From the Comparison of the binding properties of
non-glycosylated, glycosylated human leukemia inhibitory factor
(LIF) and monoclonal antibodies (mAbs) directed at gp190/LIF-receptor
Leukemia inhibitory factor
(LIF)1 is a multifunctional,
highly glycosylated soluble protein belonging to the interleukin-6 (IL-6) subfamily of helical cytokines (also including IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1)) (1-3). Signal transduction by these cytokines is proposed to
result from cytokine-mediated homodimerization of the gp130 signal
transducer (4) or heterodimerization of gp130 with another signal
transducing subunit. IL-6 first binds with low affinity (nanomolar) to
a specific IL-6R Most of the cytokines of this subfamily have been shown to exert
biological activities on various cell types, both within and outside of
the hematopoietic system (1, 2). Due to the sharing of common
transducing receptor subunits, these cytokines also show overlapping
activities. In agreement with the multiple activities elicited by LIF,
high affinity (picomolar) LIF receptors have been demonstrated on a
number of cell lines (12). In addition, several cell lines have been
described to express only low affinity (nanomolar) LIF receptors, and
it was postulated that this was linked to the expression of gp190
(LIF-R Cell Lines, Antibodies, and Cytokines--
The human myeloma
U266 cell line and the human choriocarcinoma JAR cell line were
obtained from the American Type Culture Collection (ATCC, Rockville,
MD). U266 cells were cultured in RPMI 1640 supplemented with 10% fetal
calf serum (FCS), and JAR cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 8% FCS. CHO cells stably transfected
with the full-length gp190 cDNA (10) were cultured in RPMI 1640 containing 10% FCS. The anti-human gp190 mAbs have been recently
raised against a soluble form of the human LIF-R Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Analysis--
Total RNA was extracted from human cell lines using a
guanidinium thiocyanate/phenol method (17). The first cDNA strand was synthesized using total RNA (2 µg) at 45 °C for 30 min in a
100-µl reaction mixture containing 50 mM Tris-HCl (pH
8.3), 60 mM KCl, 10 mM dithiothreitol, 5 mM MgCl2, 20 units of RNase inhibitor
(Boehringer, Mannhein, Germany), 1 mM amounts of each deoxynucleotide triphosphate (dNTP), 600 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), and 2 µg of oligo(dT) 15-mer. 1 µl of the reaction mixture was made up to
25 µl using Taq polymerase buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM
MgCl2) containing 10 pmol of each primer and 0.6 unit of
Ampli-Taq DNA polymerase (Pharmacia Biotech, Uppsala, Sweden). Amplifications were performed using a thermal cycler for 35 cycles under the following conditions: denaturation for 1 min at
94 °C, annealing for 45 s at 59 °C, and elongation for 1 min
at 72 °C using gp190 primers: 5'-CGGGATCCAGGACTGACTGCATTGCAC and
antisense: 5'-AACAGCTGTTGAATTAATATCCTTC. PCR products were separated in
a 1% agarose gel and analyzed by Southern blotting with the
3.3-kilobase pair EcoRI insert from PKCR6-gp190 plasmid (10)
as a probe. Control PCR was performed using Radioiodination of LIF and Antibodies--
The different mAbs
were radiolabeled as described (14) according to a procedure using
IODOGEN as a catalyst. The specific radioactivities obtained were in
the range of 450-900 µCi/nmol. CHO LIF and E. coli LIF
were iodinated according to the chloramine T method as described (12).
LIF was labeled at a specific radioactivity of around 15,000 µCi/nmol
for high affinity binding studies and at a specific radioactivity of
around 1,800 µCi/nmol for binding studies under low affinity
conditions.
Binding and Competition Assays--
CHO or JAR cell monolayers
were incubated with PBS containing 0.05% trypsin and 0.02% EDTA to
bring the cells in suspension, washed with FCS containing culture
medium, and resuspended in PBS containing 0.5% bovine serum albumin
(PBS-BSA). U266 cells were washed prior to resuspension in PBS-BSA.
