Deletion of V335 from the L2 Domain of the Insulin Receptor Results in a Conformationally Abnormal Receptor That Is Unable to Bind Insulin and Causes
Abstractz[+@?4, 百拇医药
An infant with Donohue’s syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the V335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the V335 receptor was capable of being immunoprecipitated with antibodies directed against the ß-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the {alpha} -subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor {alpha} -subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two ß-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.
Introductionf)cq, 百拇医药
MUTATIONS IN the insulin receptor (IR) gene lead to a variety of recognizable clinical syndromes, with the clinical phenotype broadly reflecting the severity of the receptor dysfunction (1). Thus, homozygous or compound heterozygous null mutations lead to the most severe phenotype, Donohue’s syndrome (sometimes called leprechaunism), which is usually fatal in the first year or two of life. Rabson-Mendenhall syndrome describes a somewhat less severe phenotype with frequent survival into the second decade. In these cases, homozygous missense mutations have often been reported, with these mutations severely impairing, but not totally disrupting, IR structure and function. Finally, type A insulin resistance, often presenting around puberty and compatible with survival to middle age at least, is sometimes the result of IR mutations. Where type A syndrome can be due to an IR mutation, it is often a heterozygous missense change in the intracellular tyrosine kinase domain, which inhibits the function of coexpressed wild-type receptor through a dominant negative mechanism.
Such naturally occurring mutants, found in subjects with severe insulin resistance, can aid attempts to fully understand the structure/function relationships of the IR, as, unlike systematic site-directed mutagenesis, there is a high a priori likelihood that the mutation found is significantly impairing the real biological functions of the receptor. We have detected a novel homozygous mutation, deleting valine 335 in the L2 domain of the extracellular portion of the IR in an infant with Donohue’s syndrome. Functional studies of the mutant receptor indicated that whereas it was capable of being processed and transported to the cell surface, albeit less efficiently than normal, the receptor was unable to bind insulin or autophosphorylate due to a major alteration in the conformation of the extracellular domain. Using the recently elucidated structure of the highly homologous IGF-I receptor (2), we have generated a computer model of the effect of this mutation on the structure and function of the L2 domain.
Materials and Methodsk)ekgtx, 百拇医药
Reagentsk)ekgtx, 百拇医药
Mouse monoclonal antibodies specific for the human IR (83-7, 83-14, 18-44, CT-1); D88, an irrelevant mouse monoclonal antibody (directed against TSH); and a rabbit polyclonal antibody raised against the human IR ß-subunit (Carl 10) were described previously (3, 4, 5). Mouse monoclonal antiphosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Secondary horseradish peroxidase-conjugated antibodies (goat antimouse IgG and goat antirabbit IgG) were purchased from DAKO Corp. (Copenhagen, Denmark). Monoiodo-A14 insulin was a gift from Eli Lilly \|[amp ]\| Co. (Indianapolis, IN).k)ekgtx, 百拇医药
Site-directed mutagenesisk)ekgtx, 百拇医药
All routine DNA manipulations were performed using standard protocols (6). V335 mutant receptors were reconstructed using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. All constructs were in the pRc.CMV expression vector (Invitrogen, Leek, The Netherlands), and the mutation was confirmed by sequencing before use.
Transient transfection of Chinese hamster ovary (CHO) cellsr$, 百拇医药
CHO cells were maintained at 37 C in Nutrient F-12 Ham’s medium (Sigma-Aldrich, Poole, UK) supplemented with 10% fetal calf serum, 105 U/liter penicillin, 0.1 g/liter streptomycin, and 2 mM glutamine. Transfections were performed on cells grown in 6- or 24-well plates using the appropriate cDNA and Fugene 6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions. All experiments were performed 24 h after transfection.r$, 百拇医药
Immunoprecipitation and Western blottingr$, 百拇医药
Cells were rinsed once with ice-cold PBS and solubilized in 10 µl lysis buffer/1 cm2 confluent cells [50 mM HEPES (pH 7.4), 150 mM NaCl, 30 mM NaF, 10 mM Na4P2O7, 10 mM EDTA, 1 mM Na3VO4, 1 µg/ml antipain, pepstatin and leupeptin, 2.5 mM benzamidine, 0.5 mM phenylmethylsulfonylfluoride, and 1% Triton X-100]. Cleared cell lysates were immunoprecipitated with IR-specific monoclonal antibodies or an irrelevant monoclonal antibody by mixing the lysates for 3 h at 4 C with antibody prebound to 2.5 mg protein A-agarose (IgG2 antibodies) or protein G-agarose (IgG1 antibodies). Western blotting was performed essentially as described previously (1). Briefly, washed immunoprecipitates or total cell lysates were resolved by 6% SDS-PAGE, transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA), and probed with anti-IR antibody (1:8000 Carl 10), followed by horseradish peroxidase-conjugated secondary antibody (1:5000). Proteins were visualized using enhanced chemiluminesence (Amersham Pharmacia Biotech, Little Chalfont, UK).
Biotinylation of cell surface IRs-9w, 百拇医药
CHO cells expressing wild-type (WT) or V335 mutant IRs were grown in six-well plates. Cell surface proteins were biotinylated using the enhanced chemiluminesence protein biotinylation module (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. After cell lysis, IRs were immunoprecipitated with monoclonal 18-44 antibody and analyzed by 6% SDS-PAGE, followed by electroblotting as described above. Membranes were then probed with either horseradish peroxidase-conjugated streptavidin (1:1500) or antibody (1:8000 Carl 10) as described above for detection of biotinylated or total IRs, respectively.-9w, 百拇医药
Insulin binding-9w, 百拇医药
Cells in 24-well plates were washed twice with PBS before the addition of 250 µl [125I]insulin (50–100 pM, 30,000 dpm) in binding buffer [100 mM HEPES (pH 7.8), 120 mM NaCl, 1 mM EDTA, 15 mM sodium acetate, 1.2 mM MgSO4, 10 mM glucose, and 1% BSA] for 4 h at 4 C. Cells were then washed twice with ice-cold PBS and solubilized in 0.03% sodium dodecyl sulfate for determination of cell-bound radioactivity in a -counter (1282 Compugamma CS Universal Counter, LKB Wallac, Inc., Turku, Finland). Insulin binding to solubilized receptors was essentially carried out as described previously (3). Briefly, solubilized receptors (10 µl) were incubated with [125I]insulin (50–100 pM, 30,000 dpm) for 18 h at 4 C (250 µl final volume). Receptor-bound radioactivity was then determined by precipitation with polyethylene glycol 6000. In both cases, nonspecific binding was determined in the presence of 1 µM unlabeled insulin.
