAs encoding the receptor for murine G-CSF were isolated by expression screening of a mouse myeloid leukemia NSF-60 cell library. The deduced amino-acid sequence shows that the G-CSF-R is an 812. amnio-acid polypeptide with a single transmembrane domain. The extraceUular domain contains a 220.amino-acid region very similar to rat prolactin receptor. The cytoplasmic domain shows significant similarity to parts of the cytoplasmic domain of murine IL-4-R.
growth
factor
receptor
superfamily
D/Andrea,
Fasman,
Lodish
10.
IDZERDA Rl, MARCH CJ, MOSLEY B: Human interleukin 4 receptor confers biologic responsiveness and defines a novel receptor superfamily. J Erp Med 1990, 171&l-873. A murine T cell line transfected with human IL-4R cDNA becomes responsive to human IL-4, demonstrating that the encoded protein contains sufficient information to transmit the IL-4 signal across the cell membrane. l
11. 00
HATAKEYAMA M, MOIU H, Dor T, TANTGUCHI T: A restricted cytoplasmic region of interleukin-2 receptor P-chain is essential for growth signal transduction but not for ligand biding and internalization. Cell 1989, 59:837+X45. By in vifro mutagenesis of the IL-2R P-chain cDNA, these authors identify a critical domain of the cytoplasmic tail of the IL-2R, ~75, that is essential for signal transduction. This domain contains a section of 50 amino acid residues that has 35% amino acid identity with a comparable section of the EPO-R 12.
LI J.P, D’ANDREA AD, LODISH HF, BALTIMORE D: Activation of cell growth by binding of Friend spleen focus-forming virus coprotein to the etythropoietin receptor. Nufure Ezc? % 3~762-764. The experiments presented demonstrate that the gp55 glycoprotein physically interacts (co-immunoprecipitates) with the EPO-R and that this interaction induces a mitogenic signal to the co-transfected cells. l
D’ANDREA AD, SZKLUT PJ, LODISH HF, ALDERMAN EM: Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies. Blood 1990, 75:874-880. Four monoclonal antibodies against EPO are characterized by their ability to block binding of EPO to its receptor. The two monoclonal artibodies that block binding also neutralize an EPO-dependent ceU line expressing the recombinant EPO-R. 13. 0
14.
SMITH KA The interleukin 2 receptor. Annu Ret, Cell Biol 1989, 5~397425. This very readable review describes many of the early experiments that biochemically defined the lL-2R and its signal transduction mechanisms. 0
D’ANDRFA AD, FA~MAN GD, L~DISH HF: Erythropoietin recepfor and interleukin receptor P-chain: a new receptor family. Cell 1989, 58:1023-1024. The EPO-R and IL-2R P-chains can be aligned on the basis of amino acid identity and on the basis of hydrophobic stretches of amino acids. Common sequences in the cytoplasmic domains suggest a common mechanism of signal transduction for members of this receptor famiIy.
TAGA T, HBI M, HIRATA Y. YAMASAJU K, YASUDAWA K, MATXJDA T. HIRANO T, KISHL~~OTO T: Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 1989, 58:573581. This paper indicates that the complex of IL-~ and IL-6R can interact with a non.ligand-binding membrane glycoprotein. gp130. extracellulady and thereby provide the IL-~ signal. VE~UET~F 4 BOOKMAN 1MA, HORAK EM, BOLEN JB: The CD4 16. l and CD8 T celI surface antigens are associated with the internal membrane tyrosine-protein kinase p56 Ick. Cell 1988. 55:301-308. The p56 Ick is specifically modulated with either CD4 or CD8 follow. ing antibody-mediated crosslinking of these molecules and a large fraction of the rotaI cellular Ick protein can be co-immunoprecipitated with these surface glycoproteins.
9. em
17. 0
8.
l e
BAZAN JF: A novel family of growth factor receptors: a common biding domain in the growth hormone, prolactin, erythropoietin, and IL-6 receptors, and the p75 IL2 receptor P-chain. Rio&m Bi@ys Res Commun 1989, l&1:788-793. By sequence and structural-pattern matching techniques, a new receptor family is revealed. The author discusses the evolutionary and Functional implications of this broad, mosaic receptor relationship, with particular reference fo possible structural resemblances between the cog nate growth factors.