Binding experiments were carried out in PBS-BSA as described previously
(12). Cells (1×106/well in 96-well round-bottomed plates)
were incubated with increasing concentrations of labeled LIF or
anti-gp190 mAb in a final volume of 50 µl. Nonspecific binding was
evaluated by including a 100-fold excess of the respective unlabeled
cytokine or antibodies. Incubation was carried out under agitation for
90 min (equilibrium conditions) at 4 °C. Cell bound (B) and unbound
(F) fractions were separated by centrifugation through a layer of
dibutyl-phthalate (90%) and parrafin oil (10%). Regression analysis
of the binding data was accomplished using a one- or two-site
equilibrium binding equation (Grafit, Erithacus Software,
Staines, United Kingdom).
Groupe de Recherche
Cytokines/Récepteurs/Transduction,
CNRS-UMR 5540,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit showed that most of the low affinity (nanomolar) receptors
expressed by a variety of cell lines are not due to gp190. These
receptors bind glycosylated LIF produced in Chinese hamster ovary cells (CHO LIF) (Kd = 6.9 nM) but not
Escherichia coli-derived LIF or CHO LIF treated
with endoglycosidase F. CHO LIF binding to these receptors is neither
affected by anti-gp190 mAbs nor by anti-gp130 mAbs and is specifically
inhibited by low concentrations of mannose 6-phosphate (Man-6-P)
(IC50 = 40 µM), suggesting that they could be
related to Man-6-P receptors. The identity of this LIF binding
component with the Man-6-P/insulin-like growth factor-II receptor
(Man-6-P/IGFII-R) was supported by several findings. (i) It has a
molecular mass very similar to that of the Man-6-P/IGFII-R (270 kDa);
(ii) the complex of LIF cross-linked to this receptor is
immunoprecipitated by a polyclonal anti-Man-6-P/IGFII-R antibody; (iii)
this antibody inhibits LIF and IGFII binding to the receptor with
comparable efficiencies; (iv) soluble Man-6-P/IGFII-R purified from
serum binds glycosylated LIF (Kd = 4.3 nM) but not E. coli LIF. The potential role of
Man-6-P/IGFII-R in LIF processing and biological activity is
discussed.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit and the complex then recruits and
homodimerizes two gp130 subunits for signaling. A final hexameric
complex of two molecules each of IL-6, IL-6R
, and gp130 has been
proposed (5). Similarly, specific IL-11R
subunits have also been
identified (6, 7) and the IL-11/IL-11R
complex is also proposed to
induce homodimerization of gp130 for signaling. Other members of this
cytokine family can induce heterodimerization of gp130 with gp190, a
signaling molecule initially identified as the low affinity (nanomolar)
LIF receptor (or LIF-R
) (8). CNTF recruits gp130 and gp190 together
with a third cytokine specific receptor chain (CNTF-R
). LIF and OSM
have been proposed to require only gp130 and gp190 to form a common
signaling complex designated type I OSM receptor. Another type of OSM
receptor, not used by LIF, has been described (type II OSM receptor).
It involves the recruitment of gp130 with a recently identified gp180
transducing molecule also called OSM-R
(9). Cross-linking studies
have also suggested that another receptor subunit besides gp130 and gp190 might participate in the structure of the LIF receptor (10). Similarly, a cytokine specific receptor chain has been described by
cross-linking in the case of CT-1 (11).
) in the absence of gp130 (12). Evaluation of this hypothesis
was, however, hampered by the lack of anti-LIF-R
antibodies. In this
study, with the recent availability of such anti-LIF-R
mAbs (13,
14), we show that the low affinity LIF receptor expressed by a number
of cell lines is unrelated to gp190. This receptor binds LIF through
its carbohydrate moieties and displays biochemical, immunochemical, and
functional features, indicating that it is identical to the mannose
6-phosphate/insulin-like growth factor II receptor
(Man-6-P/IGFII-R).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(gp190) and their
initial characterizations were described elsewhere (13, 14). Polyclonal
antibodies against gp190 or gp130 were purchased from R&D Systems
(Minneapolis, MN). Anti-gp130 mAb BR3 was a kind gift from Dr John
Wijdenes (Diaclone, Besançon, France). LIF (CHO LIF) was purified
from serum-free conditioned medium of CHO cells transfected with a
full-length cDNA encoding for human LIF as described (12).
