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Department of Molecular Cell Biology (J.A.M., G.C.M.V.D.Z.), Leiden University Medical Centre, 2333 AL Leiden, the Netherlands; Department of Endocrinology (J.A.M.), Vrije Universiteit Amsterdam and Department of Pediatrics (E.D.), Vrije Universiteit-Medisch Centrum, 1007 MB Amsterdam, the Netherlands; Duncan Guthrie Institute of Medical Genetics (E.S.T.), Yorkhill NHS Trust, G61 1BD Glasgow, United Kingdom; Institute of Child Health (H.K., T.T., M.Y.-A.), University of Istanbul, 34390 Istanbul, Turkey; Department of Clinical Genetics (W.J.K.), Erasmus Medical Centre, 3015 GE Rotterdam, the Netherlands; and Hopital Erasme (F.F.), Universite Libre de Bruxelles, B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Dr. J. A. Maassen, Department of Molecular Cell Biology, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. E-mail: j.a.maassen{at}lumc.nl.
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Abstract |
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Introduction |
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2ß2-subunit structure and is encoded by a single gene on chromosome 19 (2, 3). Processing of the primary -ß gene product yields the mature insulin receptor. Next to insulin receptors, most cells also express IGF-I receptors with similar structure and function. How these receptors precisely function is not known because the three-dimensional (3D) structures of these receptors have been only partially resolved. The cytosolic portion of the insulin receptor contains the tyrosine kinase domain, and its 3D structure has been elucidated by x-ray diffraction both in the unphosphorylated and Tyr-phosphorylated state (4, 5). In addition, the 3D structure of a part of the extracellular region of the homologous IGF-I receptor has been determined (6). Homozygous or compound-heterozygous mutations in the insulin receptor gene are found in patients with syndromes of severe insulin resistance (7). Leprechaunism (OMIM 246200) is the most extreme form of the various insulin resistance syndromes related to mutations in the insulin receptor. The clinical presentation of these patients, including a lack of sc fat, decreased muscle mass, and the inability to properly regulate blood glucose levels, are consistent with a complete loss of cellular insulin action. No precise genotype-phenotype correlation has yet been established in patients with severe insulin resistance. This is partly attributable to the paucity of studies in which in vitro investigations of the patient’s insulin receptors have been undertaken. It has recently been reported, however, that the degree of impairment of insulin binding by the cells of patients with severe insulin resistance is inversely correlated with the duration of the patients’ survival (8). Moreover, in patients whose insulin receptor mutations do not lead to a complete loss of insulin receptor function, milder syndromes of insulin resistance are reported, such as the Rabson-Mendenhall syndrome (OMIM 262190) and type A insulin resistance (OMIM 147670) (7, 8, 9). The less severe phenotype of these patients is believed to result from the retention of some functionality by these mutant insulin receptors.
Biochemical analyses of the various mutations seen in patients with insulin-resistant syndromes provides insight into the residues of the insulin receptor that are critical for correct functioning and processing of the receptor. Furthermore, by studying multiple patients with the same mutation, insight can be obtained into what extent the genetic background is an important modulator of phenotypic expression of insulin receptor gene mutations.
The current study describes an analysis of the insulin receptor gene in five patients with insulin resistance syndromes. Both previously described and novel mutations were identified, including both missense and nonsense changes. In the case of the novel missense mutations, their effects on insulin receptor function were examined by expressing the mutant insulin receptors in Chinese hamster ovary (CHO) cells, which have a low background of endogenous insulin receptors. This approach confirms that the observed mutations indeed impair the functionality of the insulin receptor and are thus responsible for the insulin resistant phenotype.
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Materials and Methods |
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For Western blot analysis of total cell lysate, approximately 1 million cells were lysed in 300 µl sodium dodecyl sulfate gel sample buffer. Protein concentration was determined using the Bradford reagent. Experiments on transfected cell lines were performed at least in duplicate and repeated on an independent clone, again in duplicate.
For insulin receptor substrate (IRS-1) phosphorylation, confluent cultures of CHO cells were stimulated with insulin at indicated concentrations for 10 min at 37 C. Approximately 5 million cells were lysed in 1 ml ice-cold radioimmunoprecipitation assay buffer (30 mM Tris-HCl, pH 7.50, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, 1 mM Na-orthovanadate, 10 mM NaF, 1 mM phenylmethyl sulfonylfluoride). Nuclei were removed by centrifugation and 200 µl of lysate were subjected to immune precipitation by antibodies against IRS-1 as indicated by the supplier. Immune complexes were isolated using protein A Sepharose 6MB (Amersham Pharmacia, Roosendaal, The Netherlands) and analyzed by Western blot.
