0021-972x/92/7502-0609$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol. 75, No. 2 Printed

in U.S.A.

Expression of Type I, but not Type II Insulin-Like Growth Factor Receptor on Both Undifferentiated and Differentiated HT29 Human Colon Carcinoma Cell Line* MARYSE M. REMACLE-BONNET, FRANCOISE L. GARROUSTE, JACQUES L. MARVALDI, AND Laboratoire d’lmmunopathologie France; and the Centre National Marseille Cedex 3, France

JEAN CHANTAL GILBERT

M. CULOUSCOUt, RABENANDRASANA, J. POMMIER

(J.M.C., F.L.G., G.J.P.), Facultti de Mkdecine, 13385 Marseille Cedex 5 de la Recherche Scientifique (M.M.RB., CR., J.L.M.), URA 202, 13331

the competitive binding curves with each one of these radioligands suggests that HT29 cells express both a classical type I IGF receptor (about 6,00O/cell; KdIoF.1 = 0.48 nmol) and a variant one whose YIGF-II binding is not blocked by olIR-3 (about 15,00O/cell; KdloF.,, = 4.0 nmol). Endocytosis studies of specific cell-bound ‘Y-IGF-I or YIGF-II suggest that ligand interaction with the classical, but not the variant, binding site is only able to induce receptor internalization. An identical IGF receptors pattern is observed with HT29-D4 clonal cells induced to differentiate by culture in a glucose-free medium. (J Clin Endocrinol Metab 75: 609-616, 1992)

ABSTRACT The HT29 human colonic carcinoma cell line secretes insulin-like growth factor (IGF)-II. We have examined these cells for expression of IGF receptors. Competitive binding assays as affinity cross-linking experiments using ‘251-IGF-II fail to reveal type II IGF receptors at the cell surface. In contrast, cross-linking studies with either lz51-IGF-I or I?-IGF-II reveal an M, 135,000 protein that follows a peptide binding specificity characteristic of the a-subunit of the type I IGF receptor. However, ‘251-IGF-II binding to this receptor is not inhibited at 4 C by olIR-3, a monoclonal antibody to the type I IGF receptor. Analysis of

T

HE HT29 human colonic adenocarcinoma cell line constitutes an interesting inductible-cell differentiation model to obtain some insight into the connections between cellular differentiation and secretion of growth factors by colonic cancer cells. HT29 cells, as the clonal cell line HT29-D4, can be indeed induced to differentiate either by replacing glucose by galactose (1, 2) or by adding suramin (3) [a drug known to block autocrine loops by neutralizing the endogenous expression of both the growth factor and its receptor (4)] in the culture medium. Suramin-induced HT29 cell differentiation is observed in the presence or not of fetal calf serum (FCS) (5) that suggeststhat endogenous growth factors are involved in this process.The differentiated cells are highly polarized and display two clearly distinguishable plasma membrane domains: an apical brush border domain with well organized microvilli, mature junctional complexes (2, 6) and a restricted carcinoembryonic antigen localization (7); a basolateral membrane domain where typical molecules are segregated, e.g. HLA classI molecules,vasoactive intestinal peptide receptors (6, 7). In addition, these differentiated cells generate electrically active leakproof monolayers when cultured on a permeable substratum (8). Received September 9, 1991. Address all correspondence and requests for reprints to: Dr. Gilbert J, Pommier, Laboratoire d’Immunopathologie, Facultk de Mkdecine, 27 Bd J. Moulin, 13385 Marseille Cedex 5, France. *This work was supported by grants from “Association pour la Recherche sur le Cancer” and “Ligue Nationale Francaise contre le Cancer.” t Present address: Oncogen, 3005 First Avenue, Seattle, Washington 98121.