Recombinant Escherichia coli-derived human LIF (E. coli LIF) was obtained from PeproTech, Inc. (Rocky Hill, NJ).
Recombinant Sf21-derived human IL-9 (insect IL-9) was from R&D
Systems. Natural MG63-derived human IL-6 (osteosarcoma IL-6) was from
Sigma. A natural form of LIF was purified from A375 melanoma cells by
affinity chromatography on polyclonal anti-LIF antibodies coupled to
agarose beads (Affi-Gel, Bio-Rad). Its concentration was determined by
a specific enzyme-linked immunosorbent assay as described (15).
Monosaccharides were from Sigma. Affinity-purified rabbit polyclonal
antibodies against Man-6-P/IGFII-R were a kind gift from Dr. Stuart
Kornfeld (Washington University, St Louis, MO). Control antibody was a
rabbit polyclonal antibody (purified IgG) raised against human
haptoglobin (Sigma). Soluble Man-6-P/IGFII-R (extracytoplasmic domain)
was purified from bovine serum by affinity chromatography on
phosphomannan as described (16).
-actin primers: 5'-GGCTACAGCTTCACCACCAC and antisense: 5'-GCACTGTGTTGGCGTACAGG to check
RNA integrity.
Affinity Cross-linking and Immunoprecipitations-- Cross-linking was carried out as described (12). Cells were incubated with 5 nM radiolabeled LIF. After washing, cross-linking was accomplished through the addition of 1 mM ethylene glycolbis(succinimidyl succinate) (Pierce). Cells were then lysed in 10 mM Tris buffer, pH 7.4, containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mM iodoacetamide. The cell lysate was centrifuged and the resulting pellet solubilized in 125 mM Tris buffer, pH 6.8, containing 1 mM phenylmethylsulfonyl fluoride, 3 mM EDTA, 10 mM sodium fluoride, and 0.1% SB14 (solubilization buffer). Solubilized samples were submitted to SDS-PAGE (3-9% acrylamide gradient) under reducing condition. Radiolabeled bands were visualized by autoradiography (PhosphorImager, Molecular Dynamics Inc., Sunnyvale, CA). For immunoprecipitations, cross-linked and solubilized cell samples were heated at 90 °C before adding polyclonal antibodies and protein A-agarose beads (Bio-Rad). After incubating under agitation overnight at 4 °C, agarose beads were centrifuged, washed in solubilization buffer, and submitted to SDS-PAGE and autoradiography as described above.
Surface Plasmon Resonance Studies-- These experiments were performed with the BIACore 2000 optical biosensor (BIACore, Uppsala, Sweden). Man-6-P/IGFII-R extracytoplasmic domain purified from bovine serum was covalently coupled through primary amino groups to a carboxymethyl dextran flow cell (CM5, BIACore) as recommended by the manufacturer. The coupling reaction was carried out for 7 min at a flow rate of 5 µl/min and at a protein concentration of 0.1 mg/ml in 10 mM acetate buffer, pH 5.0. The level of immobilization obtained was 13,000 resonance units corresponding to a surface concentration of 13 ng/mm2. A control dextran flow cell was prepared by blocking carboxymethyl groups with ethanolamine. Binding of CHO LIF, E. coli LIF, or IGFII was assayed at different concentrations from 5 nM to 1 µM in Hepes-buffered saline (BIACore) and at a flow rate of 10 µl/min. Association was monitored for 10 min before initiating the dissociation phase for another 10 min with Hepes-buffered saline buffer. Regeneration of the flow cells was achieved with 5 mM HCl for 30 s. The resonance signal measured on the control flow cell was subtracted from the signal measured on the experimental flow cell. The resulting sensorgrams were analyzed using the BIAEvaluation (BIACore) software.
