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Division of Endocrinology, Diabetes, and Bone Diseases (K.I., S.Y., D.L.), Department of Medicine, The Mount Sinai School of Medicine, New York, New York 10029; Institute of Pediatric Endocrinology (A.T., V.P.), Endocrinology Research Center, Moscow 117036, Russian Federation; Engelhardt Institute of Molecular Biology (P.R., P.S.), Moscow 119991, Russian Federation; and State Research Center for Medical Genetics (S.T.), Russian Academy of Medical Sciences, Moscow 115478, Russian Federation
Address all correspondence and requests for reprints to: Derek LeRoith, Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine, The Mount Sinai School of Medicine, New York, New York 10029.
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Abstract |
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Objective: The objective of the study was to explore the mechanism whereby the R481Q mutation may be causative in growth retardation.
Patients: A 13-yr-old girl with short stature was studied for functional analysis of the R481Q mutation in the IGF-IR.
Results: Two members of a family who showed intrauterine and postnatal growth retardation, with increased serum IGF-I levels, demonstrated a substitution of arginine for glutamine at 481 (R481Q) in the IGF-IR. This mutation results in the formation of an altered fibronectin type III domain within the 
-subunit. NIH-3T3 fibroblasts that overexpress the human wild-type or R481Q mutant IGF-IR demonstrated normal cell surface ligand binding by 125I-IGF-I binding assay. However, the fold increase of IGF-I stimulated tyrosine phosphorylation of the IGF-IR ß-subunit as well as downstream activation of ERK1/2 and Akt was reduced in cells overexpressing the mutant receptor. Additionally, basal and IGF-I-stimulated levels of cell proliferation were also reduced in cells overexpressing the mutant receptor.
Conclusion: Our results demonstrate that NIH-3T3 cells overexpressing a mutant form of the Igf1r gene, in which arginine at 481 is substituted by glutamine, lead to reduced levels of the fold increase of IGF-IR ß-subunit phosphorylation as well as ERK1/2 and Akt phosphorylation and was accompanied by decreased cell proliferation. These results are postulated to be the cause of intrauterine and postnatal growth retardation in the described patients.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits, which form the binding site for the IGFs, and two transmembrane ß-subunits with intrinsic tyrosine kinase activity. Upon ligand binding, tyrosine residues in the ß-subunits of IGF-IR undergo autophosphorylation. This results in the binding of signaling proteins to this cytoplasmic domain. Adaptor proteins such as the insulin receptor substrate protein family (insulin receptor substrate 1–4) and Shc proteins bind and transmit signals downstream of the receptor via the Ras/Raf/MAPK pathway and the phosphatidylinositol 3-kinase/Akt pathway, which then mediate cell proliferation, metabolism, and cell survival (1, 2, 3). The structure of the IGF-IR and the insulin receptor (IR) is similar, but their physiological roles are distinct. IGF-I signaling pathways regulate mainly proliferation and survival of the cell, whereas insulin signaling pathways play important roles in metabolism (4, 5, 6). There are a number of well-described mutations in the IR affecting its biological actions (7). In contrast, functional analyses of mutations in the IGF-IR are few (8, 9). Compound Arg108Gln/Lys115Asn substitution, which is located at the leucine-rich repeats 1 (L1) region of the IGF-IR, causes changes in the amino acid charge, which leads to reduced ligand-receptor binding (10). Arg709Gln mutation at the cleavage site of the IGF-IR precursor affects the processing of proIGF-IR to mature IGF-IR (11). Glu1050Lys substitution in the intracellular kinase domain causes a significant reduction of Igf1r phosphorylation and downstream signaling (12). Arg59stop codon causes a truncated IGF-IR (13). All these mutations in the IGF-IR were associated with growth retardation to varying degrees in humans. There are some studies using animal models of IGF-IR mutations: a null mutation of the Igf1r gene in mice causes severe prenatal growth retardation and death at birth from respiratory failure (14). Heterozygous Igf1r knockout mice do not die at birth, but they show dwarfism and metabolic disorders, and, compared with wild-type animals, they have increased longevity (15). Thus, the IGF-I/IGF-IR signaling pathway plays important roles in embryogenesis and postnatal growth in both humans and mice.