Previous reports have shown that the HT29 cell line releasesinto culture medium a number of growth factors including transforming growth factor-a, transforming growth factor-p, platelet derived growth factor, and insulinlike growth factor (IGF)-like molecules (9, 10). We have recently reported the secretion of high amounts (about 6 ng/ lo6 cells) of IGF-II entirely complexed to heterogeneousIGF binding proteins (IGFBP) (11) that represent isoforms from the same IGFBP-4 class(12). Recent data suggest a role for IGFs in several neoplastic processes. Enhanced levels of messenger RNA for IGF-II and/or secretion of elevated amounts of this peptide have been reported in a wide variety of tumors (for review, seeRef. 13) including human colorectal tumors (14, 15). IGF-I and IGF-II are multifunctional molecules that show insulin-like anabolic effects and promote the stimulation of cell proliferation and/or cell differentiation (16). IGFs trigger their biological effects by primarily interacting with specific receptors present on the target responsive cell (16, 17). The type I IGF receptor is an heterotetramer of a2 P2 form structurally similar to the insulin receptor and expressing a tyrosine-specific protein kinase activity. By contrast, the type II IGF/mannose-6-phosphate receptor is structurally unrelated and composed of a single polypeptide chain without kinase activity. An additional complexity in determining the actions of IGFs in many cells is the overlapping ligand binding specificities of these receptors (16, 17). Moreover, evidence from a number of previous studies has suggested that most of the biological actions of both IGF-I and IGF-II are mediated via the type I IGF receptor only (16, 17). To further investigate a possiblerole of endogenous IGF-

609

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REMACLE-BONNET

610

II in the induction of HT29 cell differentiation, we have investigated the properties of specific IGF receptors on these cells both in an undifferentiated and a differentiated state. Only the type I IGF receptor is detected at the HT29 cell surface whatever the state of cellular differentiation. In addition, the use of the monoclonal antibody to the type I IGF receptor, CXIR-3(18), allows to identify two structurally homologous and immunologically distinct type I IGF binding sitesthat differ by their ability to endocyte the ligand. Such an observation might have important biological consequences. Materials

Recombinant IGF-I and ‘251-IGF-I (ZOO rCi/pg) were purchased from Amersham (Arlington Heights, IL). Recombinant IGF-II was purchased from Bachem (Heidelberg, Germany) and radioiodinated using chloramine-T method to a specific activity of 150-200 pCi/pg. Bovine insulin was from Sigma Chemical Co. (St. Louis, MO). We purchased (uIR-3 from Oncogene Science (Manhasset, NY). Disuccinimidyl suberate (DSS) was obtained from Pierce Chemical Co. (Rockford, IL). Reagents for gel electrophoresis were from Bio-Rad Laboratories (Richmond, CA). Ca’+Free Eagle’s medium was supplied by GIBCO (Grand Island, NY). RPM1 containing 20 mmol N-2 hvdroxvethvlpiperazine-N’-2 ethanesulfonic acid (RPM1 HEPES), Dulbecco’s’modikeh Eagle’s medium (DMEM), FCS, ‘and other cell culture reagents were from Flow Laboratories (McLean. VA). Tissue culture flasks and multiwell plates were purchased from Costar ‘(Cambridge, MA). All other reagents were of-analytical grade.

conditions

The human colonic adenocarcinoma cell line, HT29 (19), a gift from Dr. A. Zweibaum (INSERM, U. 178, Paris, France), and HT29-D4 cells, a clone derived from the parental HT29 cell line (2), were grown in DMEM containing 25 mmol glucose and 10% FCS as previously reported (11). To induce the differentiation, HT29-D4 clonal cells were cultured in glucose-free DMEM supplemented with 5 mmol galactose and 10% dialyzed FCS as previously described (2).

IGFs binding

studies

This was done as described elsewhere (11). Briefly, HT29 cells were cultured in 16.mm tissue-culture wells, At confluent density, cell culture trays were placed on ice 30 min before and throughout the assay. Cell monolayers were washed three times in serum-free DMEM, then incubated in a total vol of 0.2 mL binding medium (RPMI-HEPES containing 0.1% BSA, 0.1 FM potassium iodide, and adjusted to pH 7.0) for 2 h at 4 C (equilibrium binding conditions) with either ‘251-IGF-I or ‘251-IGF-II with or without various concentrations of unlabeled IGF-I, IGF-II, insulin, or (uIR-3. In some experiments, cell monolayers were preincubated in binding medium 2 h at 4 C or 37 C in the presence or not of (uIR-3 prior to initiating the binding assay. At the end of incubation, cells were washed three times with cold phosphate buffered saline (PBS) containing 0.1% BSA, then lysed in the same buffer containing 1% Triton X-100 and 10% glycerol and cell-associated radioactivity was counted in a y-counter. Nonspecific binding, determined as the radioactivitv , bound in the oresence of 2 ug/mL unlabeled IGF-I or IGF-II, was subtracted from to’tal binding to dbyain specific binding. Nonspecific binding represented about 5% and 20% of total binding of ‘251-IGF-I and ‘251-IGF-II, respectively. In each experiment, replicate wells were used to determine cell number. Binding data were analyzed by using the EBDA/LIGAND computer program (20, 21) distributed by Biosoft (Cambridge, UK).