15.
l
SORENSEN PH, MUI AL, KRYSTAL G: Interleukin-3 stimulates the tyrosine phosphorylation of the 140 kD interleukin-3 receptor. J Biol Cbem 1989, 264:19 25319 258. This paper suggest that, of the IL-3 receptor subunits, only the 140 kD receptor protein is tyrosine phosphorylated after IL-3 binding. 18. WAIDMANN TA The multi-subunit interleukin-2 receotor. . Annrr Ra* B&hem 1989, 58~875911. 0 This review describes the biochemistry of the IL-2R and provides an excellent discussion of the role of this receptor in the pathogenesis of various disease states.
651
C.D. Fasmant,
H.F. Lodish*
tDepartment of Biochemistry, Brandeis institute, Cambridge, Massachusetts, Waltham, Massachusetts, and li Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA Current
Opinion
in Cell Biology
University,
1990, 2:64&651
Introduction
Extracytoplasmic
KQhin the last year, several new growth factor receptor complementary DNAs (cDNAs) have been isolated, primarily using expression cloning strategies. Analysis of the predicted amino acid sequences of these receptors by sequence and structural-pattern matching techniques has revealed a new growth factor receptor superfamily. The superfamily includes l&and-binding subunits of the receptors for the hematopoietic growth factors erythropoietin (EPO) [I], interleukin (IL)-2 [2], IL-3 [3], IL-4 [4], ~-6 (Yamasaki et al, Science 1988, 241:825-828), IL.-7 [ 51, granulocyte-macrophage colony-stimulating factor (GM-CSF) [6] an d granulocyte colony-stimulating factor (G-CSF) [7], as well as the receptors for growth hormone (Leung et a~!, Nature 1987, 330:537-5431, and prolactin (Boutin et al, Cell 1988, 53~69-77).
Investigators have recently noted other striking similarities among the growth factor receptors [9,10]. All are type 1 membrane-spanning proteins and all are synthesized with a cleaved signal sequence. In the extracytoplas mic domain, members of the receptor superfamily have four cysteines spaced approximately 9, 27, and 15 amino acids apart and also a Trp-Ser-X-Trp-Ser motif positioned just outside the membrane. More recently, other conserved amino acids, including several proline residues, have been observed as part of the extracytoplasmic domains of these receptors [ 51. In contrast, it is possible to identify differences among the receptor types, which may be important for determining the degree of relatedness. The IL-7R lacks two of the conserved cysteines. On the basis of conserved amino acids, the growth hormone receptor and prolactin receptor are more related to each other than to other members of the receptor superfamily. The mature IL6R has a Trp-Ser-X-Trp-Ser motif further from the transmembrane region and, in addition, has an extracellular 86-amino-acid N-terminus that is unrelated to this cytokine receptor family but instead related to the immunoglobulin superfamily. The IL.-3R has an internal duplication of part of the extracytoplasmic domain (Fig. 1). Finally, although the EPO-R and IL-2R P-chains can be aligned on the basis of a second hydrophobic region in the extracytoplasmic region, the other members of the superfamily do not contain this region.
Even prior to their cloning, the hematopoietic growth factor receptors appeared to have several features in common. The receptors are found at low density on the cell surface (generally less than 1000 binding sites per cell) and, in several cases, display multiple subunits, shown by crosslinking analysis, and multiple affinities, shown by equilibrium binding analysis. Although no consistent biochemical mechanism of signal transduction was apparent, occupancy of only a small fraction of the cell surface receptors appeared to initiate a mitogenic signal. Whether the receptor-mediated signal resulted in a proliferative or a differentiative response frequently depended on the concentration of growth factor present or on the characteristics of the particular cell line employed. After publication of the primary amino acid sequences of EPO receptor (R) and IL-2R, we noted gross structural similarities between their P-chains [8]. Both polypeptides are type 1 membrane-spanning proteins, approximately 500 amino acids in length with a single, central transmembrane domain and a cytoplasmic tail that is rich in proline, glutamate, and aspartate. An alignment of these polypeptides revealed approximately 35% amino acid identity in a region of 130 amino acids that included the transmembrane domain [8].