Receptor autophosphorylation]0[4+$, 百拇医药
Transfected cells were serum-starved for 6 h before stimulation with 100 nM insulin for 2 min at 37 C, followed by rinsing with ice-cold PBS and solubilization in lysis buffer. Cell lysates were then analyzed by Western blotting as described above using the mouse monoclonal antiphosphotyrosine antibody 4G10 (1:10,000) to detect tyrosine-phosphorylated receptor.]0[4+$, 百拇医药
Results]0[4+$, 百拇医药
Clinical history and molecular genetic diagnosis]0[4+$, 百拇医药
The patient was a male born to consanguineous parents of Turkish origin. He was delivered prematurely at 34 wk gestation because of intrauterine growth retardation and had a birth weight of 1.7 kg. Examination revealed the typical features of Donohue’s syndrome: elfin-like face, depressed nasal bridge, low-set large ears, hirsutism, acanthosis nigricans, muscular wasting, and lack of sc fat.]0[4+$, 百拇医药
Typical to the course of the disease, over the next few weeks he failed to thrive and had hyperglycemia with blood glucose levels up to 30 mM. He had no ketoacidosis. Initially he was treated with insulin, and at the age of 2 months had gained weight up to a maximum of 2.8 kg. His blood glucose levels were still up to 24 mM. Nonfasting serum insulin levels were enormously elevated (35,713 pmol/liter). He was then started on recombinant IGF-I treatment (0.4 mg/kg, given three times). This resulted in a much improved blood glucose profile and improved well-being. However, he suffered a febrile episode (of unknown cause), which was accompanied by hyperglycemia, and he died at the age of 3 months.
We undertook sequencing of all 22 exons of the IR in the patient, his parents and his 2-yr-old brother. This revealed a single amino acid deletion, a valine residue at position 335, which is located in the {alpha} -subunit (exon 4; Fig. 1, A and B). The patient was homozygous for this mutation, and both his parents and brother were heterozygous. Of interest, there was no family history of diabetes. The parents showed a mild degree of hyperinsulinemia, but were glucose tolerant during a glucose tolerance test (Table 1).;afj8#', 百拇医药
fig.ommitteedfig.ommitteed;afj8#', 百拇医药
Figure 1. A, Sequencing trace showing the proband’s homozygous deletion of Val335. Alignment of sequences (-f, forward; -r, reverse) from exon 4 of the IR gene adjoining the valine at position 335 in the proband and family members. Deletion of GTC (Val335) is present in all family members, and typical for deletions/insertions on one allele only (heterozygosity), the DNA sequencing becomes unreadable downstream (-f) and upstream (-r) of the deletion/insertion, whereas homozygosity is a symmetrical change, with no consequences for reading the DNA sequence. A, Adenine; T, thymine; C, cytosine; G, guanine. B, Agarose gel electrophoresis of BslI-generated restriction fragments of exon 4 of the IR gene containing V335(GTC). Deletion of the trinucleotide GTC generates a new restriction site: WT, 262 bp + 101 bp; heterozygous mutant, 262 bp +168 bp+ 94 bp; homozygous mutant, 168 bp + 101 bp + 94 bp. The 101 and 94 bp run as one band. Lanes 1 and 6, Father; lanes 2 and 7, mother; lanes 3 and 8, brother; lanes 4 and 9, proband; lanes 5 and 10, unrelated subject; lane 11, 100-bp molecular marker.
fig.ommitteedfig.ommitteed9, 百拇医药
Table 1. Clinical characteristics of the family members9, 百拇医药
Expression of WT and V335 mutant receptors in CHO cells9, 百拇医药
To assess the level of expression of the mutant receptor, equal amounts of cDNA encoding WT and mutant receptor were transiently transfected into CHO cells. Western blotting of cell lysates with anti-IR antibody (Fig. 2) showed similar amounts of WT and mutant proreceptor, but levels of mature processed receptor (as indicated by the ß-subunit) in the mutant expressing cells were only about one third of those in the WT-expressing cells, suggesting a partial impairment in proreceptor processing. The mobility of the ß-subunit was also altered, running slightly slower than WT, suggesting that its slightly greater mass might be due to altered glycosylation.9, 百拇医药
fig.ommitteedfig.ommitteed9, 百拇医药
Figure 2. Western blot analysis of expression of WT and V335 mutant IR. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. The indicated amounts of total cell lysate from a WT transfection (lanes 1–5), two different V335 transfections (lanes 6 and 7), and a mock transfection (lane 8) were resolved by 6% SDS-PAGE and analyzed for IR proreceptor (upper panel) and mature ß-subunit (lower panel) by Western blotting using a rabbit polyclonal IR antibody (1:8000; Carl 10) as described in Materials and Methods. Data are representative of three independent experiments.
The presence of the V335 IR on the cell surface was investigated by cell surface biotinylation. Consistent with the results of Western blotting, mature mutant receptor was detectable at the cell surface, but at about 30% the level of WT receptor (Fig. 3).s%, http://www.100md.com
fig.ommitteedfig.ommitteeds%, http://www.100md.com
Figure 3. Detection of biotinylated cell surface IRs. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Proteins at the cell surface were biotinylated, and IRs were immunoprecipitated from the indicated volumes of cell lysate using the IR-specific monoclonal antibody 18-44 and were analyzed by 6% SDS-PAGE, followed by electroblotting as described in Materials and Methods. Membranes were then probed with either HRP-streptavidin (1:1500) to detect biotinylated cell surface IR (left panel) or a rabbit polyclonal IR antibody (1:8000; Carl 10) to detect total IR (right panel). Data are representative of two independent experiments.s%, http://www.100md.com
Insulin binding and receptor autophosphorylations%, http://www.100md.com
Because at least some processed V335 receptors are expressed at the cell surface, the ability of mutant receptors to bind insulin was first examined in intact cells (Fig. 4A. Additionally, given that mutant IRs have been described (7) that do not bind insulin at the cell surface, but which do on solubilization, the ability of solubilized V335 receptors to bind insulin was also examined (Fig. 4B). Although, as expected, WT receptors bound insulin well, mutant receptors showed no significant binding compared with mock-transfected cells in either paradigm. Not surprisingly therefore, cells expressing the V335 receptor showed no evidence of ligand-induced receptor autophosphorylation (Fig. 5).