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RESULTS |
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U266 cells Express a LIF Receptor That Does Not Involve gp190-- In the course of studies investigating the expression of gp190, gp130 and LIF receptors by various cell lines, a surprising observation was that the plasmocytoma cell line U266 expressed LIF receptors but did not bind anti-gp190 mAbs. Indeed, as shown in Fig. 1A, U266 cells display a single class of low affinity binding sites for glycosylated, CHO-derived LIF (24,200 sites/cell, Kd = 6, 9 nM) but no detectable binding of the anti-human gp190 mAb 1B4. This is in sharp contrast with Fig. 1B, which shows that CHO cells transfected with human gp190 bind CHO LIF and 1B4 mAb with similar maximal binding capacities (11,500 and 11,700 sites/cell, respectively). Five other anti-gp190 mAbs, which have previously been shown to react with separate epitopes in addition to the one recognized by 1B4 (13, 14), also bound to gp190-CHO cells without detectable binding to U266 cells (data not shown). In agreement with previous results (18), U266 cells expressed 790 binding sites for the anti-gp130 mAb BR3 (Kd = 0.68 nM, Fig. 1A).
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The LIF Receptor on U266 Binds Glycosylated LIF but Not Non-glycosylated LIF-- Another surprising observation was that U266 cells did not bind non-glycosylated, E. coli-derived LIF (Fig. 1A). This again was in sharp contrast to gp190-transfected CHO cells, which bound the non-glycosylated cytokine with a stoichiometry (11,200 sites/cell) similar to that for the glycosylated species (11,700 sites/cell) (Fig. 1B). Additional experiments showed that treatment of glycosylated LIF with Endoglycosidase F, an enzyme that removes the N-linked carbohydrates, completely abrogated LIF binding to U266 cells, while leaving its binding to gp190-CHO cells unaffected (data not shown).
We then analyzed whether E. coli LIF could compete for CHO LIF binding. As indicated in Fig. 3A, CHO LIF binding to U266 cells was not affected by excess amounts of E. coli LIF, whereas similar amounts of E. coli LIF completely abolished CHO LIF binding to gp190-CHO cells (Fig. 3B). Fig. 3C shows dose-response curves for inhibition of CHO LIF binding to U266 cells. E. coli LIF has no effect on this binding over a wide concentration range. As expected, unlabeled CHO LIF competes with its labeled counterpart with an IC50 (12 nM) in agreement with the Kd of CHO LIF binding to U266 cells (Kd = 6.9 nM; see Fig. 1A). A glycosylated form of LIF immunopurified from cell culture supernatants of the A375 human melanoma cell line also competed with CHO LIF binding with efficiency similar to that for unlabeled CHO LIF. In contrast, natural glycosylated human IL-6 (purified from MG63 osteosarcoma cells) or glycosylated recombinant human IL-9 derived from insect cells were without effect on CHO LIF binding. These data reinforce the notion that the LIF receptor of U266 cells is unrelated to gp190. They further demonstrate that it specifically binds glycosylated forms of LIF, and suggest N-linked carbohydrate involvement in this binding.A Large Number of Cell Lines Express Low Affinity LIF Receptors Similar to Those Expressed by U266 Cells-- The JAR cell line has been previously shown in our laboratory to express both low and high affinity receptors for glycosylated LIF (10, 12, 14). As shown in Fig. 4A, the binding of glycosylated CHO LIF to JAR cells is characterized by a curvilinear Scatchard plot, which is resolved into two linear components: 2,400 high affinity sites with a Kd of 140 pM and 40,000 low affinity sites with a Kd of 14,2 nM. In contrast, non-glycosylated E. coli-derived LIF was found to bind to JAR cells with a linear Scatchard plot indicating the presence of a single class of high affinity binding sites (Kd = 112 pM) with a maximal capacity (2,300 sites/cell) being comparable to that of high affinity CHO LIF binding sites. Similarly, the anti-gp190 mAbs 1B4 bound to JAR cells with a single class of binding sites (Kd = 0.22 nM) and a maximal binding capacity (2,330 sites/cell) comparable to the number of E. coli LIF binding sites or CHO LIF high affinity binding sites and far less than the number of low affinity CHO LIF binding sites. Similar results were found with other anti-gp190 antibodies directed at separate epitopes (data not shown). These results indicated that most of the low affinity LIF receptors on JAR cells were not able to bind E. coli LIF and were different from gp190. The number of gp130 molecules on JAR cells, as measured by the binding of BR3 mAb, was in excess of the number of anti-gp190 mAb binding sites (8,200 sites/cell, Kd = 0.77 nM).