Western blot analysis
Proteins were resolved by 7% PAGE using the Laemmli protocol and transferred to polyvinylidene difluoride membrane (Immobilon, Millipore, Bedford, MA). The membrane was used as indicated by the supplier. The membrane was incubated with antibody, washed, and bands visualized using appropriate HRP-conjugated secondary antibodies, obtained from Promega (Madison, WI). Detection was by enhanced chemoluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
Insulin binding to cultured fibroblasts was carried out as described previously (10, 11). In brief, skin fibroblasts from all the patients were examined for the presence of high-affinity insulin-binding sites at the cell surface by performing insulin binding with 30 pM radiolabeled insulin (A14-monoiodinated insulin, Amersham Pharmacia) in the absence and presence of excess (1 µM) unlabeled insulin. For comparison, representative control fibroblasts and fibroblasts from leprechaunism patient G. were included. The latter patient is homozygous for a Leu233Pro mutation that fully aborts receptor processing, as described previously (11). Insulin binding was carried out at 20 C and 4 C to determine whether changes in the internalization rates of the insulin receptor could be involved in the generation of a reduced number of binding sites. Binding data obtained at both temperatures were, however, comparable.
Statistical analysis
Data were analyzed with an independent-samples t test using SPSS 10.0 (SPSS Inc., Chicago, IL). Curves represent fits to data by nonlinear regression analysis using GraphPad Prism 2.01 (GraphPad Software, Inc., San Diego, CA).
The numbering of insulin receptor mutations is according to Ebina et al. (2).
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Results |
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The parents from this patient originate from Morocco (M). She presented at the age of 17 yr with hirsutism, primary amenorrhea, acanthosis nigricans, and manifest diabetes mellitus (hemoglobin A1c 13.3%). Correction of her hyperglycemia by insulin administration proved extremely difficult, suggesting the presence of severe insulin resistance. Fibroblasts were cultured and insulin-binding assays showed an approximately 90% reduction in high-affinity insulin-binding sites (Fig. 1
). DNA analysis showed the patient to be homozygous for a CGC-to-CAC mutation leading to an Arg252His substitution. Expression of this mutant insulin receptor in CHO cells showed that the mutation leads to the predominant formation of -ß-proreceptor (Fig. 2A).
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This patient was born to Turkish parents and showed typical dysmorphic characteristics of leprechaunism with acanthosis nigricans. An insulin determination showed a value of 1800 pmol/liter. The patient died at the age of 9 months. A fibroblast culture was grown for determination of insulin receptor function and DNA extraction. High-affinity insulin-binding sites on fibroblasts were markedly reduced (by 
70%). The patient was homozygous for a Ser323Leu mutation (TCG-TTG), previously identified in two patients with Rabson-Mendenhall syndrome.
Leprechaunism patient L-K
This patient was born to parents originating from the Kurdistan (K) region. Clinical signs of leprechaunism were present, such as hirsutism, acanthosis nigricans, and extreme hyperinsulinism (19,500 pmol/liter), and the patient died at the age of 9 months. The patient’s maternal insulin receptor allele contained an Arg1092Trp mutation (CGG-TGG) and the other insulin receptor-allele harbored a nonsense mutation at Arg897 (CGA-TGA). This mutation apparently resulted from a novel germ line mutation because the father did not carry this mutation in his leukocyte DNA. Paternity testing confirmed the biological relationship.
Leprechaunism patient L-S
This patient was born to Caucasian parents in Scotland (S). He died at the age of 3 months. The patient showed the clinical characteristics of leprechaunism, including marked hirsutism and elevated serum insulin (1446 pmol/liter). Fibroblasts were cultured and analyzed for the presence of high-affinity insulin-binding sites. Only a partial reduction (45%) in the number of high-affinity binding sites was present (Fig. 1
). The patient was found to be homozygous for an Asn431Asp mutation (AAC-GAC), and both his father and mother were carriers for this mutation.
The Asn431Asp mutant receptor was expressed in CHO cells. Insulin binding was increased when the mutant receptor was expressed but not to the level seen with expression of wild-type insulin receptors in these cells (data not shown). Furthermore, Western blot analysis using an antibody recognizing an epitope on the ß-subunit, showed an elevated level of -ß proreceptors, in addition to processed ß-chains when compared with CHO cells with similar expression levels of wild-type insulin receptors (Fig. 2C). In parental CHO cells, expression levels of endogenous insulin receptor are very low and not detected during the exposure time as applied (Fig. 2C). We also determined the ability of this receptor mutant to undergo insulin-induced Tyr-phosphorylation of the ß-chain at various insulin concentrations using anti-phosphotyrosine antibodies. The Asn431Asp mutant receptor ß-chain did undergo an insulin dose-dependent Tyr-phosphorylation (Fig. 3). The maximal level of ß-chain Tyr-phosphorylation was reduced to about 35%, compared with cells with similar expression levels of wild-type insulin receptors as judged from the quantitated data in Figs. 4A and 5. No Tyr-phosphorylation of endogenous insulin receptors in parental CHO cells was detected under the conditions applied. Only overexposure of the Western blot did result in a vague signal (Fig. 3A).