fig.ommitteedfig.ommitteed/, http://www.100md.com
Figure 4. Insulin binding studies. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Cells were serum-starved for 6 h before measuring [125I]insulin binding to intact cells (A) or 10 µl solubilized receptors (B), as described in Materials and Methods. Results are expressed as a percentage of WT binding. Nonspecific binding measured in the presence of 1 µM unlabeled insulin has been subtracted. Data are the mean ± SEM of three independent experiments with two separate transfections for V335 in each experiment./, http://www.100md.com
fig.ommitteedfig.ommitteed/, http://www.100md.com
Figure 5. Western blot analysis of insulin-stimulated receptor autophosphorylation. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Cells were serum-starved for 6 h and then stimulated with (+) or without (-) 100 nM insulin for 2 min at 37 C. Total cell lysates (2.5 and 10 µl from a WT transfection, 10 µl from two different V335 transfections and a mock transfection) were resolved by 6% SDS-PAGE and analyzed for total IR ß-subunit (upper panel) or tyrosine-phosphorylated ß-subunit (lower panel) by Western blotting using a rabbit polyclonal IR antibody (1:8,000; Carl 10) or an antiphosphotyrosine antibody (1:10,000 4G10), respectively, as described in Materials and Methods. Data are representative of two independent experiments.
Studies of the conformational effects of the V335 mutation using monoclonal antibodiesi{g]4y, http://www.100md.com
To test whether the V335 mutation affects the conformation of the IR, a panel of IR-specific monoclonal antibodies, recognizing different epitopes on the receptor (3, 4, 8), were used to immunoprecipitate receptors from cells transfected with either WT or V335 IR cDNA. Two antibodies (83-7 and 83-14) recognize epitopes on the -subunit (the cysteine-rich domain and the Fn0 domain, respectively) and are conformation specific, reacting poorly with denatured protein (4, 8). Two other antibodies (CT1 and 18-44) react with regions on the ß-subunit (exon 22 at the extreme intracellular C terminus and exon 12 in the extracellular portion of the ß-subunit, respectively) (4). The results from a typical experiment are shown in Fig. 6. The processed WT receptor (as shown by the ß-subunit) was immunoprecipitated to a similar extent by all four IR-specific monoclonal antibodies, but not by an irrelevant monoclonal antibody (N). Because V335 receptors are less efficiently processed than WT (Fig. 2), we used 4 times as many receptors in the immunoprecipitation. Despite this and although immunoprecipitation was not completely abolished, the mutant receptor was less efficiently precipitated by the antibodies targeted toward the -subunit (7, 14) than those toward the ß-subunit (CT and 44; Fig. 6) This suggests that the V335 mutation causes a conformational change within the {alpha} -subunit that affects the accessibility of the epitopes to which these antibodies are targeted. In contrast, the accessibility of the ß-subunit epitopes appears relatively unaffected by the mutation. This is evidenced by the fact that the ß-subunit antibodies are able to immunoprecipitate WT and mutant receptors to a similar extent.
fig.ommitteedfig.ommitteedws, 百拇医药
Figure 6. Studies of the conformational structure of the V335 IR using monoclonal antibodies. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Cell lysates (15 µl WT, 60 µl V335) were immunoprecipitated with an irrelevant monoclonal antibody (N) or IR-specific monoclonal antibodies recognizing the subunit, 83-7 (7 ), 83-14 (14 ) or ß-subunit, CT-1 (CT), and 18-44 (44). Immunoprecipitates were resolved by 6% SDS-PAGE and analyzed for insulin proreceptor (left panel) and mature ß-subunit (right panel) by Western blotting using a rabbit polyclonal IR antibody (1:8000; Carl 10) as described in Materials and Methods. Data are representative of two independent experiments.ws, 百拇医药
The more intense signal seen from the proreceptor of the mutant receptors is accounted for by the fact that, as previously stated, we used 4 times as many receptors in the immunoprecipitation to compensate for the decreased level of processing in the mutant receptor.ws, 百拇医药
Discussion
We describe a novel mutation, V355, in the human IR that in homozygous form causes the most severe phenotype human insulin resistance, Donohue’s syndrome. Our studies demonstrate the complex molecular mechanisms by which it leads to such marked receptor dysfunction.$z|7p/, 百拇医药
V335 is the first reported homozygous single residue deletion in the extracellular domain. Its importance is reflected by the severity of the clinical phenotype as well as the consequences reflected in the results of our functional studies. It is only partially processed, to a level 30% that of WT. It has a slightly higher molecular mass than WT, suggesting altered glycosylation. However, this is unlikely to be the cause of the functional differences we have uncovered. Although V335 is very near to a glycosylation site positioned at 337, detailed mutation studies by Elleman et al. (9) show that alteration of the residue at 337 (in combination with other glycosylation sites) has no effect on either processing, cell surface expression, or autophosphorylation.
Any processed V335 receptor is, however, able to reach the cell surface. Despite this, it is unable to bind insulin whether at the cell surface or when solubilized. Insulin-stimulated autophosphorylation is consequently absent. Our experiments using the monoclonal antibodies indicate that the {alpha} -subunit is conformationally altered, but that the ßsubunit appears to remain intact.7^, 百拇医药
Molecular modeling is compatible with these findings. The recently determined quaternary structure of the whole receptor together with bound insulin (10) made it possible to establish the mutual position and interaction of the individual domains and to propose a self-consistent model of IR function (11). Based on these and the known or modeled three-dimensional structure of the homologous IGF-I and IR-related protein (2, 12), we were able to derive a model of the IR, as shown in Fig. 7. Using this model we were able to deduce the putative position of valine 335 (red ribbon) within the L2 domain (blue). Together with its backbone hydrogen-bonded partner valine 311 (pink ribbon) it initiates the first five-stranded parallel ß-sheet. It is thus located just after the flexible hinge that connects the cysteine-rich and L2 domains. The strand containing V335 then crosses to the second parallel ß-sheet, providing the rigid scaffold for the two ß-sheets faces of the L2 domain. Deletion of this residue, which is therefore in such a critical position, would seriously affect the tertiary and quaternary structures of the receptor as a whole by causing the collapse of the initial corner of the ß-sheet in L2, destabilizing cysteines 302 and 323 (yellow) that form one of the disulfide bridges, altering the mutual positions of the two ß-sheets, and lengthening and increasing the flexibility of the hinge between the cysteine-rich domain and L2.
