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Endocrinology Vol. 142, No. 3 1090-1097
Copyright © 2001 by The Endocrine Society

ARTICLES

Identification of a Type II Insulin-Like Growth Factor Receptor in Fish Embryos1

Eva Méndez, Josep V. Planas, Juan Castillo, Isabel Navarro and Joaquim Gutiérrez

Departament de Fisiologia, Facultat de Biologia, D. III Universitat de Barcelona, 08028 Barcelona, Spain

Address all correspondence and requests for reprints to: Joaquim Gutiérrez, Departament de Fisiologia, Facultat de Biologia, D. III Universitat de Barcelona, Avenida Diagonal 645, 08028 Barcelona, Spain. E-mail: joaquim{at}porthos.bio.ub.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether fish have an insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6-P) receptor similar to that of mammals, we have performed binding, cross-linking, and immunoprecipitation experiments with wheat-germ-agglutinin- and mannose 6-phosphate (M6-P)-affinity-purified receptor preparations from fish embryos. In both receptor preparations, IGF-II binding was specific, because labeled IGF-II could only be completely displaced by cold IGF-II but not by IGF-I or insulin. Labeled IGF-II bound to a protein with a molecular mass of approximately 250 kDa, which could be immunoprecipitated with an antibody against the rat IGF-II receptor. IGF-II stimulated tyrosine kinase activity in wheat germ agglutinin preparations and was more potent than insulin or IGF-I, but neither peptide stimulated tyrosine kinase activity in M6-P preparations. Two fish cell lines (CHSE-214 and EPC) were used to confirm the IGF-II binding data obtained in the receptor preparations, revealing the presence of highly specific IGF-II binding and the absence of insulin binding. Furthermore, a decrease of the IGF-I receptors on the cell surface did not alter IGF-II binding in EPC cells. In conclusion, we have detected the presence of IGF-II/M6-P receptors in fish embryos that are similar in structure and specificity for their ligand to those found in mammals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE GROWTH FACTOR (IGF)-I and IGF-II are mitogenic polypeptides structurally related to proinsulin. The liver provides the major source of circulating IGFs (1), which have an endocrine action; and both IGF-I and IGF-II are locally produced in several tissues, exerting a paracrine or autocrine function (2).

IGFs are involved in several biological processes, such as growth, development, and metabolism (3). In rodents, IGF-I predominates in postnatal and adult life, and its production is principally controlled by GH. On the other hand, IGF-II is produced in embryonic, fetal, and early neonatal life; and its production seems to be independent of GH regulation (4, 5).

In mammals, whereas insulin and IGF-I are known to exert their biological responses through specific receptors, IGF-II has been shown to act through the type I IGF receptor (6, 7) and through the insulin receptor (8, 9, 10). Insulin and IGF-I receptors are heterotetrameric glycoproteins of 350 kDa, which function as tyrosine kinases. On the other hand, the type II IGF receptor, identified as the cation-independent mannose-6-phosphate (M6-P) receptor (11, 12, 13), is structurally unrelated to the IGF-I and insulin receptor, being a single-chain glycoprotein of approximately 250–270 kDa (3, 14, 15). This receptor can bind IGF-II with high affinity, and IGF-I with less affinity, and it does not bind insulin (11). The result of IGF-II binding to the type II IGF receptor is only partially known, but it seems that the IGF-II/M6-P receptor is involved in IGF-II clearance (3, 16). In support of this hypothesis, mouse embryos lacking the type II IGF receptor have elevated serum levels of IGF-II and show accelerated somatic growth in the fetal period (17).

Among nonmammalian vertebrates, the presence of IGFs has been reported in birds (18, 19), reptiles, amphibians (20), and fish (21, 22, 23); and their structure seems to be highly conserved. For example, salmon and human IGF-I differ only in 14 out of 70 amino acids (21). Rainbow trout IGF-I and IGF-II proteins (61% homology between each other) share a 79.6% and 64.8% homology with human IGF-I and IGF-II, respectively (22).

The expression of IGF-I and IGF-II throughout fish development seems to be developmentally regulated. Several studies have detected high levels of expression of IGF-II in early embryogenesis (23, 24), followed by a decrease in the levels of expression of IGF-II and an increase in the levels of expression of IGF-I (25, 26). These results may indicate that both IGF-I and IGF-II play a role during early development of teleosts, as in other vertebrates.