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, B and C, shows the concentration-dependent stimulation of IRS-1 Tyr-phosphorylation and ERK activation, respectively, by insulin. Parental CHO cells, cells overexpressing wild-type and Asn431Asp mutant insulin receptors, were simultaneously included in these experiments. Figure 4, B and C, shows the quantitated results. The mutant insulin receptor is able to stimulate Tyr-phosphorylation of IRS-1 to nearly the same level as seen with cells expression wild-type insulin receptors. Stimulation is already detectable at 0.1 nM insulin. It is also seen that at the highest insulin concentration (100 nM), insulin stimulates Tyr-phosphorylation of IRS-1 in the parental CHO cells. When ERK phosphorylation is considered (Fig. 4C), it is seen that the Asn431Asp mutant is also able to activate this pathway though much less active, compared with wild-type insulin receptors. No significant increase in ERK activation by insulin was detected when using the parental CHO cell line. Its should be noted that this cell line has a high basal level of ERK phosphorylation. The migration pattern of the Tyr-phosphorylated ß-subunit of the insulin receptor on the Western blot in the case of the mutant Asn431Asp receptor was also slightly different from the pattern seen with wild-type insulin receptors, suggesting differences in posttranslational modification (data not shown). Leprechaunism patient L-E
This patient, born to Dutch Caucasian parents near the city of Ede (E), died at the age of 3 months, with clinical characteristics of leprechaunism, including hirsutism. The patient developed severe cardiomyopathy, and laboratory values showed increased circulating insulin levels (above 6000 pmol/liter). Insulin-binding studies on cultured fibroblasts showed reduced insulin binding (Fig. 1). DNA sequence analysis showed the patient to be a compound heterozygote. The paternal allele showed a novel missense mutation resulting in a Leu93Gln mutation (CTG-CAG), but the maternal allele revealed a nonsense mutation changing Tyr1122 into a stop codon (TAC-TAA). The Leu93Gln mutation was introduced into the insulin receptor cDNA, and the mutant insulin receptor was expressed in CHO cells for analysis of receptor function. Figure 2B shows that the mutant insulin receptor appears as proreceptor without detectable cleavage into - and ß-subunits.
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Discussion |
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. For comparison, leprechaunism patient from the city of Geldermalsen (L-G) is included. This patient is homozygous for a Leu233Pro mutation, which completely blocks processing and transport of insulin receptors to the cell surface (10, 11).
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The Arg252His mutation in this type A resistant patient has been described previously in a Japanese patient with type A syndrome (12). Both our studies using transfected cell lines and the studies by the Japanese group show that this mutation interferes with receptor processing (Fig. 2A) and surface expression (data not shown) Also, an Arg252Cys mutation has been identified in a type A patient with similar consequences for insulin receptor processing (13). The consequence of the Arg252His mutation on receptor processing as seen in transfected cell lines is also reflected by the very low insulin binding to primary fibroblasts from the patient type A-M. This reduction in insulin binding values is as severe as that observed in leprechaunism patient G, who had a similar processing and transport defect because of a Leu233Pro mutation (10, 11). It appears, however, that patients with mutations at position 252 do not develop leprechaunism. It is unclear why a similar reduction in insulin binding results in the type A syndrome in the case of mutations at codon 252 and in leprechaunism in the case of other mutations. One explanation is provided by the observation that expression of the Arg252His mutant in cells does allow some remaining insulin-induced signaling (either via the mutant insulin receptor or endogenous IGF-I receptors) as reflected by an increase in ERK phosphorylation in response to 50 nM insulin in fibroblasts from patient type A-M. In contrast, expression of leprechaun-related mutant insulin receptors (such as Leu233Pro) reduced insulin-induced signaling by a dominant effect of the mutant insulin receptor on signaling via endogenous IGF-I receptors (data not shown).
Patient 99RD219
This patient with leprechaunism has a homozygous mutation in the insulin receptor at position 323, changing the small, hydrophilic side chain of Ser into the bulky, hydrophobic side chain of Leu. This mutation has been previously described in two patients with Rabson-Mendenhall syndrome (14, 15). This syndrome resembles leprechaunism but is less severe in its clinical presentation. The mutation does not interfere with expression of insulin receptors on the cell surface. However, these receptors have a low binding affinity for insulin. Monoclonal antibodies specific for the extracellular domain of the insulin receptor were, nevertheless, able to activate this mutant receptor (14). Therefore, it is feasible that if a patient with this receptor mutant does have autoantibodies against insulin receptors, the mutant receptors may undergo a constitutive, low-level activation, preventing the patient from developing leprechaunism and, instead, predisposing to Rabson-Mendenhall syndrome. In the absence of such antibodies, these patients would be predicted to develop leprechaunism.