fig.ommitteedfig.ommitteed\0|pzz, 百拇医药
Figure 7. Computer-generated molecular model of the L2 domain. The L2 domain (blue) is connected by a hinge (magenta) to the cysteine-rich domain (gray). The position of the V335 residue and its hydrogen-bonded partner V322 are highlighted as red and pink ribbons, respectively. All disulfide bridges are in yellow; the backbone of the cysteines next to V335 are also colored yellow. Some of the mutations detected in insulin-resistant patients are represented as balls: from top to bottom those affecting insulin binding are red: S323L (21 ), N435S (27 ), and K433E (28 ); those affecting processing are green: F382V (24 ) and W412S (22 ). The figure was produced with Protein Data Bank coordinates of the IGF-I receptor (29 ).\0|pzz, 百拇医药
The putative insulin-binding site is mainly formed by L1, the cysteine-rich domain and the C-terminal tail of the {alpha} -chain. Alanine scanning of the NH2-terminal domain of the {alpha} -subunit (13) and the C-terminal domain (14) have revealed several key residues within these domains that are critical for hormone binding. In the N-terminal domain it appears that mutation of Arg14, Asn15, and Phe64 produced large decreases in binding affinity; the remaining residues tested produced less marked effects. Scanning of the C-terminal domain revealed that the mutation of four residues (Thr704, Phe705, Glu706, and His710) produced large decreases in affinity that were too great to be quantified, and three other amino acids (Tyr708, Leu709, and Phe714) also produced very large decreases in affinity when mutated to alanine.
More recently, studies by Kristensen et al. (15) have attempted to investigate the minimum length of the {alpha} -subunit required for insulin binding. They report that when a fragment consisting of the first 255 amino acids is mixed with the CT (C-terminal) domain in a soluble peptide form, insulin binding is restored. The importance of the C-terminal tail was further demonstrated by the work of Surinya et al. (16). By using IR fragments containing all four of the amino-terminal domains fused to different fragments coded by exon 10, they showed that the precise positioning of this domain critically determines the insulin binding affinity.e, 百拇医药
The importance of the cysteine-rich domain was shown by work carried out by Yip et al. (17). Using receptors labeled with radioactive insulin photoprobes, they isolated a 23-kDa fragment that contained the putative insulin-binding site, and mutational studies revealed this to contain the cysteine-rich domain within it.e, 百拇医药
Despite the fact that the L2 domain is not directly in the insulin binding pocket, it is no less important in helping to confer the receptor’s ability for high affinity ligand binding. L2 anchors the protruding insulin pocket in the main body of the IR and transmits the signal to the interior of the cell by pushing the kinases of each monomer together for mutual activation. Schumacher et al. (18) used IR and IGF-I receptor chimeric receptor constructs to define the sites of high affinity insulin binding. Their work revealed that one of the two major structural determinants required for such binding was indeed located in L2 between amino acids 325 and 524.
Although L2 has not been directly probed for insulin binding by alanine scanning mutagenesis, the naturally occurring mutations described to date clearly indicate its importance in this function as well as receptor processing and its transport to the cell surface. Figure 7 shows the mutations found in the L2 domain only. V335 is crucial for both functions, whereas the other mutations described to date are implicated in one or other of them. Apart from V335, all other mutations in L2 are located in the connecting loops outside the rigid secondary structure elements. These will therefore only affect local side-chain interactions, as opposed to the global structural change that the deletion of V335 has been shown to cause with such drastic clinical consequences.cu, 百拇医药
Although multiple nonsense and missense mutations in the {alpha} -subunit have been found in patients with Donohue’s and Rabson-Mendenhall syndromes, deletions of single amino acids have rarely been reported. Jospe et al. (19) described a 3-bp deletion in exon 2 leading to a deletion of the lysine residue in position 121. This was found in a female patient with Donohue’s syndrome (patient Rochester). The mutation was present in heterozygous form in this subject; the presumed second mutated allele was not found. Initially, functional studies of the 121 suggested that, unlike V335, cell surface expression and insulin binding were normal. Later investigations (20) revealed that this receptor did, in fact, have altered insulin binding, but that this was temperature sensitive. When measured at 21 C, binding was similar to that in the WT, but at 37 C, insulin binding was 50% WT binding.
There have also been several other descriptions of naturally occurring substitution mutations in the extracellular domain. The nearest of these to V335 is the substitution of serine for leucine at position 323. This was found in a patient with Rabson-Mendenhall syndrome. Functional studies by Krook et al. (21) showed that unlike V335, processing was normal. However, insulin binding was abolished, as was subsequent ligand-stimulated autophosphorylation.{e5+, 百拇医药
Another mutation described by Van de Vorm (22) resulted in a substitution of tryptophan at position 412 into serine. This mutation in the L2 domain was found in a patient with Donohue’s syndrome in a homozygous form. When studied in COS1 cells, this was found to be retained intracellularly as the 210-kDa precursor. However, it retained its insulinbinding activity in cross-linking experiments and was able to undergo autophosphorylation.{e5+, 百拇医药
Maasen et al. (23) investigated a substitution mutation at position 233 of the receptor, which was transfected into CHO cell lines. This leucine to proline mutation was found in a homozygous form in a patient with Donohue’s syndrome. Here, receptor processing was defective; the mutation prevented proreceptor cleavage. In a similar manner to {Delta} V335, the mutant receptor was also poorly precipitated by two antibodies targeted to the extracellular domain, indicating that it, too, caused a conformational change, such as to alter the epitopes for these antibodies. However, unlike {Delta} V335, this receptor though was not transported to the cell surface. When solubilized, it was also unable to bind insulin or undergo autophosphorylation.
Accili et al. (24) described another mutation in L2 that was found in two sisters who were both homozygous for the same mutation. Experiments in transfected NIH-3T3 cells revealed that this substitution of phenylalanine to valine at position 382 affected posttranslational processing and transport to the cell surface.qv+*n$7, http://www.100md.com
There have been several other naturally occurring mutations within the L2 domain (25, 26, 27), but unlike these and the ones described above, {Delta} V335 is the only single residue homozygous deletion within the extracellular domain of the IR to have been implicated in causing severe insulin resistance. Its position within the L2 domain appears to be critical in keeping the structure of the extracellular domain intact. Therefore, in its absence, proreceptor processing is impaired and insulin binding is abolished, and clinically it results in the most severe phenotype of the insulin resistance spectrum.qv+*n$7, http://www.100md.com
Acknowledgmentsqv+*n$7, http://www.100md.com
Recombinant human IGF was kindly provided by Chiron Corp. (Emeryville, CA).