In fish, receptors for insulin and IGF-I have been detected in different tissues (24) and during early stages of development (25). Maestro and co-workers (27) found that, throughout early development of trout, IGF-I receptors were more abundant than insulin receptors. The binding of insulin and IGF-I to their receptors seems to be specific, thus, demonstrating the existence of two separate receptors from very early stages of development. Both receptors possess tyrosine kinase activity (TKA) and are structurally similar to their mammalian counterparts (28).

The cation-independent M6-P receptor has been identified in birds (29, 30, 31, 32, 33) and amphibians (34, 35); however, contrary to that in mammals, the avian or the amphibian M6-P receptor was reported to be unable to bind IGF-II, suggesting that IGF-II could bind to other receptors. Recently, Yandell and co-workers (36) have suggested that the acquisition of the IGF-II binding site on this receptor may have occurred relatively late in evolution.

In this study, we have identified and characterized, for the first time in nonmammalian vertebrates, IGF-II receptors in embryos of a teleost (brown trout, Salmo trutta).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Wheat germ agglutinin (WGA), bound to agarose, was purchased from Vector Laboratories, Inc. (Burlingame, CA). Human recombinant IGF-I was a gift from Chiron Corp. (Emeryville, CA), and human recombinant IGF-II was purchased from Peninsula Laboratories, Inc. Europe Ltd. (Merseyside, UK). Human recombinant 3-125I IGF-II, human recombinant 3-125I IGF-I, both with a specific activity of 2000 Ci/mmol and [32{gamma}P]-ATP with a specific activity of 5000 Ci/mmol, were purchased from Amersham Pharmacia Biotech Europe GmBH (Barcelona, Spain). Disuccinimidyl suberate (DSS) was purchased from Pierce Chemical Co. (Rockford, IL). IGF-IIR, the monoclonal antibody against the rat IGF-II receptor, was from Transduction Laboratories, Inc. (Lexington, KY), and FBS was purchased from Cansera International Inc. (Ontario, Canada). All other cell culture and chemical reagents were purchased from Life Technologies, Inc. (Barcelona, Spain) and Sigma-Aldrich Corp. Quimica, S.A (Alcobendas, Madrid, Spain), respectively.

Animals and experimental design
Brown trout (Salmo trutta) eggs, embryos, and juvenile fish muscle, obtained from the fish farm Piscifactoría de Bagá (Barcelona, Spain), were sampled throughout two reproductive cycles (1996–1998). Before hatching, samples were collected at two stages: 1st and 5th weeks of life (newly laid eggs and organogenesis stage, respectively). After hatching, fish were sampled during the 9th week of life (yolksac larvae). In addition, 1-yr-old fish (juvenile) were sampled for skeletal muscle. To remove the skeletal muscle, juvenile fish were killed by a cranial blow and strips of white lateral muscle were rapidly excised and frozen in liquid nitrogen. All other samples were frozen directly in liquid nitrogen. Samples were stored in liquid nitrogen until purification of receptors.

Partial purification of glycoprotein receptors
Two different procedures were used for samples with and without yolk.

Samples with yolk. Partial purification of receptors, removing yolk by sequential centrifugations, was performed as previously described by Maestro and co-workers (27). Frozen samples (25–30 g for samples before hatching, and 7 g for 9-week-old embryos) were defrosted in Tris-HCl buffer (25 mM Tris-HCl, 5 mM CaCl2, pH 7.6) and homogenized with a POLYTRON. The homogenate was centrifuged at 600 x g for 10 min at 4 C. The supernatant was recovered and centrifuged at 40,000 x g for 30 min at 4 C. Both pellets obtained were resuspended in a buffer containing 25 mM HEPES, 4 mM EDTA, 4 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM bacitracin, 1 mM leupeptin, 1 mM pepstatin, and 25 mM benzamidine, pH 7.6. Solubilization of the membranes was performed by adding Triton X-100 to a final concentration of 2% and stirring for 1 h at 4 C. The resuspension was centrifuged at 150,000 x g for 90 min at 4 C, and the supernatant was recycled three times through a column containing 1 ml WGA bound to agarose. The resin was washed with 70 ml of a buffer containing 25 mM HEPES and 0.1% Triton X-100, pH 7.6. Receptors were eluted from the WGA column with the same buffer supplemented with 0.3 M N-acetyl-D-glucosamine.