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Biochemical and Immunochemical Identification of the LIF Receptors-- In order to identify the molecular features of the LIF receptors expressed by U266, CHO-190, and JAR cells, cross-linking experiments were carried out with iodinated CHO LIF. The cross-linked species were immunoprecipitated with anti-LIF, anti-gp130, or anti-gp190 antisera and resolved by SDS-PAGE under reducing conditions (Fig. 5).
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Glycosylated LIF Binding to U266 Cells and Low Affinity Binding to JAR Cells Are Inhibited by Mannose and Mannose 6-Phosphate-- The fact that LIF receptors expressed by U266 cells were able to bind glycosylated LIF but not its non-glycosylated (E. coli) or deglycosylated (endoglycosidase-F-treated) isoforms, prompted us to investigate the potential competing effects of monosaccharides on this binding.
Fig. 6A shows that CHO LIF binding to U266 cells is completely and dose-dependently inhibited by D-mannose, D-mannose 6-phosphate (Man-6-P), and D-glucose 6-phosphate (Glc-6-P), whereas it remains insensitive to similar concentrations of D-galactose and D-glucose. N-Acetylglucosamine was also inactive (not shown). The effect of Man-6-P was much more pronounced than that of mannose or Glc-6-P; its half-maximal inhibitory effect was observed at a concentration of 40 µM as compared with 58 and 14 mM for mannose and Glc-6-P, respectively. Control experiments showed that CHO LIF or E. coli LIF binding to gp190-CHO cells were not affected by any of the monosaccharides (Fig. 6B and data not shown).
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Phosphate Groups on Glycosylated LIF Are Required for Binding to U266 Cells-- In view of the high inhibitory effect of Man-6-P as compared with mannose on LIF binding to U266 cells, we investigated whether CHO LIF contained phosphate groups that could be involved in binding. This was evaluated by competition experiments. Fig. 6D shows that treatment of unlabeled CHO LIF with alkaline phosphatase completely inhibited its ability to compete with iodinated CHO LIF binding to U266 cells. In contrast, alkaline phosphatase-treated CHO LIF retained a strong (>80%) inhibitory effect on iodinated CHO LIF binding to gp190-CHO cells.
The U266 LIF Receptor Is Biochemically and Immunochemically Related to the Man-6-P/IGFII Receptor-- The high inhibitory effect of Man-6-P on LIF binding to U266 cells as well as the high molecular mass of the receptor in cross-linking studies prompted us to investigate whether the LIF receptor on these cells could be related to the cation-independent mannose 6-phosphate receptor (CI-MPR), a type I membrane glycoprotein with an Mr of 275,000. This receptor is also a receptor for insulin-like growth factor II (IGFII) (20). In Fig. 7A, it is shown that affinity-purified immunoglobulins raised against the Man-6-P/IGFII-R are able, as does anti-LIF antiserum, to immunoprecipitate the radiolabeled LIF-receptor cross-linked complex solubilized from U266 cells.
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Soluble Man-6-P/IGFII-R Purified from Serum Binds Glycosylated but Not Non-glycosylated LIF-- In order to provide a direct proof that the Man-6-P/IGFII-R is a receptor for LIF, a natural soluble form of this receptor purified from bovine serum was covalently coupled to a dextran matrix and assayed for its ability to bind LIF by the technique of surface plasmon resonance. Fig. 8 shows clear association and dissociation curves for IGFII and CHO LIF (500 nM). Experiments performed at different concentrations (between 5 nM and 1 µM) of the factors showed that the increases in resonance signals were dose-dependent (data not shown). In sharp contrast, no association of E. coli LIF at similar concentrations (500 nM in Fig. 8) could be observed. Furthermore, Man-6-P (5 mM) strongly inhibited the ability of CHO LIF to interact with immobilized Man-6-P/IGFII-R, whereas it did not affect binding of IGFII (not shown). The kinetic and equilibrium parameters derived from analysis of the sensorgrams depicting IGFII and CHO LIF binding are also shown in Fig. 8. The equilibrium dissociation constant calculated for CHO LIF (Kd = 4.6 nM) is very similar to the value obtained by equilibrium binding studies on U266 cells (Kd = 6.9 nM). IGFII has a slightly higher affinity than CHO LIF (Kd = 1.4 nM), mainly because of a more rapid association constant (kon).