Cytoplasmic
region
region
homology
homology
Analysis of the cytoplasmic domain sequences reveals no clues about signalling mechanisms. The cytoplasmic domains differ greatly in length (Fig. l), and no homologies with protein kinases or with sequences found at phosphorylation acceptor sites for protein tyrosine kinases or protein kinase C are observed. Within the superfamily, there is a subfamily that includes the EPO, K-2, K-3, and IL-4 receptors (Fig. 1); aJ have a high percentage of serine residues and proline residues in their cy toplasmic region, suggesting that they use similar signal
Abbreviations ECF-epidermal CM-CSF-granulocyte-macrophage
648
growth factor colony-stimulating
@ Current
cDNA--complementary factor; IL-lnterleukin;
Biology
DNA; EPO-qhropoietin; R-receptor; SFW-spleen
Ltd ISSN 0955-0674
focus-forming
virus.
A new
hematopoletlc
growth
tactor
receptor
supertamlly
U’AncWea,
tasmafi,
Lodlsh-
IL-4R
Fig. 1 Schematic representation of the hematopoietic growth factor receptor superfamily. Cl-C4 denote the four conserved cysteine residues of the extracytoplasmic domain. Note C2 and C4 are absent from the IL-7R. The black box in the extracytoplasmic domain denotes the conserved Trp-Ser-X-Trp-Ser motif, which is absent from the growth hormone receptor (CRH). The cytoplasmic domains of the receptors differ greatly in length. The cytoplasmic domains of the EPO-R, IL-2R P-chain, IL-3R, and IL-4R are related (shaded box) and may function through common signaling mechanisms.
transduction mechanisms. Significantly, Hatakeyama et al. [ll ] have recently shown that the conserved proximal region of the cytoplasmic domain of the IL-2R P-chain is sufficient to signal IL-2-dependent growth of Ba/F3 lymphocytes, and we have similar results with the EPO-R (D’Andrea, unpublished observation). Some of the receptors of the superfamily have multiple representatives differing in the length of the cytoplasmic tail, presumably as a result of alternative splicing. The IL-4R can even exist as a truncated, non-membrane anchored polypeptide [4]. On the basis of the structural similarities among these receptors, several testable hypotheses can be generated. First, the structural homologies of the receptor superfamily may extend to structural homologies among the growth factors themselves. The crystal structure of growth hormone reveals an antiparallel, four-helix bundle core, which might lead us to expect a similar tertiary structure for other growth factors such as EPO or IL-~, although these factors have no amino acid homologies. Second, the conserved Trp-Ser-X-Trp-Ser motif proximal to the membrane may serve some effector function that
is common to all receptor family members, such as the binding of a shared second subunit important for signal transduction. Implications
for signal
transduction
In addition to the shared structural features of the receptor superfamily, the receptors may use a common mechanism of signal transduction. For instance, EPO-R, stably expressed in the IL3-dependent cell line Ba/F3, confers EPO dependence upon these cells [ 12,131. Also, expression of the human IL-4R in the IL-2-dependent cell line CTLL, confers IL-4 dependence. Care must be taken in evaluating these results; for example, epidermal growth factor (EGF) receptor, a receptor with a tyrosine kinase catalytic domain, confers EGF responsiveness on IL-3-dependent cells, even though there is no structural similarity between the cytoplasmic domains of the EGF-R and the IL-3R (Pierce et al, Science 1988, 239:628-631). One way to understand fully the signal transduction by the cytokine receptors will be through an understand-
d b4Y
650
Membranes
ing of the additional receptor subunits. Some cytokine receptor members already have well defined second subunits. For instance, the IL-2R P-chain (~75) forms heterodimeric complexes with the p55 (Tat) subunit, even in the absence of IL-2. The p55 subunit is not required for signalling, but does increase the aifinity of the IL-2R complex for IL-2 (see [l4] >. In contrast, the IL-6R binds to a second subunit, gp130, only in the presence of IL-6 [ 151. That gp130 subunit is the signal transducer is suggested by the observation that a soluble, extracytoplasmic truncated form of the IL-GR, rebound to IL-~, will promote growth when bound to the gp130 subunit. The association of cytokine receptors with critical signal transducing polypeptides, such as gp130, fundamentally distinguishes them from the EGF family of receptors which contain an