Patient L-K
This patient with leprechaunism is a compound heterozygote with a stop codon at position 897, leading to a nonfunctional truncated insulin receptor that lacks the transmembrane domain. In the other allele, an Arg1092Trp mutation is present. Mutations at codon 1092 have previously been reported to result in a loss of tyrosine kinase activity of the receptor without affecting insulin binding (16). Consistent with this is our observation that the fibroblasts showed significant insulin binding (30% of control).
Patient L-S
This patient has a novel Asn431Asp mutation. In the partially resolved 3D structure of the homologous IGF-I receptor, this residue is conserved and is part of an Asn-ladder that stabilizes a staircase structure in the L2 lobe of the -subunit of the receptor (6). It is likely that changing Asn into Asp leads to destabilization of this structure. In line with this is the observation that the processing of this mutant receptor is less efficient. Fibroblasts from the patient still show substantial binding (60%), indicating that this mutation does not disrupt the structure of the insulin-binding site. Remarkably the ability of insulin binding to induce ß-chain autophosphorylation is reduced to 35–40%, indicating also an impaired transmission of the insulin-binding signal to activation of the Tyr-kinase. Furthermore, the Tyr-phosphorylated ß-subunit of this mutant receptor showed slight abnormalities in its mobility on Western blots, suggesting some alterations in its posttranslational modification, the nature of it being under investigation.
Thus, we are dealing in this case with a mutation that partially inhibits two properties of the insulin receptor, i.e. processing and autophosphorylation. Upon expression of the Asn431Asp mutant receptor into CHO cells, it was found in multiple clonal cell lines that the receptor is able to induce substantial levels of Tyr-phosphorylation of IRS-1 (Fig. 3B) and concomitant phosphatidylinositol-3-kinase association (data not shown). When ERK phosphorylation is considered, the mutant receptor shows a strongly impaired activation of this signaling intermediate, the maximal response being about 20% of the response seen with wild-type insulin receptors, the ED50 shifting from about 0.5–5 nM insulin. It remains, however, mysterious that in the case of mutations at position 252, in which a much more pronounced defect in insulin receptor function and processing is induced, the phenotype of the patient is less severe, compared with the situation observed for the Asn431Asp mutant. Currently we are analyzing time- and concentration-dependent activation of multiple signaling intermediates in relation to insulin-induced mitogenicity and stimulation of glycogen synthesis.
Patient L-E
This patient is a compound heterozygote with a premature stop codon in one allele, leading to a truncated insulin receptor lacking the tyrosine kinase domain. The other allele contains a novel missense mutation changing the nonpolar side chain of Leu-93 into the polar side chain of Gln.
The 3D structure of the L1 lobe, within the N-terminal region of the IGF-I receptor, possesses six parallel ß-strands (6). On the inner surface, Leu-87 of the IGF-I receptor, corresponding to Leu-91 of the insulin receptor, is in close proximity to the hydrophobic side chains of Leu-55 and other hydrophobic residues. It is likely that this hydrophobic interaction stabilizes the structure of the L1 lobe. The substitution of Leu by Gln disrupts this stabilizing interaction. The consequent change in conformation will affect correct disulfide bond formation during maturation, resulting, in transfected CHO cells, in the appearance of predominantly unprocessed -ß-proreceptors which, in general, do not reach the cell surface.
In conclusion, we observe in these five patients that the number of high-affinity insulin-binding sites is reduced on their fibroblasts but that the magnitude of the reduction does not correspond to the severity of the clinical phenotype (Table 1). In another group of patients with insulin resistance, a relation between binding defect and clinical appearance was seen (8). In particular, the type A patient from Morocco has almost no specific insulin binding, like the reference leprechaunism patient G. In contrast, the fibroblasts of the Scottish patient with leprechaunism L-S have binding values of about 60% of controls. Furthermore, the magnitude of the biochemical defect when the mutant receptor is expressed in CHO cells does not correlate to the severity of the clinical phenotype. This is particularly striking for the Asn431Asp mutant. In addition, the phenotypic expression of a mutation can depend on the genetic or environmental background, as illustrated for the Ser323Leu mutation. Together, these observations illustrate that a detailed prediction of the clinical phenotype on the basis of biochemical data is currently not possible. Additional studies are in progress to explore in more detail the unexpected signaling behavior of the Asn431Asp mutant.
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Acknowledgments |
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Footnotes |
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Abbreviations: CHO, Chinese hamster ovary; 3D, three-dimensional; IRS-1, insulin receptor substrate.
Received January 6, 2003.
Accepted May 16, 2003.
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-subunit of the human insulin receptor: trial of insulin-like growth factor I injection therapy to enhance insulin sensitivity. J Clin Endocrinol Metab 80:3662–3667[Abstract]
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