Juvenile fish muscle samples. Processing of the samples and semipurification of receptors were performed at 4 C, following the method of Párrizas and co-workers (37). Briefly, frozen samples (7–8 g) were macerated in a cooled mortar and then homogenized in the HEPES buffer used for pellet resuspension of the samples with yolk. The homogenate was then solubilized by adding Triton X-100 to a final concentration of 2%. Further processing of samples was conducted as described for samples with yolk.

Purification of IGF-II/M6-P receptors
Solubilized membranes were obtained as described above. After the ultracentrifugation (150,000 x g for 90 min at 4 C), the supernatant was subjected to affinity chromatography, following the method of Dahms and co-workers (38), with some modifications. Briefly, the supernatant was recycled three times through a column containing 1 ml M6-P bound to agarose. The resin was washed with 15 ml of a buffer containing 50 mM imidazole, 150 mM NaCl, and 5 mM sodium {beta}-glycerophosphate, pH 7. Receptors were eluted from the column with the same buffer supplemented with 5 mM M6-P.

Ligand binding assays in receptor preparation
Binding assays were performed by an adaptation of the method of James and co-workers (39). Forty microliters of the WGA eluate and 80 µl of the M6-P eluate (concentrated 5- to 10-fold by centrifugation at 5000 x g for 15 min at 4 C in filtron 30K Microsep Centrifugal Concentrators), corresponding to 10–30 µg glycoproteins and approximately 6 µg of IGF-II/M6-P receptors, respectively (measured by the Bradford Protein Assay, Bio-Rad Laboratories, S.A, Madrid, Spain), were incubated for 16 h at 4 C in 30 mM HEPES buffer containing 0.1% BSA and 100 U/ml bacitracin (pH 7.6), with increasing concentrations of unlabeled insulin, IGF-I, and IGF-II (from 0.0125–100 nM porcine insulin or human recombinant IGF-I and IGF-II) and the radiolabeled ligand at tracer concentrations (25 pM). Specificity of the IGF-II binding sites was determined by displacing labeled IGF-II with increasing concentrations of heterologous peptide (cold insulin or IGF-I). Semipurified receptors and purified IGF-II/M6-P receptors were precipitated by bovine {gamma}-globulin (0.08%) and polyethylene glycol (10.4% wt/vol) and centrifugation at 14,000 x g for 7 min at 4 C. Nonspecific binding was estimated as 125I-ligand bound in the presence of 100 nM unlabeled hormone and subtracted from the total counts, to give specific binding values. Specific binding data were analyzed on Scatchard plots. The program Sigma Plot (SPSS Science, Chicago, IL) was used for the mathematical fitting with a sigmoidal function of displacement curves and the EC50 values (concentration of cold peptide that displaced 50% of tracer) were calculated with the program GraphPad Software, Inc. Inplot (Harvey J. Matulsky, CA). Each binding experiment was performed in duplicate at least four times using semipurified or purified receptors from each different developmental stages.

Ligand binding assays in fish cell lines
The salmon embryo cell line (CHSE-214), obtained from the American Type Culture Collection (Manassas, VA), was maintained in DMEM containing 10%(vol/vol) FBS, penicillin (100 U/ml), and streptomycin (100 U/ml). The cells were grown and used for experiments as monolayers at 21 C in a 5% CO2 atmosphere (40). The carp epitelioma cell line (EPC), from the European Collection of Cell Cultures (Wiltshire, UK), was maintained and used for experiments in the same conditions as the CHSE-214 but at 25 C (41).

Confluent monolayers in 24-well plates (approximately 7 x 105 cells/well) were washed twice, over 2 h, in 0.1 M HEPES, 0.12 M NaCl, 5 mM KCl, 1.2 mM MgSO4, 8 mM glucose, 0.5% BSA (binding buffer, pH 7.6) and were incubated for 4 h at 4 C in 0.5 ml of the same buffer containing radiolabeled ligand (25 pM), in the presence or absence of unlabeled insulin, IGF-I, or IGF-II. Subsequently, the monolayers were washed twice with the binding buffer and burst open with 0.5 N NaOH for the determination of radioactivity and protein content.

To demonstrate the presence or absence of IGF-binding proteins (IGFBPs), labeled IGF-I was displaced by IGF-I and des(1, 2, 3) IGF-I, an analog of IGF-I that exhibits low affinity for most of the IGFBPs.