intramolecular tyrosine kinase in their cytoplasmic domain. Further, the expression of the additional cytokine receptor subunits may be developmentally regulated. For instance, expression of Tut is induced by IL-2 binding to the p75 subunit of the IL-2 receptor, thus increasing the sensitivity of the cells to low concentrations of IL-2 [ 14,181. This could be an important cellular mechanism to control the particular stage of differentiation at which a cell is responsive to a growth factor. Several lines of evidence suggest that tyrosine phosphotylation is a component of the receptor superfamily signalling mechanism. The IL2R, bound to IL-2, activates cellular tyrosine kinase activity and the IL-2R p-chain (~75) is itself phosphorylated in response to IL-2 binding (Mills et aA, J Biol cbem 1330, 265:3561-3567). It is possible that the cytokine receptors are non-covalently associated with a second tyrosine kinase subunit in a similar marner to the association of CD4 with the tyrosine kinase, 1cK [16]. Also, IL-3 stimulates the tyrosine phosphorylation of a 140 kD protein that may be a subunit of the IL-3 receptor [ 171. While a role for phosphorylation is supported by these experimental data, other second messengers for signal transduction have apparently been ruled out by studies with IL-2R [ 181. According to several independent investigations (reviewed ln [14,18]), the IL-2. mediated mitogenie signal does not involve cyclic AMP or phosphatidylnositol hydrolysis or elevations of Ca2+. Given the structural similarities of these growth factor receptors, any molecular insight into one particular receptor member, such as its subunit structure, the means of generation of multiple affiities, or the method of signalling, should be generalizable to the other family members. Such an insight is provided by the recent observation that the glycoprotein, gp55, encoded by the Friend spleen focus-forming virus (SFFV) binds to and stimulates the EPO-R. It is well known that a class of viruses, the mink cell focus-forming viruses, promote cellspecific proliferation; the gp55, encoded by SFFV, can itself induce EPO-independent proliferation of erythroblasts and its expression accounts for the early stage of Friend erythroleukemia - that is, the Friend SFFV transforms erythroblasts to a growth-factor-independent state by producing an envelope protein capable of activating the EPO receptor directly We have recently shown a physical and functional interaction between the EPO-R
and the gp55 envelope protein [ 121. It is therefore possible that analogous retroviruses could transform other cell types using, for example, the IL-2R or the IL-3R. An unusual aspect of the EPO system concerns receptor metabolism. The EPO-R is synthesized as a 64kD species with one N-linked high mannose sugar. Within 30-60mi1-1 of manufacture, much of this protein has passed through the Golgi apparatus, and emerges as a mature 66kD species containing complex oligosaccharides. Surprisingly, most of the fully processed 66kD EPO-R is not found on the cell surface; presumably, it is en route either from the trunsGolgi to the cell surface or from the cell surface to the lysosome. The 66kD receptor has a half-life of only 60 min. We predict that other hematopoetic growth factor receptors will have a similar cell surface distribution and metabolism (Yoshimura et al, Proc Nati Acud Sci USA 1!990,87:413H143). Importantly, when the SFFV gp55 is co-expressed with EPO-R, very little mature EPO-R is generated; most newly made EPO-R remains in the endoplasmic reticulum, bound to gp55, and has a half-life of approximately 2 h. This raises the intriguing possibility that a growth-promoting signal from the gp55/EPO-R complex is generated from within the cell, and not from the plasma membrane. Conclusion
A new growth factor receptor superfamily has been identified on the basis of various extracytoplasmic and cytoplasmic amino acid sequence homologies. There is currently little data available on specific second messengers which may be involved in the signal transduction by the receptors in this superfamily. Several members of the superfamily, including the EPO-R, the IL-2R and the IL-3R, have multiple receptor ailinities and multiple subunits. We have recently demonstrated that a retroviral envelope protein, the gp55 of the Friend SFFV, directly activates the EPO-R This raises the intriguing possibility that other members of the receptor superfamily are activated by an analogous mechanism. Annotated reading l
.* 1.