Redistribution

of specific HT29 cell-bound

lz51-IGFs

HT29 cell monolayers were first incubated with the radioactive ligand for 2 h at 4 C. The specific cell associated radioactivity was determined

JCE & M .1992 Voll.5.No2

in control wells in triplicate as described above and used as a reference for the calculations. In test wells, the cells were washed three times with PBS containing 0.1% BSA and further incubated in 0.5 mL prewarmed binding medium at 37 C. At the indicated times, the cells were cooled rapidly on ice. The supernatant and two cell-washes with 0.5 mL icecold PBS containing 0.1% BSA were harvested and counted to determine the “released ‘251-IGF.” Next, the cells were incubated for 5 min at 4 C with 1 mL 0.2 mol acetic acid, 0.5 mol NaCl, pH 2.5, then washed with the same buffer in order to remove “cell surface-bound ‘?-IGF.” Finally, the cells were solubilized and the radioactivity of the lysates reflecting the “internalized ‘?-IGF” counted. All values were corrected to represent specific binding and expressed as a percentage of the control cell associated radioactivity.

Affinity

and Methods

Materials

Cell lines and cell culture

ET AL.

cross-linking

analysis

Undifferentiated HT29 or differentiated HT29-D4 confluent cell monolayers were obtained in place multiwell dishes. To access also the basolateral domain of the HT29-D4 differentiated cells, the tight junctions were opened by treatment of the cells with Ca’+-free Eagle’s medium at 37 C for 1 h as previously described (22) before affinity labeling of cell receptors. Cell monolayers were washed three times with cold binding medium, then incubated for 2 h at 4 C in 1 mL of the same medium containing about 500,000 cpm/well of either ‘251-IGF-I or lz51IGF-II in the presence or not of various unlabeled peptides or (uIR-3. Subsequently, the cells were washed three times in binding medium without BSA at 4 C, after which 0.5 mL 0.5 mmol DSS in the same buffer was added. After 20 min at 4 C, the reaction was quenched with 1.5 mL 0.1 mol Tris-HCl, pH 7.4, containing 1 mmol sodium EDTA and the cells were left for a further 20 min at 4 C, then the liquid was discarded. After washing once more with the buffer quench, the cells were solubilized in 200 PL electrophoresis sample buffer composed as 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, 62 mmol Tris-HCl, pH 6.8. The samples were boiled for 10 ruin and submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 5% polyacrylamide slab gel according to the method of Laemmli (23). The gels were dried and autoradiographed using RX Fuji x-ray film in the presence of intensifying screens at -80 C for a minimum of 20 days.

Results Affinity

labeling

of IGF receptors

in HT29

cells

To determine the nature of IGF receptors at the HT29 cell surface, lz51-IGF-I and ‘251-IGF-II were affinity cross-linked to confluent cell monolayers and analyzed by SDS-PAGE under reducing conditions (Fig. 1). Cross-linking of ‘251-IGF-I to HT29 cells demonstrated a predominant radioactive complex with an apparent M, 135,000 (Fig. lA, lane a). An additional complex exhibiting a weakly autoradiographic intensity with an apparent M, 250,000-260,000 was also detected (Fig. lA, lane a). The labeling of these specieswas totally inhibited in the presence of 50 nmol unlabeled IGF-I (Fig. lA, lane b) or 10 pg/mL aIR-3 (Fig. lA, lane e), markedly inhibited in the presence of 50 nmol unlabeled IGF-II (Fig. lA, lane c), and slightly inhibited by high amounts (1.5 pmol) of unlabeled insulin (Fig. 1A, lane d). The labeling of both bands was coordinately diminished by coincubation with the various competitors. Thus, the 135,000 speciesidentified at the HT29 cell surface has both the molecular weight and the ligand specificity of the a-subunit of the type I IGF receptor, whereas the 250,000-260,000 complex probably corresponds to the chemically cross-linked dimer of the IGF-I receptor a-subunit (16, 17, 24). Affinity cross-linking of ‘251-IGF-II to HT29 cell monolayers was also performed and compared with the results