For internalization experiments, confluent monolayers in 60-mm plates (1 x 107 cells/plate) were preincubated with or without 1 µM IGF-I, for 60 min at 4 C, with the binding buffer, and further incubated with radiolabeled IGF-I or IGF-II (25 pM) for 4 h at 4 C. Then, the binding assay was performed as described above.

Cross-linking
Affinity cross-linking experiments were performed by the method of Waugh and co-workers (42),with some modifications, using DSS as a cross-linker. WGA-enriched preparations (50 µg, about 1500 fmol) were incubated overnight at 4 C with or without IGF-II, insulin, and IGF-I (400 nM, final concentration) and labeled hormone (100 pM) in HEPES 60 mM, pH 7.6. Subsequently, samples were incubated with DSS (1 mM) on ice for 15 min. The cross-linking reaction was stopped by adding one third of total volume of Laemmli sample buffer three times concentrated (LSB x 3) (300 mM Trizma base, 60% glycerol, 6% SDS wt/vol; ±1.15% DL-dithiothreitol, 0.004% bromophenol blue). The samples were heated to 95 C for 5 min. They were then subjected to polyacrylamide 7.5% gel electrophoresis performed in the presence of SDS (SDS-PAGE). Labeled IGF-II, linked to the insulin, IGF-I ,and IGF-II receptors, was detected by autoradiography using Kodak X-OMAT AR films (Amersham Pharmacia Biotech) and Cronex Lightning Plus enhancing screens.

Immunoprecipitation
IGF-II/M6-P receptors were cross-linked to 125I-IGF-II and immunoprecipitated. Briefly, different receptor preparations (solubilized membranes, WGA, and M6-P) were first concentrated 5- to 10-fold with Filtron 30K and then incubated for 16 h at 4 C with 125I-IGF-II (25 pM) in binding buffer (pH 7.6). After the overnight incubation, cross-linking was carried out with 1 mM DSS on ice for 15 min. The reaction was stopped by adding 10 mM TrisHCl. Subsequently, receptor preparations were reduced with 20 mM DL-dithiothreitol and incubated with or without the antibody against the rat IGF-II receptor (3.7 µg/tube) for 8 h at 4 C, with agitation. The labeled IGF-II/M6-P receptors were then immunoprecipitated by the aid of ProteinA-Sepharose, eluted, and subjected to SDS-PAGE under reducing conditions (7.5% acrylamide). Radiolabeled bands were visualized by autoradiography using Kodak X-OMAT AR films and Cronex lightning Plus enhancing screens.

TKA
TKA was determined according to James and co-workers (36), with some modifications. Receptor preparations (8–12 µg) were preincubated for 16 h at 4 C with cold insulin, IGF-I, or IGF-II (60 nM, final concentration) in HEPES buffer containing 100 mM MgCl2, pH 7.4. Receptor samples were then incubated with 50 µM [32{gamma}P]-ATP for 10 min to allow autophosphorylation. Synthetic substrate poly(Glu:Tyr; 4:1) was added to a final concentration of 0.25 mg/ml and, after a further incubation for 30 min, the reaction was stopped by transferring samples to filter paper squares (Whatmann 3MM) and soaking them in 10% trichloroacetic acid containing 10 mM sodium pyrophosphate. Paper squares were counted in a scintillation counter, and results were expressed as radioactivity incorporated into the substrate: percent of phosphorylation above basal level (without added hormone).

Statistical analysis
Differences among groups were analyzed by ANOVA followed by a test based on the least significant differences between means using the STATGRAPHICS System Version 5.0. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ligand binding properties
The characteristics of IGF-II binding were studied in WGA semipurified receptor preparations, in affinity-purified IGF-II/M6-P receptor preparations, and in a cell culture system.

WGA receptor preparations. Glycoprotein content in semipurified receptor preparations from trout embryos varied with the developmental stage. We detected 10.47 ± 1.6 µg glycoprotein per gram of initial tissue for 1-week-old embryos, 16.44 ± 3.2 for 5-week-old embryos (eyed eggs), and 130 ± 12 µg for 9-week-old embryos (yolksac larvae). Muscle of juvenile fish (1 yr old) yielded 147 ± 3.14 µg per gram of initial tissue.