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DISCUSSION |
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This paper demonstrates the existence of a novel type of low affinity receptor for LIF. This receptor is different from gp190 (also designated as the low affinity LIF receptor) or gp130. It binds glycosylated LIF but not E. coli-derived LIF, and this binding involves N-linked carbohydrates containing Man-6-P residues. Its identity with the Man-6-P/IGFII-R is strongly supported by biochemical, functional, and immunological findings.
Binding of CHO LIF to the Man-6-P/IGFII-R was not due to a special glycosylation state of LIF produced in the non-human CHO cell line, as glycosylated LIF naturally produced by the human melanoma cell line A375 was also found to react with the Man-6-P/IGFII-R. It also appeared to be cytokine-specific, as IL-9, a cytokine that, like LIF, is also heavily glycosylated (21), as well as natural human IL-6, also a glycosylated protein (22), did not interfere with CHO LIF binding to Man-6-P/IGFII-R when tested at similar concentrations.
Mannose, Man-6-P, and Glc-6-P, but not galactose, glucose, or N-acetylglucosamine, inhibited LIF binding to Man-6-P/IGFII-R in a dose-dependent fashion. Whereas the inhibitory effects of mannose and Glc-6-P developed at non-physiological concentrations, Man-6-P was active at much lower concentrations (IC50 of 40 µM), an observation that has initially raised the hypothesis that the LIF receptor on U266 cells could be a Man-6-P receptor (MPR). Two distinct MPRs have been characterized and their cDNAs cloned (23, 24). One is a type I integral membrane glycoprotein with a molecular mass of 275 kDa. This receptor binds Man-6-P-containing ligands independent of divalent cations and has thus been called the cation-independent (CI)-MPR. This receptor has subsequently been demonstrated to bind IGFII (25) and is now referred to as the Man-6-P/IGFII-R. The other receptor is also a type I integral membrane glycoprotein with a molecular mass of 46 kDa. Unlike the first one, it requires divalent cations for optimal binding of Man-6-P-containing ligands and is referred to as the cation-dependent (CD)-MPR. Strong experimental evidence suggests that the LIF receptor expressed by U266 cells is identical to the Man-6-P/IGFII-R. (i) A polyclonal anti-LIF antibody immunoprecipitates a 310-kDa band corresponding to one molecule of LIF cross-linked to a receptor of 270 kDa; (ii) a polyclonal antibody raised against Man-6-P/IGFII-R immunoprecipitates a similar 310-kDa LIF-receptor complex; (iii) this anti-Man-6-P/IGFII-R antibody inhibits binding of CHO LIF to U266 cells with an efficiency comparable to its ability to inhibit IGFII binding. Finally, a direct proof for the LIF binding capacity of Man-6-P/IGFII-R is provided by the demonstration that purified soluble Man-6-P/IGFII-R binds CHO LIF with an affinity (Kd = 4.6 nM) similar to that measured on U266 cells (Kd = 6.9 nM), while being unable to bind E. coli LIF.
We have previously described the expression of high and low affinity receptors for CHO LIF by various cell lines (12). Subsequent reports have shown that gp190, when expressed alone, behaves as a low affinity LIF receptor (8, 26). It was therefore assumed that low affinity LIF receptors expressed by numerous cell lines reflected gp190 molecules in excess to gp130 at the cell membrane. In this study, we show that most of the low affinity LIF receptors expressed by various tumor cell lines do not correspond to excess gp190 molecules but behave like the Man-6-P/IGFII/LIF receptor identified on U266 cells. This was demonstrated in detail with the JAR cell line. This cell line was found to express low affinity LIF receptors (over 40,000) far in excess of the number of gp190 (2,500) as well as gp130 (8,000) molecules. These low affinity LIF receptors displayed the same characteristics as the U266 LIF receptors with respect to CHO LIF versus E. coli LIF binding, inhibition by anti-gp130 and anti-gp190 mAbs, inhibition by monosaccharides, LIF cross-linking, and reactivity with the anti-Man-6-P/IGFII-R antibodies.