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EXPRESSION

OF IGF RECEPTORS

0A

1251- IGF -I

Mr x 10’ 3

ON HT29 CELLS

Mr x10

-3

611

0B

1251-IGF-ll / 1. \

200-

\ 11697669

a

bed

e

a

bed

e

FIG. 1. Affinity cross-linking of 1251-IGF-I and ‘251-IGF-II to HT29 cells. HT29,cell monolayers were incubated 2 h at 4 C with ‘251-IGF-I (A) or ‘261-IGF-II (B) (lanes a) and various amounts of unlabeled competitors: 50 nmol IGF-I (lanes b), 50 nmol IGF-II (lanes c), 1.5 rmol insulin (lanes d). 10 &mL arIR-3 (lanes e). Cross-linking was done with 0.5 mmol DSS. Samples were solubilized, reduced, and analyzed by SDS-PAGE (5% gei), then autoradiographed.

obtained with ‘251-IGF-I (Fig. 1B). Although affinity crosslinking of ‘251-IGF-II to membrane-associated type II IGF receptor would result in formation of a M, 250,000-260,000 complex under reducing conditions (16, 17), Fig. lB, lane a, shows that such a radioactive band was not apparent even in greatly overexposed autoradiographs. However, a labeled band was observed at an electrophoretic mobility corresponding exactly to the samemolecular weight as that of the a-subunit of the type I IGF receptor identified by ‘251-IGF-I affinity labeling, i.e. M, 135,000 (Fig. lB, lane a). The autoradiographic intensity of this band represented about 3050% of the one obtained with cross-linking of lz51-IGF-I, depending on the experiment. This band disappeared after coincubation with 50 nmol of unlabeled recombinant IGF-I or IGF-II (Fig. lB, lanes b and c), and was partially inhibited by 1.5 pmol insulin (Fig. lB, lane d). In contrast to IGF-I, the binding of radiolabeled IGF-II to the M, 135,000 complex was not affected by aIR-3 at a concentration as high as 10 pg/mL (Fig. lB, lane e). CompetitiveIGF binding studiesin HT29 cells To further study the binding characteristics of IGF receptors associated with HT29 cells, lz51-IGF-I and ‘251-IGF-II binding to cells was examined in competitive binding experiments. Figure 2 shows that the binding of ‘251-IGF-I was competed for by unlabeled IGF-I (IQ,,, = 8 X 10-l’ mol) and by IGF-II with approximately 6-fold lower potency (ICso= 5 x lo-’ mol). Virtually all the specific binding of ‘251-IGF-I was displaceablein the presence of an excessof cold IGF-I

or -11,i.e. 1.0 X 10m7mol. Insulin had little effect on lz51-IGFI binding up to a concentration of 1.0 X 10m7mol and the maximum inhibition of the binding was only 20% at 2.5 X 10v7 mol. Inhibition of ‘251-IGF-I binding carried out in the presence of (uIR-3 was never complete. The half-maximal inhibition was observed at a concentration of 1.5 pg/mL, whereas the maximal inhibition obtained with an antibody concentration as high as 25 pg/mL was only 75% of the specific binding (Fig. 2). Analysis of these binding data by LIGAND revealed a linear plot consistent with a single class of high affinity binding sites with an apparent dissociation constant (Kd) of 4.8 f 0.2 X 10-l’ mol and a total number of type I IGF receptor of approximately 6,00O/cell (Fig. 2, inset). The relative competition curves generated with tracer amounts of ‘251-IGF-II are shown in Fig. 3. They were also consistent with a binding of radiolabeled IGF-II to the type I IGF receptor in that unlabeled IGF-I was the most potent competitive inhibitor, followed by unlabeled IGF-II (IC50 = 3 X lo-’ mol) and insulin (a maximal inhibition of 35% was achieved with 0.25 pmol). Although IGF-II was able to totally inhibit ‘251-IGF-II binding, the maximal inhibition observed with IGF-I was only 40-50% at a concentration as high as 5 X 10m7mol. This suggeststhat ‘251-IGF-II bound to an additional component which is specific for IGF-II and distinct from the type I IGF receptor. However, the more striking difference in the competitive displacement curves generated bY lz51-IGF-I and ‘251-IGF-II binding to HT29 cells was the almost complete incapacity of aIR-3 to alter the binding of ‘251-IGF-II. The maximal inhibition observed was only 5% of the specific binding at an antibody concentration of 25 pg/

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REMACLE-BONNET

612

FIG. 2. Competitive binding curves of ‘Y-IGF-I to HT29 cells. Cell monolayers were incubated with 0.15 nmol “‘1-IGFI and graded amounts of unlabeled IGFI (O), IGF-II (0), insulin (W), or LuIR-3 (A). After a 2-h period incubation at 4 C, specific cell-associated binding was determined as described in Mater& and Methods and expressed as a percentage of binding in the absence of any competitor. Each point represents the mean of three duplicate experiments. SD was always inferior to 5%. Inset, Scatchard plot of IGF-I binding analyzed by the LIGAND program. The solid line is the computer-generated best fit for a onesite binding model.