In this study, we demonstrate the clinical and functional analysis of a substitution of an arginine for glutamine at amino acid 481 in the Igf1r gene, which was found in two members of the affected family, together with the molecular biological impact of the mutation. For this purpose, we established cells that overexpress either wild-type or mutant IGF-IR to evaluate the cellular function of these mutant receptors. Here we show that NIH-3T3 cells that overexpress the mutant IGF-IR exhibit an alteration in receptor phosphorylation and consequently phosphorylation of downstream signaling molecules such as MAPK and Akt as well as alterations in cellular proliferation in response to IGF-I.
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Materials and Methods |
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Genomic DNA was extracted from peripheral leukocytes by standard procedures. Total RNA was isolated from whole blood using PAXgene blood RNA kit (QIAGEN, Hilden, Germany). The sequence of the IGF-IR cDNA was amplified in two fragments (
2 kb each) by RT-PCR using total RNA from whole blood as template. The amplified products were purified and directly sequenced using an automated DNA sequencer (model 310; Applied Biosystems, Foster City, CA). Exon 7 of the Igf1r gene was individually amplified by PCR using genomic DNA as template. Sequences of oligonucleotides used for amplification and sequencing were deduced from the published Human Genome sequence (http://genome.ucsc.edu). GenBank IGF-IR cDNA entry with accession number NM_000875 was used as a reference sequence for analysis of mutations and numbering of nucleotides. Codon numbering is given for mature IGF-IR protein. All clinical studies were approved by the review board at the Endocrinological Research Center (Moscow, Russian Federation), and written informed consent was obtained from the parents.
Materials
The antibodies used for immunoblotting including anti-IGF-IR ß-subunit (C-20 and N-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p42/44 MAPK, anti-p42/44 MAPK, anti-phospho-Akt, and anti-Akt antibodies were purchased from Cell Signaling Technology (Danvers, MA). Antiphosphotyrosine antibody (4G10) was from Upstate (Lake Placid, NY). The anti-IGF-IR monoclonal antibody (Ab-1) used for immunoprecipitation was from Calbiochem (Darmstadt, Germany). 125I-IGF-I was purchased from Amersham Bioscience (Pittsburgh, PA). Recombinant human IGF-I was a gift from Genentech Inc. (San Francisco, CA).
Construction of plasmids and cell culture
Construction of the plasmid that encodes the wild-type human IGF-IR was described elsewhere (16). To create the mutant construct, the 5' IGF-IR cDNA amplicon obtained from the patient’s blood by RT-PCR was digested with NarI and BglII restriction enzymes, and the resulting 389-bp fragment was subcloned into the corresponding part of the wild-type full-length IGF-IR cDNA construct. R481Q mutation eliminated a unique KpnI restriction site, which was used for selection of mutated clones. Integrity of the subcloned PCR fragment was confirmed by sequencing.
NIH3T3 fibroblasts were transfected with either wild-type or mutant IGF-IR plasmids, and cells stably overexpressing these exogenous mutant human receptors were cultured under the selection by Geneticin (Invitrogen, Carlsbad, CA). Parental cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 95% air-5% CO2 at 37 C. All transfected cells were cultured under the identical conditions in the presence of 0.25 g/ml Geneticin.
Immunoprecipitation and Western blot
Cells were seeded in 60-mm culture dish and cultured to 100% confluence. These cells were starved overnight and stimulated with IGF-I at various concentrations or time intervals as indicated in Results and the legends to the figures. Cells were then collected and subjected to immunoprecipitation and Western blot as previously described (17).
IGF-I binding assay
Cells were seeded and cultured in 12-well culture plates to 100% confluency. The cells were then washed once with cold PBS and incubated with 125I-labeled IGF-I with various concentrations of unlabeled IGF-I for 5 h at 4 C. Cells were washed and lysed in 0.2 N sodium hydroxide for 1 h at 37 C. Radioactivity of the cell lysates was measured using a 
-scintillation counter.