Received August 5, 2002.3!]z2o, 百拇医药
Accepted for publication November 4, 2002.3!]z2o, 百拇医药
References3!]z2o, 百拇医药
Longo N, Wang Y, Smith SA, Langley SD, DiMeglio LA, Giannella-Neto D 2002 Genotype-phenotype correlation in inherited severe insulin resistance. Hum Mol Genet 11:1465–14753!]z2o, 百拇医药
Ward CW, Garrett TP, McKern NM, Lou M, Cosgrove LJ, Sparrow LG, Frenkel MJ, Hoyne PA, Elleman TC, Adams TE, Lovrecz GO, Lawrence LJ, Tulloch PA 2001 The three dimensional structure of the type I insulin-like growth factor receptor. Mol Pathol 54:125–1323!]z2o, 百拇医药
Soos MA, Siddle K, Baron MD, Heward JM, Luzio JP, Bellatin J, Lennox ES 1986 Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. Biochem J 235:199–2083!]z2o, 百拇医药
Prigent SA, Stanley KK, Siddle K 1990 Identification of epitopes on the human insulin receptor reacting with rabbit polyclonal antisera and mouse monoclonal antibodies. J Biol Chem 265:9970–99773!]z2o, 百拇医药
Rau H, Kocova M, O’Rahilly S, Whitehead JP 2000 Naturally occurring amino acid substitutions at Arg1174 in the human insulin receptor result in differential effects on receptor biosynthesis and hybrid formation, leading to discordant clinical phenotypes. Diabetes 49:1264–1268
Sambrook J FE, Maniatis T 1989 Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Pressu:v}$, 百拇医药
Hart LM, Lindhout D, Van der Zon GC, Kayserilli H, Apak MY, Kleijer WJ, Van der Vorm ER, Maassen JA 1996 An insulin receptor mutant (Asp707 –> Ala), involved in leprechaunism, is processed and transported to the cell surface but unable to bind insulin. J Biol Chem 271:18719–18724u:v}$, 百拇医药
Zhang B, Roth RA 1991 A region of the insulin receptor important for ligand binding (residues 450–601) is recognized by patients’ autoimmune antibodies and inhibitory monoclonal antibodies. Proc Natl Acad Sci USA 88:9858–9862u:v}$, 百拇医药
Elleman TC, Frenkel MJ, Hoyne PA, McKern NM, Cosgrove L, Hewish DR, Jachno KM, Bentley JD, Sankovich SE, Ward CW 2000 Mutational analysis of the N-linked glycosylation sites of the human insulin receptor. Biochem J 347:771–779u:v}$, 百拇医药
Luo RZ, Beniac DR, Fernandes A, Yip CC, Ottensmeyer FP 1999 Quaternary structure of the insulin-insulin receptor complex. Science 285:1077–1080
Ottensmeyer FP, Beniac DR, Luo RZ, Yip CC 2000 Mechanism of transmembrane signaling: insulin binding and the insulin receptor. Biochemistry 39:12103–121127r(;.5x, 百拇医药
Marino-Buslje C, Martin-Martinez M, Mizuguchi K, Siddle K, Blundell TL 1999 The insulin receptor: from protein sequence to structure. Biochem Soc Trans 27:715–7267r(;.5x, 百拇医药
Williams PF, Mynarcik DC, Yu GQ, Whittaker J 1995 Mapping of an NH2-terminal ligand binding site of the insulin receptor by alanine scanning mutagenesis. J Biol Chem 270:3012–30167r(;.5x, 百拇医药
Mynarcik DC, Yu GQ, Whittaker J 1996 Alanine-scanning mutagenesis of a C-terminal ligand binding domain of the insulin receptor {alpha} subunit. J Biol Chem 271:2439–24427r(;.5x, 百拇医药
Kristensen C, Andersen AS, Ostergaard S, Hansen PH, Brandt J 2002 Functional reconstitution of insulin receptor binding site from non-binding receptor fragments. J Biol Chem 277:18340–183457r(;.5x, 百拇医药
Surinya KH, Molina L, Soos MA, Brandt J, Kristensen C, Siddle K 2002 Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies. J Biol Chem 277:16718–16725
Yip CC, Hsu H, Patel RG, Hawley DM, Maddux BA, Goldfine ID 1988 Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor {alpha} -subunit. Biochem Biophys Res Commun 157:321–329%, 百拇医药
Schumacher R, Soos MA, Schlessinger J, Brandenburg D, Siddle K, Ullrich A 1993 Signaling-competent receptor chimeras allow mapping of major insulin receptor binding domain determinants. J Biol Chem 268:1087–1094%, 百拇医药
Jospe N, Zhu J, Liu R, Livingston JN, Furlanetto RW 1994 Deletion of 3 basepairs resulting in the loss of lysine-121 in the insulin receptor {alpha} -subunit in a patient with leprechaunism: binding, phosphorylation, and biological activity. J Clin Endocrinol Metab 79:1294–1302%, 百拇医药
Liu R, Zhu J, Jospe N, Furlanetto RW, Bastian W, Livingston JN 1995 Deletion of lysine 121 creates a temperature-sensitive alteration in insulin binding by the insulin receptor. J Biol Chem 270:476–482%, 百拇医药
Krook A, Soos MA, Kumar S, Siddle K, O’Rahilly S 1996 Functional activation of mutant human insulin receptor by monoclonal antibody. Lancet 347:1586–1590
van der Vorm ER, Kuipers A, Kielkopf-Renner S, Krans HM, Moller W, Maassen JA 1994 A mutation in the insulin receptor that impairs proreceptor processing but not insulin binding. J Biol Chem 269:14297–143026*#, 百拇医药
Maassen JA, Van der Vorm ER, Van der Zon GC, Klinkhamer MP, Krans HM, Moller W 1991 A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface. Biochemistry 30:10778–107836*#, 百拇医药
Accili D, Frapier C, Mosthaf L, McKeon C, Elbein SC, Permutt MA, Ramos E, Lander E, Ullrich A, Taylor SI 1989 A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes. EMBO J 8:2509–25176*#, 百拇医药
Barbetti F, Gejman PV, Taylor SI, Raben N, Cama A, Bonora E, Pizzo P, Moghetti P, Muggeo M, Roth J 1992 Detection of in insulin receptor gene by denaturing gradient gel electrophoresis. Diabetes 41:408–4156*#, 百拇医药
Longo N, Langley SD, Griffin LD, Elsas LJ 1995 Two mutations in the insulin receptor gene of a patient with leprechaunism: application to prenatal diagnosis. J Clin Endocrinol Metab 80:1496–15016*#, 百拇医药
Kadowaki T, Kadowaki H, Rechler MM, Serrano-Rios M, Roth J, Gorden P, Taylor SI 1990 Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. J Clin Invest 86:254–2646*#, 百拇医药
Kadowaki T, Bevins CL, Cama A, Ojamaa K, Marcus-Samuels B, Kadowaki H, Beitz L, McKeon C, Taylor SI 1988 Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science 240:787–7906*#, 百拇医药
Garrett TP, McKern NM, Lou M, Frenkel MJ, Bentley JD, Lovrecz GO, Elleman TC, Cosgrove LJ, Ward CW 1998 Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor. Nature 394: 395–399(S. George A. Johansen M. A. Soos H. Mortensen S. Gammeltoft V. Saudek K. Siddle L. Hansen and S. O’)
An infant with Donohue’s syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the V335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the V335 receptor was capable of being immunoprecipitated with antibodies directed against the ß-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the {alpha} -subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor {alpha} -subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two ß-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.