Binding characteristics of insulin, IGF-I, and IGF-II in 5-week-old embryos are shown in Table 1Go. Binding for IGF-II was higher than that for IGF-I (1.2-fold) and higher than insulin binding (20-fold). This binding ratio among insulin, IGF-I, and IGF-II was maintained in all stages studied (data not shown). Labeled IGF-II bound the semipurified receptor preparations with affinity similar to that of IGF-I and higher (2.4-fold) to that of insulin.


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  		Table 1. Binding characteristics of insulin, IGF-I, and IGF-II in semipurified receptor preparations from 5-week-old embryos obtained by WGA-agarose affinity chromatography

 
The characteristics of IGF-II binding in four developmental stages (1st, 5th, and 9th week of development in whole embryos, and white muscle of 1-yr-old fish) were studied (Fig. 1Go). IGF-II binding was detectable in all the stages analyzed, and elevated binding values were found during the 5th week of development (77.9% per 20 µg glycoprotein). The affinity of the binding was similar in all the stages studied [dissociation constant (Kd) values ranged from 0.13–0.16 nM]. Receptor number (Ro) varied with development, being 106.7 ± 26 fmol/mg glycoprotein eluted for 1-week-old embryos, 1157 ± 285 for 5-week-old embryos, 444 ± 31 for 9-week-old embryos, and 57 ± 3 for 1-yr-old fish.



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  		Figure 1. Changes in percentage of IGF-II-specific binding per 20 µg glycoprotein in four developmental stages: first week of development; fifth week; ninth week; 1-yr-old fish. Data are mean ± SE of at least four different semipurifications, each performed in duplicate. Different letters indicate significantly (P < 0.01) different values among groups.

 
The ability of cold IGF-II, IGF-I, and insulin to displace 125I-IGF-II in receptor preparations from 5-week-old embryos is shown in Fig. 2Go. Displacement of labeled IGF-II with unlabeled IGF-II was achieved at lower concentrations than with IGF-I and insulin. The EC50 value for IGF-II was 15-fold lower than that for IGF-I (0.4 ± 0.15 and 6 ± 0.36 nM, respectively). Insulin was not able to displace labeled IGF-II at any concentration tested (from 0.01–100 nM).



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  		Figure 2. Specificity of IGF-II binding in 5-week-old embryos: 125I-IGF-II displaced by increasing concentrations of unlabeled IGF-II (•), IGF-I ({circ}), and insulin ({blacktriangledown}). Binding values are expressed as percentage of maximum binding. Data are mean ± SE of four separate receptor semipurifications, each performed in duplicate.

 
IGF-II/M6-P receptor preparations. The glycoprotein content of affinity-purified IGF-II/M6-P receptor preparations was 0.30 ± 0.01 µg per gram of initial tissue for 1-week-old embryos and 2.82 ± 0.6 µg for 9-week-old embryos.

The IGF-II binding characteristics of the affinity-purified IGF-II/M6-P receptors were studied in 9-week-old embryos (Fig. 3Go). Specific binding for IGF-II was 2 ± 0.26%, whereas IGF-I binding percentage in these preparations ranged from undetectable values to 0.5%, and insulin was not detectable (data not shown). The number of IGF-II/M6-P receptors was lower than the number of insulin and IGF-I receptors, as shown in Table 1Go (5-fold and 36-fold, respectively). IGF-II/M6-P receptors bound labeled IGF-II with high affinity, similar to that found in WGA semipurified receptor preparations and to that of IGF-I for its receptor, but higher than the affinity of insulin for the insulin receptor (Table 1Go). Binding characteristics of IGF-II/M6-P receptors were obtained by Scatchard analysis, which revealed the presence of a single class of high-affinity binding sites. The specificity of the receptors was evidenced by competitive displacement and the inability of unlabeled IGF-I and insulin to completely displace labeled IGF-II: unlabeled IGF-I (100 nM) displaced 47% of labeled IGF-II, whereas unlabeled insulin did not displace labeled IGF-II (even at 100 nM). However, cold IGF-II was able to completely displace labeled IGF-II at a concentration of 100 nM. In those preparations, EC50 for IGF-II was 1250-fold higher than that for IGF-I (0.8 nM and 1 µM, respectively).