Human LIF contains seven potential N-glycosylation sites (27), of which four have been shown to be functional (28). In LIF purified from the human HSB2 T lymphoma cell line, N-linked carbohydrates have been described to account for about 20 kDa in the molecular mass (43 kDa) of the cytokine. O-Linked glycosylations were also demonstrated, which accounted for about 1-2 kDa (29). There are three main types of N-linked sugar chains in glycoproteins: those of the oligomannosidic type with two to nine linked mannose, those of the N-acetyllactosaminic type with terminal sialic acid residues, and those of the mixed type (30). Our results suggest that oligomannosidic and/or mixed type sugar chains on the LIF molecule are specifically involved in the interaction with the Man-6-P/IGFII-R, and indicate that at least some of these side chains contain terminal Man-6-P residues.
Crystallization of LIF and site-directed mutagenesis studies have allowed a three-site model for receptor binding to be proposed (31, 32). In this model, site II formed by the N-terminal part of helix A and the C-terminal part of helix C contacts the cytokine receptor module of gp130 molecule, whereas site I (beginning of AB loop and C-terminal half of helix D) and site III (C terminus of CD loop) contact, respectively, the C-terminal and N-terminal cytokine receptor modules of gp190. In this model, it can be noticed that none of these sites contain potential N-glycosylation sites. Most of the N-glycosylation sites are distributed on parts of the LIF molecule that are exposed to solvent (1 site near N terminus, 1 site on helix A, 2 sites on end of AB loop, 2 sites on helix B, 1 site on N terminus of helix C). This observation raises the possibility that the Man-6-P/IGFII-R could interact with carbohydrates on glycosylated LIF even if it is bound to gp130 and gp190.
The extracellular region of the Man-6-P/IGFII-R is composed of 15 homologous repeat units with an average length of 147 amino acids (25). Whereas repeats 1-3 and 7-9 each contain one Man-6-P binding determinant involved in binding of Man-6-P-containing ligands (33, 34), the binding site of IGFII has been localized on repeat 11 (35), indicating that IGFII and Man-6-P binding sites are distinct. Some investigators have shown that high molecular weight Man-6-P-containing lysosomal enzymes can compete with IGFII for binding to the Man-6-P/IGFII-R (36). These results could suggest some overlap between the binding sites or merely reflect steric hindrance. The present work shows that whereas Man-6-P abrogates glycosylated LIF binding, glycosylated LIF and IGFII do not compete for binding, in agreement with the notion of non-overlapping binding sites.
One major question that arises from this work concerns the role of the
Man-6-P/IGFII-R in LIF biological action. Diverse functions have been
attributed to the Man-6-P/IGFII-R (20). The majority (90%) of this
receptor is expressed within endosomal compartments, where its first
role is to bind the newly synthesized lysosomal enzymes via their
Man-6-P-containing N-linked oligosaccharides and to divert
these phosphorylated ligands from the secretory pathway for subsequent
sorting to endosomes and lysosomes. This receptor is also present at
the plasma membrane where it endocytoses secreted lysosomal enzymes. It
has also been shown to have a role in the clearance and activation of
hormones and growth factors. The Man-6-P/IGFII-R is able to internalize
IGFII, resulting in lysosomal degradation of this factor (37). It binds
the precursor form of TGF- (latent TGF-
) through Man-6-P
residues, leading to the activation of this precursor form into
biologically active TGF-
(38, 39). It also binds the
Man-6-P-containing hormone proliferin, a prolactin-related murine
protein (40), as well as porcine thyroglobulin (41) and may participate
in the capture and degradation of these hormones in lysosomes. By this
ability to activate TGF-
, a potent growth inhibitor for most cell
types, to promote degradation of IGFII, a potent growth factor, and to endocytose proteolytic enzymes involved in extracellular matrix degradation, the Man-6-P/IGFII-R has been considered as a tumor suppressor, and recent findings have strengthened this concept by
showing that Man-6-P/IGFII-R allelic loss is an early event in the
etiology of cancer (42). LIF is a growth and differentiation factor
that has been shown to be produced by various cell types and to be
active on a large spectrum of cellular targets. In addition to its
demonstrated roles in hematopoiesis, inflammation, lipid metabolism,
bone homeostasis, and embryogenesis, LIF can also regulate tumor
growth and metastasis (1, 2). By its capacity to bind to the Man-6-P
binding sites, glycosylated LIF could modulate TGF-
activation, lysosomal enzyme trafficking, and extracellular matrix degradation.