ET

AL.

JCE & M. 1992 Vol75.No2

ElouND(~mdllO~ce,,s,

0

n

l!

I 9 PEPTIDES f 0.1

1’0

I 8 CONCENTRATION

(-LOG

I 7 M)

1

I 1

10 OOR-3(pg/ml)

FIG. 3. Competitive binding curves of ‘251-IGF-II to HT29 cells. Cell monolayers were incubated with 0.15 nmol ‘251-IGF-II and graded amounts of unlabeled IGF-I (O), IGF-II (0), insulin (H), or olIR-3 (A). After a 2-h period incubation at 4 C, specific cell-associated binding was determined as described in Muterials and Methods and expressed as a percentage of binding in the absence of any competitor. Each point represents the mean of three duplicate experiments. SD was always inferior to 7%. zn.Wt, scatchard plot of IGF-II binding analyzed by the LIGAND program. The solid curue is the computer-generated best fit for a two-site binding model, and the two binding sites are indicated by the dotted lines.

0 10

9

b

+

PE PTI DE S CONCENTRATION

I

1

(-LOG

1’0

o(IA-S(pJ,rLlP

mL (Fig. 3), a concentration which inhibited 75% of the binding of “‘1-IGF-I (Fig. 2). Thus, ‘251-IGF-II appears to bind to a site on the tvpe I IGF receptor that is not recognized by aIR-3 antibody. The Scatchard $ot derived from th< IGFII binding data was curvilinear (Fie. 3. inset). The curve xv analyzed by LIGAND had been fit by a two-sites binding model. The dissociation constants and binding capacities were Kdl = 4.0 + 0.5 X lop9 mol and 15,000 sites/cell for the high affinity binding site and Kd2 = 1.47 + 0.2 X 10e7

k b

mol and approximately 400,000 sites/cell for the low affinity binding site. Temperature-dependent

inhibitory

effect

of

CUZR-3

The inability of aIR-3 to significantly inhibit ‘251-IGF-II binding at 4 C to HT29 cells was also observed when the cells were preincubated with this antibody for 2 h at 4 C before initiating the binding test as described above (Fig.

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EXPRESSION Temperature dependence of the inhibitory effect of aIR-3 on ‘251-IGF-I and ‘251-IGF-II binding to HT29 cells. Cell monolayers were preincubated in the binding medium with or without graded amounts of aIR-3 for 2 h either at 4 C (A) or at 37 C (B). If necessary, the culture trays were cooled on ice for 30 min, then the cells washed three times and incubated with 0.15 nmol of either ‘Z51-IGF-I (0) or ‘251-IGF-II (A). Specific cell-associated binding was determined as described in Materials and Methods and expressed as a percentage of the binding obtained with cells either preincubated at 4 C (A) or at 37 C (B) with the binding medium alone. The data plotted represent the result of a representative experiment made in triplicate. SD was always inferior to 5%.

OF IGF RECEPTORS

ON HT29 CELLS

613

FIG. 4.

i

0:1

l-0

i

0:1

lb

o( IR-3(pg/mll

4A). In contrast, the preincubation of HT29 cells with aIR-3 for 2 h at 37 C in the binding medium strongly increased the inhibitory activity of this monoclonal antibody on ‘251-IGFs binding. Figure 4B, indeed, shows that such a preincubation allowed to inhibit more than 90% of ‘251-IGF-I binding and about 45% of ‘251-IGF-II binding to HT29 cells, respectively, with respect to the *251-IGFs control binding obtained when

the cells were preincubated at 37 C with the medium alone. Thus, at this temperature olIR-3 was found to be as potent as unlabeled IGF-I to inhibit both 12’I-IGF-I and ‘251-IGF-II binding to HT29 cells (cf. Fig. 4B with Figs. 2 and 3). Internalization