Proliferation assay
Cells were seeded in 60-mm dishes at the concentration of 15 x 104 cells and cultured in DMEM supplemented with 1% fetal calf serum and 0.1% BSA with or without IGF-I. Culture medium was changed every day. The cells were detached using Trypsin-EDTA, and the number of cells was counted with a hematocytometer on each day.
Statistical analysis
Comparison of cell proliferation was performed by two-way ANOVA using StatView 4.5 software (SAS, Cary, NC).
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Results |
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The patient was a 13.6-yr-old girl who had severe short stature. She was born after 41 wk gestation as the first child of a nonconsanguineous marriage. Her birth length was 41 cm [–4.9 SD score (SDS)], and birth weight was 2100 g (–3.1 SDS). Her early psychomotor development was normal and there were no remarkable findings in her medical history. The height of her father was 160 cm (–2.2 SDS) and her mother 128 cm (–5.7 SDS). There were other family members with short stature on the maternal side, with final height SDS as low as –6.1. The patient’s younger brother was born at term with a birth length of 50 cm and birth weight of 3000 g and was reported to have a height of 86 cm (–1.2 SDS) at 2.5 yr old. The aunt was 45 yr old and her height was 128 cm (–5.7 SDS). She had no obvious medical problems. She apparently had normal pubertal development with menarche at 13 yr of age. Several maternal siblings died in the neonatal period due to unknown causes. The family belongs to a small ethnic group living in Dagestan, a state in the Russian Federation in the Eastern Caucasus. Consanguinity is common for this population; however, there was no proven consanguinity in this family (Fig. 1
). Her school performance was above average.
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height SDS = 0). Blood chemistry analysis in this patient showed normal basal levels of cortisol, TSH, free T4, prolactin, LH, FSH, and estradiol, for her age and pubertal stage. The basal GH level measured on one occasion was 7.4 ng/ml. During a clonidine-stimulation test, GH rose from a basal level of 0.4 to 10.6 ng/ml. Her basal IGF-I level was elevated at 404.3 ng/ml (165–300 ng/ml), and 4 d of GH treatment (0.033 mg/kg·d) produced no further rise (354.2 ng/ml). Basal IGFBP-3 level was 73.1 mg/liter (3.5–35 mg/liter) and it rose slightly after GH treatment (93.4 mg/liter).
Genetic analysis
RT-PCR and subsequent direct sequencing of IGF-IR cDNA fragments revealed a heterozygous G to A substitution at position 1577 that resulted in an arginine (CGG) to glutamine (CAG) codon substitution of residue 481 (R481Q) in both the patient and her aunt. The existence of this mutation both in the patient and her aunt was further confirmed by PCR and sequencing of exon 7 using genomic DNA as template. Arginine at position 481 phylogenetically is highly conserved. This residue corresponds to the N-terminal fibronectin type III domain of IGF-IR and is located in the proximity of the first disulfide bond at cysteine 514 between the two -subunits of the protein.
Overexpression of the mutant IGF-IR in NIH-3T3 cells
To study the effect of the point mutation in the IGF-IR on IGF-I-stimulated signaling pathways, we constructed NIH-3T3 fibroblasts that overexpress either the wild-type or mutant human IGF-IR. The plasmid that contains mutant IGF-IR cDNA was constructed by site-directed mutagenesis from wild-type IGF-IR cDNA as a template. These plasmids were transfected into NIH-3T3 fibroblasts to obtain cells that stably overexpress the receptors. Western blot analyses of whole-cell lysates from these fibroblasts revealed similar expression levels of wild-type and mutant receptors. Flow cytometry analysis demonstrated that the cell surface expression of wild-type and mutant IGF-IR are similar and greater than parental NIH-3T3 cells (data not shown).