Glycosylated LIF, as already suggested for other Man-6-P-containing ligands (43) (44), might as well modulate the activity of other Man-6-P/IGFII-R binding hormones such as IGFII itself and proliferin. Proliferin has been shown to promote neovascularization (45, 46), whereas LIF inhibits endothelial cell proliferation (47). Competition between these two factors at the level of the Man-6-P/IGFII-R could be the basis of these opposite effects.
In view of its large range of biological effects, there is a need to tightly regulate the production and circulating levels of LIF. Our data suggest that the Man-6-P/IGFII-R could participate in this process by its ability to recapture LIF, leading to internalization and lysosomal degradation. Indeed, preliminary data2 indicate that LIF bound to Man-6-P/IGFII-R is internalized and degraded at 37 °C but not at 4 °C.
Another function for the Man-6-P/IGFII-R could be to capture LIF
molecules at the cell surface and favor its interaction with functional, high affinity LIF receptors containing gp130 and gp190. Such high affinity receptors have usually a low cell surface density. In contrast, low affinity Man-6-P/IGFII/LIF receptors have a much higher cell density (see Table I) and therefore could serve to increase
the local concentration LIF at the cell surface and increase the
probability of interaction of LIF with high affinity receptors. Such a
mechanism has already been proposed in the case of growth factors that
bind with low affinity to cell surface proteoglycans such as fibroblast
growth factor and TGF- (48).
The cytoplasmic domain of the Man-6-P/IGFII-R is short and devoid of tyrosine kinase activity. However, a number of reports have suggested that the Man-6-P/IGFII-R may function as a transmembrane signaling molecule (20). For example, IGFII interaction with the Man-6-P/IGFII-R has been reported to induce DNA synthesis in Balb/c 3T3 cells (49) or stimulation of K652 cell proliferation (50). Other reports have subsequently presented evidence that the Man-6-P/IGFII-R can couple to heterotrimeric G proteins (51, 52), although this finding is controversial (53, 54). Finally, a recent report has shown that either proliferin or IGFII binding to Man-6-P/IGFII-R induces endothelial cell chemotaxis through a G protein-coupled, mitogen-activated protein kinase-dependent pathway (46). Whether glycosylated LIF is also able to activate signal transduction through the Man-6-P/IGFII-R remains to be investigated.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stuart Kornfeld (Washington University, St Louis, MO) for the generous gift of anti-CI-MPR antibody. We also thank Dr. John Wijdenes (Diaclone, Besançon, France) for providing the anti-gp130 mAb BR3 and Dr. Marie-Martine Hallet (INSERM U463, Nantes, France) for the cell line CHO-190.
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FOOTNOTES |
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* This work was supported in part by INSERM, the CNRS, Association pour la Recherche contre le Cancer (ARC) Grant 6474, and Boehringer Mannheim (Penzberg, Germany).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from the Ligue Contre le Cancer de Vendée.
To whom correspondence should be addressed. Tel.:
33-2-40-08-47-47; Fax: 33-2-40-35-66-97; E-mail:
yjacques{at}inserm.nantes.fr.
The abbreviations used are:
LIF, leukemia
inhibitory factor; IL, interleukin; OSM, oncostatin M; CNTF, ciliary
neurotrophic factor; CT-1, cardiotrophin-1; CD-MPR and CI-MPR, cation-dependent and cation-independent mannose 6-phosphate
receptor; Man-6-P, mannose 6-phosphate; Glc-6-P, glucose 6-phosphate; IGFII, insulin-like growth factor II; Man-6-P/IGFII-R, mannose
6-phosphate/insulin-like growth factor II receptor; mAb, monoclonal
antibody; CHO, Chinese hamster ovary; FCS, fetal calf serum; TGF-, transforming growth factor-
; PAGE, polyacrylamide gel
electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buffered
saline; RT, reverse transcription; PCR, polymerase chain
reaction.
2 F. Blanchard, unpublished data.
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