Introductionf)cq, 百拇医药
MUTATIONS IN the insulin receptor (IR) gene lead to a variety of recognizable clinical syndromes, with the clinical phenotype broadly reflecting the severity of the receptor dysfunction (1). Thus, homozygous or compound heterozygous null mutations lead to the most severe phenotype, Donohue’s syndrome (sometimes called leprechaunism), which is usually fatal in the first year or two of life. Rabson-Mendenhall syndrome describes a somewhat less severe phenotype with frequent survival into the second decade. In these cases, homozygous missense mutations have often been reported, with these mutations severely impairing, but not totally disrupting, IR structure and function. Finally, type A insulin resistance, often presenting around puberty and compatible with survival to middle age at least, is sometimes the result of IR mutations. Where type A syndrome can be due to an IR mutation, it is often a heterozygous missense change in the intracellular tyrosine kinase domain, which inhibits the function of coexpressed wild-type receptor through a dominant negative mechanism.
Such naturally occurring mutants, found in subjects with severe insulin resistance, can aid attempts to fully understand the structure/function relationships of the IR, as, unlike systematic site-directed mutagenesis, there is a high a priori likelihood that the mutation found is significantly impairing the real biological functions of the receptor. We have detected a novel homozygous mutation, deleting valine 335 in the L2 domain of the extracellular portion of the IR in an infant with Donohue’s syndrome. Functional studies of the mutant receptor indicated that whereas it was capable of being processed and transported to the cell surface, albeit less efficiently than normal, the receptor was unable to bind insulin or autophosphorylate due to a major alteration in the conformation of the extracellular domain. Using the recently elucidated structure of the highly homologous IGF-I receptor (2), we have generated a computer model of the effect of this mutation on the structure and function of the L2 domain.
Materials and Methodsk)ekgtx, 百拇医药
Reagentsk)ekgtx, 百拇医药
Mouse monoclonal antibodies specific for the human IR (83-7, 83-14, 18-44, CT-1); D88, an irrelevant mouse monoclonal antibody (directed against TSH); and a rabbit polyclonal antibody raised against the human IR ß-subunit (Carl 10) were described previously (3, 4, 5). Mouse monoclonal antiphosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Secondary horseradish peroxidase-conjugated antibodies (goat antimouse IgG and goat antirabbit IgG) were purchased from DAKO Corp. (Copenhagen, Denmark). Monoiodo-A14 insulin was a gift from Eli Lilly \|[amp ]\| Co. (Indianapolis, IN).k)ekgtx, 百拇医药
Site-directed mutagenesisk)ekgtx, 百拇医药
All routine DNA manipulations were performed using standard protocols (6). V335 mutant receptors were reconstructed using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. All constructs were in the pRc.CMV expression vector (Invitrogen, Leek, The Netherlands), and the mutation was confirmed by sequencing before use.
Transient transfection of Chinese hamster ovary (CHO) cellsr$, 百拇医药
CHO cells were maintained at 37 C in Nutrient F-12 Ham’s medium (Sigma-Aldrich, Poole, UK) supplemented with 10% fetal calf serum, 105 U/liter penicillin, 0.1 g/liter streptomycin, and 2 mM glutamine. Transfections were performed on cells grown in 6- or 24-well plates using the appropriate cDNA and Fugene 6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions. All experiments were performed 24 h after transfection.r$, 百拇医药
Immunoprecipitation and Western blottingr$, 百拇医药
Cells were rinsed once with ice-cold PBS and solubilized in 10 µl lysis buffer/1 cm2 confluent cells [50 mM HEPES (pH 7.4), 150 mM NaCl, 30 mM NaF, 10 mM Na4P2O7, 10 mM EDTA, 1 mM Na3VO4, 1 µg/ml antipain, pepstatin and leupeptin, 2.5 mM benzamidine, 0.5 mM phenylmethylsulfonylfluoride, and 1% Triton X-100]. Cleared cell lysates were immunoprecipitated with IR-specific monoclonal antibodies or an irrelevant monoclonal antibody by mixing the lysates for 3 h at 4 C with antibody prebound to 2.5 mg protein A-agarose (IgG2 antibodies) or protein G-agarose (IgG1 antibodies). Western blotting was performed essentially as described previously (1). Briefly, washed immunoprecipitates or total cell lysates were resolved by 6% SDS-PAGE, transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA), and probed with anti-IR antibody (1:8000 Carl 10), followed by horseradish peroxidase-conjugated secondary antibody (1:5000). Proteins were visualized using enhanced chemiluminesence (Amersham Pharmacia Biotech, Little Chalfont, UK).