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  		Figure 3. Scatchard analysis of IGF-II binding to affinity-purified IGF-II/M6-P receptors in the presence of increasing concentrations of unlabeled IGF-II in 9-week-old embryos. B, Bound; B/F, bound/free. A representative experiment, from a total of six, is shown. Kd (dissociation constant, inverse of affinity of receptors, nM); %Bsp, percentage of specific binding per 20 µg glycoprotein; Ro, receptor number (binding capacity fmol per mg glycoprotein eluted). Data are mean ± SE of three experiments, each performed in duplicate. Each different determination represents the concentrated eluate resulting from at least four purifications.

 
Cell culture. To find an in vitro model system in which to further investigate the presence and characteristics of IGF-II/M6-P receptors, binding experiments were performed with two established fish cell lines, a salmon embryonic line (CHSE-214) and a carp epitelioma cell line (EPC) (Table 2Go). IGF-II binding was higher than IGF-I binding (3- to 6-fold) in the two fish cell lines, and this binding was found to be specific, given that neither insulin nor IGF-I (at a concentration of 1 µM) was able to displace labeled IGF-II. Complete competition of 125I-IGF-II took place at 500 nM IGF-II. This gave us the nonspecific binding value (1.73 ± 0.12% for CHSE-214, and 0.89 ± 0.2% for EPC). Insulin binding was not detected in any experiment analyzed. Experiments performed with des(1, 2, 3) IGF-I revealed displacement curves similar to those found with IGF-I, confirming the low presence of IGFBPs in our culture system (data not shown).


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  		Table 2. Specific binding for insulin, IGF-I, and IGF-II in two fish cell lines

 
In addition, to further demonstrate the specificity of IGF-II binding, IGF-I receptor internalization experiments, using unlabeled IGF-I, were performed with EPC cells (Fig. 4Go). IGF-I (1 µM) caused a significant reduction in the number of IGF-I receptors from the cell surface (which corresponded to an 80% decrease in IGF-I binding) but without altering IGF-II binding.



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  		Figure 4. Effect of preincubation with IGF-I (1 µM) on IGF-I- and IGF-II-specific binding in EPC cells. Data are mean ± SE of two different experiments, each performed in duplicate. Different letters indicate significantly (P < 0.01) different values among groups.

 
Cross-linking experiments
In cross-linking experiments performed with WGA preparations from 9-week-old embryos, two bands (of approximately 250 and 125 kDa) were observed (Fig. 5Go). The band with an apparent molecular mass of 250 kDa was abolished when excess of IGF-II (400 nM) was added, and it was not displaced by cold IGF-I (400 nM) or insulin (400 nM). The band of 125 kDa was displaced almost completely by cold IGF-II and IGF-I, but cold insulin was not able to displace bound 125I-IGF-II. Rat liver, used as control, gave a unique band (molecular mass, 250 kDa), corresponding to the size of the mammalian IGF-II/M6-P receptor.



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  		Figure 5. Affinity cross-linking of 125I-IGF-II to WGA receptor preparations in 9-week-old embryos. Glycoproteins were analyzed on 7.5% SDS-PAGE under reducing conditions and autoradiography. T, Total binding. Unlabeled IGF-II, IGF-I, and insulin (Ins) were also included (400 nM), to analyze the specificity of the binding. A representative experiment, from a total of five, is shown.

 
Immunoprecipitation of IGF-II/M6-P receptors
To confirm that the 250-kDa band obtained in the cross-linking experiments corresponded to an IGF-II/M6-P receptor with a functional IGF-II binding site, immunoprecipitation of the cross-linked receptor was performed with WGA preparations from 9-week-old embryos. The unique protein to which 125I-IGF-II was cross-linked and could be immunoprecipitated by the antibody against rat IGF-II receptor is shown in Fig. 6Go. Immunoprecipitation was followed by SDS-PAGE, under reducing conditions, and a 250-kDa band was detected. This band had the same molecular mass as the rat sample used as control. Complete competition for 125I-IGF-II occurred in the presence of an excess of unlabeled IGF-II (100 nM).



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  		Figure 6. Immunoprecipitation of IGF-II/M6-P receptors from 9-week-old embryos. Labeled IGF-II was cross-linked to IGF-II/M6-P receptors, followed by immunoprecipitation with a monoclonal antibody against rat IGF-II receptor. Receptors were resolved in 7.5% SDS-PAGE under reducing conditions. T, Total 125I-IGF-II bound to the IGF-II/M6-P receptors that could be immunoprecipitated; NS, nonspecific binding (by adding unlabeled IGF-II at 100 nM in the binding, before immunoprecipitation); Bl, blank (immunoprecipitation of receptors without antibody). A representative experiment, from a total of five, is shown.