Status of dimeric formation in the mutant IGF-IR
Given that the mutation localizes close to the first disulfide bond, whole-cell lysates of NIH-3T3 fibroblasts overexpressing either wild-type or mutant receptor were subjected to Western blot analysis under reducing and nonreducing conditions to evaluate the effect of the mutation on the disulfide bonds between the two ß-subunits. Under reducing conditions, two bands of 90 kDa (monomeric form of the ß-subunit) and 220 kDa (precursor receptor) were seen in both wild-type and mutant receptor-expressing cells (Fig. 2, black and white arrowheads). On the contrary, under nonreducing conditions, a band of 90 kDa, which most likely represents a ß-subunit monomeric form of the IGF-IR (Fig. 2, black arrowhead), appeared in the cells overexpressing the mutant receptors but not in cells expressing the wild-type IGF-IR. The precursor receptors whose molecular size was 220 kDa also increased in mutant cells (Fig. 2, white arrowhead). These results were reproducible using antibodies (Ab) both against the IGF-IR carboxyl terminus (Fig. 2) and the amino terminus (data not shown) in three different mutant cell clones. This result suggests that the mutant receptors have an impairment in the adjacent disulfide bond.
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To evaluate the effect of the mutation on ligand binding, IGF-I binding capacity was measured using 125I-labeled IGF-I. NIH-3T3 fibroblasts that overexpress either wild-type or mutant IGF-IR as well as parental NIH-3T3 cells were incubated with 125I-labeled IGF-I and various concentrations of unlabeled IGF-I for 5 h and then lysed in sodium hydroxide. Radioactivity of these lysates was measured by a -scintillation counter. As shown in Fig. 3, ligand binding was similar in both fibroblasts that overexpress wild-type or mutant receptors. These results indicate that the mutation in the IGF-IR does not affect its ligand binding capacity.
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Tyrosine phosphorylation levels of the IGF-IR ß-subunit after stimulation with IGF-I at various concentrations revealed that the fold increase of tyrosine autophosphorylation above basal is lower in cells that express mutant receptors, compared with cells expressing the wild-type receptor (Fig. 4A, top and bottom). Consequently, downstream signals of the IGF-IR were also affected in cells expressing the mutant receptor. The fold increase of the levels of phosphorylation of p42/44 MAPK were decreased in cells expressing the mutant receptor, compared with cells expressing the wild-type receptor (Fig. 4B, top and bottom). Similarly, the fold increase of Akt phosphorylation was significantly reduced in cells expressing the mutant receptor, compared with cells overexpressing the wild-type IGF-IR (Fig. 4C, top and bottom). A time-course study was also performed using 10 nM IGF-I. This result demonstrates that the fold increase of both phosphorylation of IGF-IR ß-subunit and p42/44 MAPK in parental and mutant cell was less than that in cells expressing wild-type receptor (data not shown). These experiments were repeated more than three times, and the results were reproducible using three different mutant cell clones for all experiments (data not shown). These results suggest that the IGF-IR downstream signaling pathways are disturbed due to the mutation in the extracellular domain of the IGF-IR.
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To study the effect of the mutation of the IGF-IR on cell proliferation in response to IGF-I, we evaluated the proliferation rate of NIH-3T3 fibroblasts that express either wild-type or mutant receptors as well as parental cells under the same culture conditions using 1% fetal bovine serum containing medium in response to 10 nM IGF-I. In the absence of IGF-I, the proliferation of cells that overexpress wild-type IGF-IR increased, compared with parental cells cultured in 1% fetal bovine serum. In contrast, cells expressing the mutant receptor exhibited a reduction in the proliferation rate. In the presence of 10 nM IGF-I, the proliferation rate increased significantly in cells overexpressing the wild-type receptor, compared with parental cells. On the other hand, cells that overexpress the mutant receptor responded poorly to the addition of IGF-I. Figure 5 shows the result on d 4 with statistic analysis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IGF-IR consists of two -subunits and two ß-subunits and forms a dimer (20). Given that these two -subunits are bound by four disulfide bonds and the first disulfide bond (Cys514) is located close to the mutation site (21), we hypothesize that the first disulfide bond may be disturbed and that there is incomplete dimerization, which results in the conformational change. In cells that overexpress the mutant IGF-IR, there are different forms of the IGF-IR seen under nonreducing conditions on Western blotting such as the monomeric form of the - and/or ß-subunit. Thus, we suggest that the point mutation detected in the IGF-IR from the patients may affect the first disulfide bridge between the two -subunits, and some of the mutant receptor become structurally unstable. According to the results of the ligand binding assay, this mutation does not affect the ligand binding capacity of the mutant IGF-IR in vitro. The ligand binding face is composed of three domains, the large domain L1, the cysteine-rich domain, and L2 (22, 23). The structural disorder caused by the postulated disturbance of the first disulfide bridge in the mutant receptor does not seem to have an affect on this ligand binding face.