of specific cell-bound

IGFs in HT29

cells

The interaction of ligands such as growth factors with high-affinity cell surface receptors is usually accompanied by the endocytosis of the ligand-receptor complexes (25). To determine whether the type I IGF receptors expressed at the cell surface in HT29 cells were able to efficiently mediate such a process, cells were first incubated with either ‘251-IGFI or ‘251-IGF-II for 2 h at 4 C, then washed three times and further incubated at 37 C. At various times, the amount of ‘251-IGF either released, bound to the cell surface (acid stripped), or internalized (acid resistant) was determined as described in Materials and Methods. Figure 5 shows that after a 2-h incubation at 37 C, about 60% of the specifically cellbound 12’I-IGF-I was found to be in an acid-resistant compartment (Fig. 5A, c) whereas about 30% of the initial cellbound radioactivity was found in the supernatant (Fig. 5A, a). Moreover, the percentage of ‘251-IGF-I that remained at the cell surface was low, being approximately 6% of the total (Fig. 5A, b). Such a kinetic is in good agreement with several previous studies indicating that IGF-I is internalized via a receptor-mediated endocytosis (26). In contrast to ‘251-IGF-I, HT29 cell-bound 1251-IGF-II behaved differently when the cells were further incubated at 37 C. A negligible amount of the initial cell-bound 1251-IGF-II (about 3%) was internalized (Fig. 5B, c) and the percent of ‘251-IGF-II remaining bound to the cell surface after a 2-h period of incubation was signifi-

cantly higher (21%) (Fig. 5B, b) as compared to 1251-IGF-I (6%) (Fig. 5A, b) whereas almost 70% of the initial cellbound ‘251-IGF-II was recovered in the supernatant. Analysis

of IGF receptors

in differentiated

HT29-D4

cells

Figure 6 shows the results of 12’I-IGF-I and ‘251-IGF-II affinity cross-linking to differentiated HT29-D4 cells. This was done after opening the tight junctions in order to access to both apical and basolateral membrane domains. The results were basically the same as those obtained with undifferentiated HT29 cells and shown in Fig. 1. A major radioactive band with an apparent M, of 135,000 was present with the two radiolabeled ligands which corresponds to the putative a-subunit of the type I IGF receptor (Fig. 6, A and B, lanes a). Labeling of this species by ‘251-IGF-I disappeared in the presence of 150 nmol recombinant unlabeled IGF-I or IGF-II (Fig. 6A, lanes b and c) or 10 pg/mL (YIR-3 (Fig. 6A, lane d), but was only slightly inhibited in the presence of 1.5 pmol insulin (Fig. 6A, lane e). Figure 6B shows that the formation of the homologous M, 135,000 complex labeled bY iz51-IGF-II (Fig. 6B, lane a) was similarly prevented by 150 nmol of either IGF-I or IGF-II (Fig. 6B, lanes b and c), was weakly inhibited by 1.5 pmol insulin (Fig. 6B, lane e), but was not inhibited at all by 10 pg/mL aIR-3 (Fig. 6B, lane 4. Discussion

In order to subsequently investigate a possible role of endogenous IGF-II in the HT29 cell differentiation process, we have here studied the expression, antigenicity, ligand specificity, and ligand-induced redistribution of IGF receptors in both undifferentiated and differentiated HT29 cells. By use of affinity cross-linking and competitive binding experiments, we were unable to detect the type II IGF receptor on the HT29 cell surface whatever the state of cell differentiation. Such a conclusion is also consistent with the incapacity of HT29 cells to endocyte specific cell-bound radiolabeled IGF-II. If present, the constitutive cycling of

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REMACLE-BONNET

614

a-released

b-Surface

IGF

Bound

IGF

/‘j~~~j

v Li

C-Internalized

OlooI

IGF

T-----l

:l/iL==A

2 TIME(h)

FIG. 5. Time course of HT29 cell-associated ‘251-IGF-I and 12”I-IGF-II redistribution. Cell monolayers were incubated with 0.3 nmol of either Y-IGF-I (A) or ‘251-IGF-II (B) for 2 h at 4 C in the absence or the presence of 1 Fmol unlabeled IGF-I or IGF-II (to determine nonspecific binding). Cells were then washed three times and incubated at 37 C (time 0 on the Fig.). At each time, lz51-IGF released in the media (a), associated to the cell surface (acid stripped) (b), or internalized (acid resistant) (c), was determined and expressed as a percentage of the initial cell-specific radioactivity measured in control cells after the first incubation at 4 C (time 0 on the Fig.). Results are the means of triplicate determinations representative of three experiments. SD was always inferior to 4%.