 
To further confirm the specificity of the IGF-II binding, IGF-II receptors were removed from the WGA preparation by passing it through a M6-P affinity column, and the fraction of the WGA eluate that was not bound to the M6-P column was then subjected to the immunoprecipitation protocol. The result was that no band of 250 kDa appeared, probably as a result of the lack of IGF-IIRs in the M6-P eluate.

Immunoprecipitation experiments performed with EPC cells also resulted in a unique 250-kDa band, giving similar data as described for WGA (data not shown).

TKA
Stimulation of TKA by insulin, IGF-I, and IGF-II was studied in WGA semipurified receptor preparations and in affinity-purified IGF-II/M6-P receptor preparations from 9-week-old embryos (Fig. 7Go). In WGA preparations, IGF-II was more potent than insulin (nearly 2-fold higher) and IGF-I (1.2-fold higher) in stimulating the phosphorylation of the exogenous substrate. However, neither IGF-II, IGF-I, nor insulin stimulated TKA when affinity-purified IGF-II/M6-P receptors were used.



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  		Figure 7. TKA in WGA (A) and M6-P preparations (B) from 9-week-old embryos. Results are expressed as percent of phosphorylation of the exogenous substrate above basal level (without added hormone). Data are mean ± SE of at least three different experiments. Different letters indicate significantly (P < 0.01) different values among groups. INS, Insulin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent studies indicate that the mammalian IGF-II/M6-P receptor is a bifunctional protein that mediates lysosomal enzyme targeting and also regulates the extracellular levels of IGF-II (2, 3, 43). Ligand binding studies demonstrate that the mammalian IGF-II receptor has a single high-affinity binding site for IGF-II and two sites for M6-P-containing proteins (11, 12, 13). However, binding and affinity cross-linking studies in chicken (29, 31, 44) and Xenopus (30, 33, 34) showed that IGF-II/M6-P receptors failed to bind IGF-II, which led the authors to suggest that the acquisition of a high-affinity binding site for IGF-II could have occurred after the separation of birds and amphibians from mammals but before the separation of metatheria and eutheria (38). Recently, Yandell and co-workers (36) have shown that the deduced amino acid sequence of the IGF-II binding site of the kangaroo IGF-II receptor is different from the eutherian counterpart and that this IGF-II binding site is of low affinity. Therefore, Yandell, and co-workers (36) postulated that the appearance of a high-affinity binding site for IGF-II in the IGF-II receptor could be a recent event specific to the eutherian lineage.

In spite of these previous reports, we have detected the presence of specific IGF-II binding in fish embryos, because IGF-II binding could not be completely displaced by either IGF-I or insulin in any of the receptor preparations tested (WGA, purified with a WGA-agarose column that binds membrane glycoproteins, such as receptors for insulin, IGF-I and IGF-II; and M6-P, purified with a M6-P affinity column that allows us to elute specifically IGF-II/M6-P receptors). In addition, labeled IGF-II bound to a protein of a molecular mass of approximately 250 kDa, as demonstrated by the cross-linking experiments, and we suggest that it represented the IGF-II/M6-P receptor, because IGF-I and insulin were unable to displace the bound labeled IGF-II. When we immunoprecipitated the cross-linked receptor with an antibody against the rat IGF-II receptor, only one protein of a molecular mass of approximately 250 kDa was detected, similar in size to its mammalian counterpart. This band could be completely displaced when an excess of IGF-II was added to the sample before immunoprecipitation, which constitutes further evidence for the presence of a type II IGF receptor in fish that binds specifically IGF-II.

Furthermore, the fact that M6-P preparations were devoid of TKA suggests that the fish IGF-II/M6-P receptor lacks the tyrosine kinase domain, similar to other vertebrate IGF-II receptors described (11, 15, 45). Our studies with WGA preparations revealed that IGF-II was more potent than IGF-I or insulin in the stimulation of TKA. Similarly, Perdue and co-workers (46) observed that IGF-II stimulated the IGF-I receptor’s protein kinase more efficiently than IGF-I, as a result of the similar ability of IGF-I and IGF-II to bind the {alpha}-subunit of the IGF-I receptor. Consistent with this, in our cross-linking experiments, it can be observed that labeled IGF-II also bound to the 130-kDa {alpha}-subunit of the IGF-I receptor and could only be completely displaced by cold IGF-I and IGF-II but not by an excess of cold insulin (34). Therefore, we suggest that, in our WGA preparations, IGF-II could be stimulating TKA mainly through the IGF-I receptor.