references
Of interest Of outstanding
and recommended
interest
D’ANDREA AD, LODL~H HP, WONG GG: Expression the murine erythropoietirt receptor. Cell 1989, &s paper describes the cloning of the cDNA encoding the mouse erythroleukemia cells. The cDNA, expressed in COS the expression of high (2OpM) and low (2OOpM) atfinity EPO crosslinked to its receptors generates two crosslinked
cloning of 57:177-285. EPO-R from cells, directs receptors. bands.
2. 0
HATAKEY~ M, Tsuw M, MJNMOTO S, KONO T, DOI T, MNATA T, MNA.C&A M, TANIGUCHI T: Interleulcin-2 receptor p-chain gene: generation of three receptor forms by cloned human a and g-chain cDNAs. Science 1989, 244:551-556. Using a monoclonal antibody directed against the lL2R g-chain, these investigators isolated the cDNA encoding the b-chain (~75) by an expression cloning strategy in COS cells. The p75 polypeptide, coexpressed with Tat (p55), generates high-afTinky receptors for IL-2. 3. l
ITOH N, YOHEHARA S, !%HREURS J, CORMAN DM, MARtNAhtANA K, ISHU A Y,wtAkt~ I, ARAI K-l MNAJ~MA A: Cloning of an
A new hematopoietic interleukin-3 receptor gene: a member of a distinct receptor gene family. Science 1990, 247:32C327. Fibroblasts &ansfected~ with IL-3R cDNA bound IL-3 with a low Alanity. No consensus sequence for a tyrosine kinase was present in the cytoplasmic domain. 4. 0
MOS~EY B, BECKMANN MP, MARCH CJ, IDZERDA RI+ GUMPEL SD, VAND~NBOS T, F~UEND D, ALPERT A, ANDERSON D, JACKSON J: The murine interleukin-4 receptor: molecular cloning characterization of secreted and membrane bound forms. Cell 1989, 59:33%348. Fluorescence-acthated celI sorting of a subclone of CTLL which expresses more than one million receptors per cell surface was accomplished. Then these cells were used as a source of messenger RNA for preparation of an expression cDNA Library and isolation of the IL-4R clone from COS cells. 5. 0
GOODY RG, FRIEND D, ZIEGLER SF, JERZY R. FAIX BA, GWPEL S, COWAN D, DOWER SK, MARCH CJ, NAMEN AE, PARK Is: Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily. Ceil 1990, 60:941-951. The binding properties and molecular size of the recombinant IL-7R were comparable to those of the native receptor. In addition, several cDNA clones were isolated that encode a secreted form of the IL-7R capable of binding IL-7 in solution. 6. 0
GEARING DP, KING JA, GOUGH NM, NICOLA NA: Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J 1989, 8~3667-3676. A cDNA clone encoding a receptor for human GM-CSF was isolated by expression screening of a library made from human placental mes senger RNA Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodlnated GM-CSF using a sensitive microscopic autoradiographic approach. 7. 0
F~KLINAYA R, ISHIX-XIKEDA E. Sflo Y, NACATA S: Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell 1990, 61:341-350. Two cDNAs encoding the receptor for murine G-CSF were isolated by expression screening of a mouse myeloid leukemia NSF-60 cell library. The deduced amino-acid sequence shows that the G-CSF-R is an 812. amnio-acid polypeptide with a single transmembrane domain. The extraceUular domain contains a 220.amino-acid region very similar to rat prolactin receptor. The cytoplasmic domain shows significant similarity to parts of the cytoplasmic domain of murine IL-4-R.
growth
factor
receptor
superfamily
D/Andrea,
Fasman,
Lodish
10.