type II IGF receptors (27) would induce its intracellular accumulation. In addition, both acid shock and suramin experiments do not evidence l&and-masked type II IGF receptors. Also, insulin is unable to promote a translocation of thesereceptors from an intracellular pool to the cell surface (our unpublished results), an ability reported in other systems (28). This absenceof HT29 cell-surface type II IGF receptors is in agreementwith a recent report indicating that receptors for IGF-I, but not for IGF-II, are present on plasma membranes from rabbit colon epithelial cells (29). In contrast, distinct type I and type II IGF receptors have been identified in the rat gastrointestinal epithelium (30) and in the human colonic epithelium (31). However, these results have been obtained with membrane preparations more or lesscontaminated by intracellular membranes.Therefore, it is likely that a great part of the identified receptors could account for the intracellular pool of type II IGF/mannose-6-phosphate receptors that represents about 90% of the total cell content at the steady state (28). However, the IEC-6 embryonic intestinal cell line derived from rat jejunal crypts expressesboth type I and type II IGF receptors judging by competitive

ET AL.

JCE & M. 1992 Vol75.No2

binding experiments done with intact cells (32). Also, in situ localization of IGF receptors using cryostat sections shows that the two types of receptors are expressedby the epithelium of the rat gastrointestinal tract (33). When compared to our findings, these latter observations raise the question whether the lack of type II IGF receptors at the HT29 cell surface is specifically associatedwith the malignant state of these cells. Although constitutively unable to express type II IGF receptors at their cell surface, we have previously reported that HT29 cells are quite able to secretesoluble forms of this receptor (34). These are likely truncated in their transmembrane and cytoplasmic domains (35). Similar soluble forms also have been found in the fetal blood circulation of several species(36, 37). Further studies will be necessary to generalize such results to other colorectal carcinoma cell lines and to define their precise physiological significance in viva. Further, our results clearly show that HT29 cells express high affinity type I IGF receptors on their surface, based on the presence of an affinity-labeled species of apparent M, 135,000, the specificity of inhibition of ‘251-IGF-I binding by unlabeled IGF-I, IGF-II, and insulin, and the inhibition of lz51-IGF-I binding by the cuIR-3 highly specific monoclonal antibody to the type I IGF receptor (18). Our findings also suggest that ‘251-IGF-II, at tracer amounts, binds to a variant binding site on the a-subunit of the type I IGF receptor at the HT29 cell surface: this radioligand is affinity cross-linked to a receptor protein identical in size and peptide binding specificity to the a-subunit of the HT29 type I IGF receptor; however, its binding is not inhibited by aIR-3. Again, the expression of both typical and variant type I IGF binding sites is not regulated in a differentiation-dependent manner. The binding plots give three times more varianf type I IGF receptors as compared to the typical ones (Figs. 2 and 3, insets). This suggeststhat the two types of binding sites are either associatedwith two subsets of type I IGF receptors having homologous structures but expressing a different antigenicity at their binding site or coexpressedby some a-subunits of the type I IGF receptors only. If the expression of such a variant type I IGF binding site by HT29 cells is in relation with the malignant transformation is, at the present time, unknown. Whatever may be, when ‘251-IGF-I in trace amounts was incubated with HT-29 cells, 75% of the labeled ligand bound to the aIR-3 sensitive type I IGF binding site (typical site) and 25% to the antibody insensitive site (variant site) (Fig. 2). Conversely, incubation of HT-29 cells with ‘251-IGF-II Ied to 95% of the trace binding to the otIR-3 insensitive type I-like IGF-I binding site and only 5% to the antibody sensitive-type I IGF binding site (Fig. 3). Several reports have previously described more than one type of IGF-I receptor (38-42). In agreement with our results, human placental and neuroblastoma cells expresstype I IGF receptors that contain two different binding sites that may be distinguished by their antigenicity toward otIR-3 (43, 44). It is interesting to note that the majority of these “&ypical” type I IGF receptors are of fetal or cancerous origin. I-IGFII binding to HT29 cells also evidences a second IGF-II binding site. However, its affinity is such (Kd = 147 nmol) that it might be considered as a nonspecific binding component. Alternatively, it might represent an IGFBP bound to the cells or merely to the tissueculture plastic (32, 45) which binds IGF-II only. Several biological effects of both IGF-I and II, especially their mitogenic effects are abolished by cuIR-3(16, 17). This