Biotinylation of cell surface IRs-9w, 百拇医药
CHO cells expressing wild-type (WT) or V335 mutant IRs were grown in six-well plates. Cell surface proteins were biotinylated using the enhanced chemiluminesence protein biotinylation module (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. After cell lysis, IRs were immunoprecipitated with monoclonal 18-44 antibody and analyzed by 6% SDS-PAGE, followed by electroblotting as described above. Membranes were then probed with either horseradish peroxidase-conjugated streptavidin (1:1500) or antibody (1:8000 Carl 10) as described above for detection of biotinylated or total IRs, respectively.-9w, 百拇医药
Insulin binding-9w, 百拇医药
Cells in 24-well plates were washed twice with PBS before the addition of 250 µl [125I]insulin (50–100 pM, 30,000 dpm) in binding buffer [100 mM HEPES (pH 7.8), 120 mM NaCl, 1 mM EDTA, 15 mM sodium acetate, 1.2 mM MgSO4, 10 mM glucose, and 1% BSA] for 4 h at 4 C. Cells were then washed twice with ice-cold PBS and solubilized in 0.03% sodium dodecyl sulfate for determination of cell-bound radioactivity in a -counter (1282 Compugamma CS Universal Counter, LKB Wallac, Inc., Turku, Finland). Insulin binding to solubilized receptors was essentially carried out as described previously (3). Briefly, solubilized receptors (10 µl) were incubated with [125I]insulin (50–100 pM, 30,000 dpm) for 18 h at 4 C (250 µl final volume). Receptor-bound radioactivity was then determined by precipitation with polyethylene glycol 6000. In both cases, nonspecific binding was determined in the presence of 1 µM unlabeled insulin.
Receptor autophosphorylation]0[4+$, 百拇医药
Transfected cells were serum-starved for 6 h before stimulation with 100 nM insulin for 2 min at 37 C, followed by rinsing with ice-cold PBS and solubilization in lysis buffer. Cell lysates were then analyzed by Western blotting as described above using the mouse monoclonal antiphosphotyrosine antibody 4G10 (1:10,000) to detect tyrosine-phosphorylated receptor.]0[4+$, 百拇医药
Results]0[4+$, 百拇医药
Clinical history and molecular genetic diagnosis]0[4+$, 百拇医药
The patient was a male born to consanguineous parents of Turkish origin. He was delivered prematurely at 34 wk gestation because of intrauterine growth retardation and had a birth weight of 1.7 kg. Examination revealed the typical features of Donohue’s syndrome: elfin-like face, depressed nasal bridge, low-set large ears, hirsutism, acanthosis nigricans, muscular wasting, and lack of sc fat.]0[4+$, 百拇医药
Typical to the course of the disease, over the next few weeks he failed to thrive and had hyperglycemia with blood glucose levels up to 30 mM. He had no ketoacidosis. Initially he was treated with insulin, and at the age of 2 months had gained weight up to a maximum of 2.8 kg. His blood glucose levels were still up to 24 mM. Nonfasting serum insulin levels were enormously elevated (35,713 pmol/liter). He was then started on recombinant IGF-I treatment (0.4 mg/kg, given three times). This resulted in a much improved blood glucose profile and improved well-being. However, he suffered a febrile episode (of unknown cause), which was accompanied by hyperglycemia, and he died at the age of 3 months.
We undertook sequencing of all 22 exons of the IR in the patient, his parents and his 2-yr-old brother. This revealed a single amino acid deletion, a valine residue at position 335, which is located in the {alpha} -subunit (exon 4; Fig. 1, A and B). The patient was homozygous for this mutation, and both his parents and brother were heterozygous. Of interest, there was no family history of diabetes. The parents showed a mild degree of hyperinsulinemia, but were glucose tolerant during a glucose tolerance test (Table 1).;afj8#', 百拇医药
fig.ommitteedfig.ommitteed;afj8#', 百拇医药
Figure 1. A, Sequencing trace showing the proband’s homozygous deletion of Val335. Alignment of sequences (-f, forward; -r, reverse) from exon 4 of the IR gene adjoining the valine at position 335 in the proband and family members. Deletion of GTC (Val335) is present in all family members, and typical for deletions/insertions on one allele only (heterozygosity), the DNA sequencing becomes unreadable downstream (-f) and upstream (-r) of the deletion/insertion, whereas homozygosity is a symmetrical change, with no consequences for reading the DNA sequence. A, Adenine; T, thymine; C, cytosine; G, guanine. B, Agarose gel electrophoresis of BslI-generated restriction fragments of exon 4 of the IR gene containing V335(GTC). Deletion of the trinucleotide GTC generates a new restriction site: WT, 262 bp + 101 bp; heterozygous mutant, 262 bp +168 bp+ 94 bp; homozygous mutant, 168 bp + 101 bp + 94 bp. The 101 and 94 bp run as one band. Lanes 1 and 6, Father; lanes 2 and 7, mother; lanes 3 and 8, brother; lanes 4 and 9, proband; lanes 5 and 10, unrelated subject; lane 11, 100-bp molecular marker.
fig.ommitteedfig.ommitteed9, 百拇医药
Table 1. Clinical characteristics of the family members9, 百拇医药
Expression of WT and V335 mutant receptors in CHO cells9, 百拇医药
To assess the level of expression of the mutant receptor, equal amounts of cDNA encoding WT and mutant receptor were transiently transfected into CHO cells. Western blotting of cell lysates with anti-IR antibody (Fig. 2) showed similar amounts of WT and mutant proreceptor, but levels of mature processed receptor (as indicated by the ß-subunit) in the mutant expressing cells were only about one third of those in the WT-expressing cells, suggesting a partial impairment in proreceptor processing. The mobility of the ß-subunit was also altered, running slightly slower than WT, suggesting that its slightly greater mass might be due to altered glycosylation.9, 百拇医药
fig.ommitteedfig.ommitteed9, 百拇医药
Figure 2. Western blot analysis of expression of WT and V335 mutant IR. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. The indicated amounts of total cell lysate from a WT transfection (lanes 1–5), two different V335 transfections (lanes 6 and 7), and a mock transfection (lane 8) were resolved by 6% SDS-PAGE and analyzed for IR proreceptor (upper panel) and mature ß-subunit (lower panel) by Western blotting using a rabbit polyclonal IR antibody (1:8000; Carl 10) as described in Materials and Methods. Data are representative of three independent experiments.