IDZERDA Rl, MARCH CJ, MOSLEY B: Human interleukin 4 receptor confers biologic responsiveness and defines a novel receptor superfamily. J Erp Med 1990, 171&l-873. A murine T cell line transfected with human IL-4R cDNA becomes responsive to human IL-4, demonstrating that the encoded protein contains sufficient information to transmit the IL-4 signal across the cell membrane. l
11. 00
HATAKEYAMA M, MOIU H, Dor T, TANTGUCHI T: A restricted cytoplasmic region of interleukin-2 receptor P-chain is essential for growth signal transduction but not for ligand biding and internalization. Cell 1989, 59:837+X45. By in vifro mutagenesis of the IL-2R P-chain cDNA, these authors identify a critical domain of the cytoplasmic tail of the IL-2R, ~75, that is essential for signal transduction. This domain contains a section of 50 amino acid residues that has 35% amino acid identity with a comparable section of the EPO-R 12.
LI J.P, D’ANDREA AD, LODISH HF, BALTIMORE D: Activation of cell growth by binding of Friend spleen focus-forming virus coprotein to the etythropoietin receptor. Nufure Ezc? % 3~762-764. The experiments presented demonstrate that the gp55 glycoprotein physically interacts (co-immunoprecipitates) with the EPO-R and that this interaction induces a mitogenic signal to the co-transfected cells. l
D’ANDREA AD, SZKLUT PJ, LODISH HF, ALDERMAN EM: Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies. Blood 1990, 75:874-880. Four monoclonal antibodies against EPO are characterized by their ability to block binding of EPO to its receptor. The two monoclonal artibodies that block binding also neutralize an EPO-dependent ceU line expressing the recombinant EPO-R. 13. 0
14.
SMITH KA The interleukin 2 receptor. Annu Ret, Cell Biol 1989, 5~397425. This very readable review describes many of the early experiments that biochemically defined the lL-2R and its signal transduction mechanisms. 0
D’ANDRFA AD, FA~MAN GD, L~DISH HF: Erythropoietin recepfor and interleukin receptor P-chain: a new receptor family. Cell 1989, 58:1023-1024. The EPO-R and IL-2R P-chains can be aligned on the basis of amino acid identity and on the basis of hydrophobic stretches of amino acids. Common sequences in the cytoplasmic domains suggest a common mechanism of signal transduction for members of this receptor famiIy.
TAGA T, HBI M, HIRATA Y. YAMASAJU K, YASUDAWA K, MATXJDA T. HIRANO T, KISHL~~OTO T: Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 1989, 58:573581. This paper indicates that the complex of IL-~ and IL-6R can interact with a non.ligand-binding membrane glycoprotein. gp130. extracellulady and thereby provide the IL-~ signal. VE~UET~F 4 BOOKMAN 1MA, HORAK EM, BOLEN JB: The CD4 16. l and CD8 T celI surface antigens are associated with the internal membrane tyrosine-protein kinase p56 Ick. Cell 1988. 55:301-308. The p56 Ick is specifically modulated with either CD4 or CD8 follow. ing antibody-mediated crosslinking of these molecules and a large fraction of the rotaI cellular Ick protein can be co-immunoprecipitated with these surface glycoproteins.
9. em
17. 0
8.
l e
BAZAN JF: A novel family of growth factor receptors: a common biding domain in the growth hormone, prolactin, erythropoietin, and IL-6 receptors, and the p75 IL2 receptor P-chain. Rio&m Bi@ys Res Commun 1989, l&1:788-793. By sequence and structural-pattern matching techniques, a new receptor family is revealed. The author discusses the evolutionary and Functional implications of this broad, mosaic receptor relationship, with particular reference fo possible structural resemblances between the cog nate growth factors.
15.
l
SORENSEN PH, MUI AL, KRYSTAL G: Interleukin-3 stimulates the tyrosine phosphorylation of the 140 kD interleukin-3 receptor. J Biol Cbem 1989, 264:19 25319 258. This paper suggest that, of the IL-3 receptor subunits, only the 140 kD receptor protein is tyrosine phosphorylated after IL-3 binding. 18. WAIDMANN TA The multi-subunit interleukin-2 receotor. . Annrr Ra* B&hem 1989, 58~875911. 0 This review describes the biochemistry of the IL-2R and provides an excellent discussion of the role of this receptor in the pathogenesis of various disease states.
651