Summarizing data on binding, cross-linking, immunoprecipitation, and TKA experiments, we can assume that only a small percentage of the total IGF-II binding found in the WGA preparations corresponds to an IGF-II/M6-P receptor. The major part of this binding probably corresponds to the binding of IGF-II to the IGF-I receptor.

From the time of fertilization, IGF-II binding was detectable, and the highest levels were observed during organogenesis, suggesting an important role for this peptide during early stages of development. This is consistent with the data available on expression of IGFs in fish, where it has been confirmed that both IGF-I and IGF-II messenger RNAs (mRNAs) are detected during early embryonic development (23, 24, 25, 26), thus suggesting that both IGF-I and IGF-II may play an important role during early development in teleosts. In mammals, a spatial and temporal coordination in the expression of IGF-II and IGF-II receptors has been reported in several embryonic tissues, suggesting a role for the receptor during the period when IGF-II is playing an important autocrine/paracrine role in development (47).

We have described that IGF-II/M6-P receptors bind IGF-II with high affinity, and this binding function for the IGF-II/M6-P receptor in lower vertebrates is compatible with the existence of two types of M6-P receptors in lower vertebrates, as demonstrated by Nadimpalli and co-workers (35). It is possible that both types of M6-P receptors could mediate the transport of lysosomal enzymes to lysosomes, but only the IGF-II/M6-P receptor would bind IGF-II. The effect of high levels of IGF-II is to increase embryo size (17), and the role of the IGF-II/M6-P receptor could be to bind and degrade IGF-II, therefore regulating the effects of the mitogenic peptide.

Experiments performed with fish cell lines allowed us to find another model where IGF-II binding was found to be highly specific. IGF-II binding was 3- to 4-fold higher than IGF-I binding, suggesting that this binding could be attributable to a type II IGF receptor. We found that, in the fish cell lines used, insulin receptors were not detectable; and when the number of IGF-I receptors from the cell surface was decreased experimentally, IGF-II binding was not affected. Moreover, the studies performed with des(1, 2, 3) IGF-I confirmed the absence or the low presence of IGFBPs in the cell culture. des(1, 2, 3) IGF-I does not bind IGFBPs; therefore, if IGFBPs were found in our culture system, des(1, 2, 3) IGF-I would not have displaced labeled IGF-I bound to IGFBPs. Given that des(1, 2, 3) IGF-I displaced IGF-I binding similarly to the unmodified IGF-I, we can conclude that IGFBPs did not significantly interfere with IGF binding in our cell culture.

The most intriguing question is why IGF-II/M6-P receptors could not be detected in previous studies (29, 30, 31, 44). We believe that this may be caused by the low number of IGF-II receptors present in the receptor preparations. In our study, we had to concentrate the receptors several fold to detect them. Similarly, Yandell and co-workers (36), working with solubilized membranes, also had to concentrate the receptor preparations 10 times to detect the kangaroo type II IGF receptors.

In conclusion, we have detected for the first time, in a nonmammalian vertebrate (the trout), specific IGF-II receptors that bind IGF-II with high affinity. Further studies are needed to establish the physiological role of IGF-II binding to the IGF-II/M6-P receptor in fish.


   Acknowledgments
 
We thank Mr. Antonino Clemente at the Piscifactoría de Bagà (Departament de Medi Natural, Generalitat de Catalunya) for providing the trout and facilities to conduct the sampling, and for his generous assistance. We thank Drs. Jorge Lloberas for his assistance with the immunoprecipitations and Simon MacKenzie for his critical review of the manuscript. We are very grateful to Chiron Corp. for providing human recombinant IGF-I.


   Footnotes
 
1 This study was supported by grants from the European Union (FAIR CT95–0174), DGICY Spain (AGF98–0325, PB98–1249, PB97–0902), CIRIT (1998 SGR-0037), and NATO (CRG 974408). Back

Received May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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