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EXPRESSION

Mr x10-3

1251-lG

OF IGF RECEPTORS

F- I \ _

0A

200-

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abcde FIG. 6. Affinity cross-linking of ‘Y-IGF-I and ‘251-IGF-II to differentiated HT29-D4 cells. Cell for 1 h at 37 C in order to open the tight junctions as described in Materials and Methods. Cells IGF-I (A) or ‘261-IGF-II (B) (lanes a) and various amounts of unlabeled competitors: 150 nmol mL (uIR-3 (lanes d), 1.5 pmol insulin (lanes e). Cross-linking, SDS-PAGE, and autoradiography

a

b

c

monolayers were treated with a Ca’+-free medium were then incubated for 2 h at 4 C with either iz51IGF-I (lanes b), 150 nmol IGF-II (lanes c), 10 bg/ were done as indicated in the Fig. 1.

responsesthrough a domain of the type I IGF receptor that is not blocked by aIR-3 (47, 48). Thus, it is possiblethat the two different type I IGF binding sitesidentified in HT29 cells may signal different IGF biological functions. our present results, as those reported by others (43, 44), Although undifferentiated and differentiated cells express showing that (wIR-3is unable to inhibit the binding of 1251an identical IGF receptors pattern, the endogenous IGF-II IGF-II to the type I IGF receptors may appear surprising. may yet interfere in the differentiation cell process. The Recently, Steele-Perkins and Roth (46) have attempted to heterogeneous IGFBP also secreted by these cells (11) may explain such a discrepancy by suggestingthat aIR-3 binds to indeed regulate IGF-II bioavailability to cell receptors, This the type I IGF receptor in such a way that it locks the receptor issue should be resolved by analyzing IGFBP molecular in a conformation which does not inhibit IGF-II binding but parameters all along this process. does inhibit the subsequent ability of the receptor to signal after IGF-II binds. We feel that this latter proposal is unlikely, however, since these authors compare the different aIR-3Acknowledgment induced alterations at different temperatures, i.e. 4 C for the We thank Ms. R. Rance for excellent technical assistance, binding experiments and 37 C for the biological assays.Our results indeed show that the binding inhibitory properties of &R-3 measuredat 4 C cannot be extrapolated to any biologReferences ical assaysmade at 37 C. At this latter temperature, ~YIR-3is quite as potent as unlabeled IGF-I to inhibit both ‘251-IGF-I 1. Pinto M, Appay B, Simon-Assman P, et al. 1982 Enterocytic differentiation of cultured human colon cancer cells by replacement and ‘251-IGF-II binding to HT-29 cells. Although the mechof glucose by galactose in medium. Biol Cell. 44:193-6. anismof such a temperature-dependent inhibition is unclear, 2. Fantini J, Abadie B, Tirard A, et al. 1986 Spontaneous and induced such an observation might contribute to give insight to dome formation by two clonal cell populations derived from a explain some if not all discrepanciesfound in the literature. human adenocarcinoma cell line. J Cell Sci. 83:235-49. It is well known that many biological functions of IGFs 3. Fantini J, Rognoni JB, Roccabianca M, Pommier G, Marvaldi J. 1989 Suramin inhibits cell growth and glycolytic activity and triggers require the internalization of the receptor-ligand complex differentiation of human colic adenocarcinoma cell clone HT29-D4. (26). If about 60% of cell-bound ‘251-IGF-I is specifically J Biol Chem. 264:10282-6. endocyted after a 2-h period at 37 C by HT29 cells, no 4. Betsholtz C. Iohnsson A, Heldin CH. Westermark B. 1986 Efficient significant endocytosis of ‘251-IGF-II at tracer concentrations reversion of Simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin. Proc Nat1 Acad Sci is observed. In these experimental conditions, the HT29 USA. 83:6440-4. variant type I IGF binding site appears, therefore, to be 5. Fantini J, Verrier B, Pit P, Pichon J, Mauchamp J, Marvaldi J, unable to mediate a biological signal that would require the 1990 Suramin-induced differentiation of the human colic adenocarinternalization of the receptor. IGFs have been found to cinema cell clone HT29-D4 in serum-free medium. Exp Cell Res. mediate in some tumor cell lines nonproliferative biological 189:109-17.

observation as a growing body of evidence suggests that most of the biological effects of IGF-II are mediated predominantly via the type I IGF receptor. In light of this assumption,

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