The reason that accounts for the discrepancy between the normal ligand binding capacity and reduced downstream signaling is as yet undefined. Signal transduction is considered to be the result of conformational changes in the receptor after binding of IGF-I to the receptor and subsequent activation of the tyrosine kinase in the ß-subunit. After this, tyrosine residues in the other ß-subunit within the heterodimer are phosphorylated. Because the first disulfide bridge is presumably impaired in the mutant receptor, this receptor may still form a heterodimer that does not interfere with ligand binding but may not be able to modify its conformation to activate the tyrosine kinase, i.e. impaired coordination of a conformational change after ligand binding (24).
One previous report described a substitution of glutamic acid for lysine at position 460 in the IR in a patient who demonstrated insulin resistance. This mutant IR showed increased stability of insulin binding at acidic conditions, disturbed dissociation of the insulin-IR complex after internalization, and impaired IR recycling and accelerated IR degradation (25). However, in the current study, we examined internalization of the mutant IGF-IR in response to IGF-I stimulation using flow cytometry analysis, and no difference was revealed between wild-type and mutant receptors (data not shown).
This mutation in the IGF-IR apparently also affected integrin-mediated cell morphology. The fibroblasts that overexpress mutant IGF-IR revealed morphological changes: their shape was flattened and less polarized as observed by phase-contrast microscope. Cell spreading was also altered in mutant cells. The mutant fibroblasts were detached more rapidly from extracellular matrix, compared with fibroblasts that overexpress the wild-type IGF-IR and parental cells as measured by cell detachment assays (data not shown). Given that IGF-I signaling is closely related to integrin signaling, it is possible that these integrin-mediated cytoskeletal reorganization was also affected by the reduction of the IGF-I signal, and these morphological changes may affect cell proliferation (26, 27, 28).
Thus, we have demonstrated clinical and functional analyses of the R481Q mutation in the IGF-IR that was observed in two relatives in one family. The postulated lack of conformational changes after ligand binding that leads to the impairment of tyrosine autophosphorylation in the IGF-IR has not been described in previous reports and still needs to be elucidated. Nevertheless, these results may account for the intrauterine and postnatal growth retardation seen in two affected family members.
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Footnotes |
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First Published Online January 30, 2007
1 K.I. and A.T. contributed equally to this study. 
Abbreviations: Ab, Antibodies; IGF-IR, IGF-I receptor; IR, insulin receptor; L, leucine-rich repeat; SDS, SD score.
Received October 26, 2006.
Accepted January 22, 2007.
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References |
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IR-3). J Biol Chem 268:2655–2661Vß3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs. Mol Endocrinol 19:1–11[Medline]Vß3 affinity by increasing the amount of integrin-associated protein that is associated with non-raft domains of the cellular membrane. J Biol Chem 277:1800–1805This article has been cited by other articles:
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 H A van Duyvenvoorde, M J E Kempers, T. B Twickler, J van Doorn, W J Gerver, C Noordam, M Losekoot, M Karperien, J M Wit, and A R M M Hermus Homozygous and heterozygous expression of a novel mutation of the acid-labile subunit Eur. J. Endocrinol., August 1, 2008; 159(2): 113 - 120. [Abstract] [Full Text] [PDF]
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M. J. E. Walenkamp, S. M. P. F. de Muinck Keizer-Schrama, M. de Mos, M. E. Kalf, H. A. van Duyvenvoorde, A. M. Boot, S. G. Kant, S. J. White, M. Losekoot, J. T. Den Dunnen, et al. Successful Long-Term Growth Hormone Therapy in a Girl with Haploinsufficiency of the Insulin-Like Growth Factor-I Receptor due to a Terminal 15q26.2->qter Deletion Detected by Multiplex Ligation Probe Amplification J. Clin. Endocrinol. Metab., June 1, 2008; 93(6): 2421 - 2425. [Abstract] [Full Text] [PDF]
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