The presence of the V335 IR on the cell surface was investigated by cell surface biotinylation. Consistent with the results of Western blotting, mature mutant receptor was detectable at the cell surface, but at about 30% the level of WT receptor (Fig. 3).s%, http://www.100md.com
fig.ommitteedfig.ommitteeds%, http://www.100md.com
Figure 3. Detection of biotinylated cell surface IRs. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Proteins at the cell surface were biotinylated, and IRs were immunoprecipitated from the indicated volumes of cell lysate using the IR-specific monoclonal antibody 18-44 and were analyzed by 6% SDS-PAGE, followed by electroblotting as described in Materials and Methods. Membranes were then probed with either HRP-streptavidin (1:1500) to detect biotinylated cell surface IR (left panel) or a rabbit polyclonal IR antibody (1:8000; Carl 10) to detect total IR (right panel). Data are representative of two independent experiments.s%, http://www.100md.com
Insulin binding and receptor autophosphorylations%, http://www.100md.com
Because at least some processed V335 receptors are expressed at the cell surface, the ability of mutant receptors to bind insulin was first examined in intact cells (Fig. 4A. Additionally, given that mutant IRs have been described (7) that do not bind insulin at the cell surface, but which do on solubilization, the ability of solubilized V335 receptors to bind insulin was also examined (Fig. 4B). Although, as expected, WT receptors bound insulin well, mutant receptors showed no significant binding compared with mock-transfected cells in either paradigm. Not surprisingly therefore, cells expressing the V335 receptor showed no evidence of ligand-induced receptor autophosphorylation (Fig. 5).
fig.ommitteedfig.ommitteed/, http://www.100md.com
Figure 4. Insulin binding studies. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Cells were serum-starved for 6 h before measuring [125I]insulin binding to intact cells (A) or 10 µl solubilized receptors (B), as described in Materials and Methods. Results are expressed as a percentage of WT binding. Nonspecific binding measured in the presence of 1 µM unlabeled insulin has been subtracted. Data are the mean ± SEM of three independent experiments with two separate transfections for V335 in each experiment./, http://www.100md.com
fig.ommitteedfig.ommitteed/, http://www.100md.com
Figure 5. Western blot analysis of insulin-stimulated receptor autophosphorylation. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Cells were serum-starved for 6 h and then stimulated with (+) or without (-) 100 nM insulin for 2 min at 37 C. Total cell lysates (2.5 and 10 µl from a WT transfection, 10 µl from two different V335 transfections and a mock transfection) were resolved by 6% SDS-PAGE and analyzed for total IR ß-subunit (upper panel) or tyrosine-phosphorylated ß-subunit (lower panel) by Western blotting using a rabbit polyclonal IR antibody (1:8,000; Carl 10) or an antiphosphotyrosine antibody (1:10,000 4G10), respectively, as described in Materials and Methods. Data are representative of two independent experiments.
Studies of the conformational effects of the V335 mutation using monoclonal antibodiesi{g]4y, http://www.100md.com
To test whether the V335 mutation affects the conformation of the IR, a panel of IR-specific monoclonal antibodies, recognizing different epitopes on the receptor (3, 4, 8), were used to immunoprecipitate receptors from cells transfected with either WT or V335 IR cDNA. Two antibodies (83-7 and 83-14) recognize epitopes on the -subunit (the cysteine-rich domain and the Fn0 domain, respectively) and are conformation specific, reacting poorly with denatured protein (4, 8). Two other antibodies (CT1 and 18-44) react with regions on the ß-subunit (exon 22 at the extreme intracellular C terminus and exon 12 in the extracellular portion of the ß-subunit, respectively) (4). The results from a typical experiment are shown in Fig. 6. The processed WT receptor (as shown by the ß-subunit) was immunoprecipitated to a similar extent by all four IR-specific monoclonal antibodies, but not by an irrelevant monoclonal antibody (N). Because V335 receptors are less efficiently processed than WT (Fig. 2), we used 4 times as many receptors in the immunoprecipitation. Despite this and although immunoprecipitation was not completely abolished, the mutant receptor was less efficiently precipitated by the antibodies targeted toward the -subunit (7, 14) than those toward the ß-subunit (CT and 44; Fig. 6) This suggests that the V335 mutation causes a conformational change within the {alpha} -subunit that affects the accessibility of the epitopes to which these antibodies are targeted. In contrast, the accessibility of the ß-subunit epitopes appears relatively unaffected by the mutation. This is evidenced by the fact that the ß-subunit antibodies are able to immunoprecipitate WT and mutant receptors to a similar extent.
fig.ommitteedfig.ommitteedws, 百拇医药
Figure 6. Studies of the conformational structure of the V335 IR using monoclonal antibodies. CHO cells were transfected with equal amounts of WT or V335 IR cDNA. Cell lysates (15 µl WT, 60 µl V335) were immunoprecipitated with an irrelevant monoclonal antibody (N) or IR-specific monoclonal antibodies recognizing the subunit, 83-7 (7 ), 83-14 (14 ) or ß-subunit, CT-1 (CT), and 18-44 (44). Immunoprecipitates were resolved by 6% SDS-PAGE and analyzed for insulin proreceptor (left panel) and mature ß-subunit (right panel) by Western blotting using a rabbit polyclonal IR antibody (1:8000; Carl 10) as described in Materials and Methods. Data are representative of two independent experiments.ws, 百拇医药
The more intense signal seen from the proreceptor of the mutant receptors is accounted for by the fact that, as previously stated, we used 4 times as many receptors in the immunoprecipitation to compensate for the decreased level of processing in the mutant receptor.ws, 百拇医药
Discussion
We describe a novel mutation, V355, in the human IR that in homozygous form causes the most severe phenotype human insulin resistance, Donohue’s syndrome. Our studies demonstrate the complex molecular mechanisms by which it leads to such marked receptor dysfunction.$z|7p/, 百拇医药
V335 is the first reported homozygous single residue deletion in the extracellular domain. Its importance is reflected by the severity of the clinical phenotype as well as the consequences reflected in the results of our functional studies. It is only partially processed, to a level 30% that of WT. It has a slightly higher molecular mass than WT, suggesting altered glycosylation. However, this is unlikely to be the cause of the functional differences we have uncovered. Although V335 is very near to a glycosylation site positioned at 337, detailed mutation studies by Elleman et al. (9) show that alteration of the residue at 337 (in combination with other glycosylation sites) has no effect on either processing, cell surface expression, or autophosphorylation.
Any processed V335 receptor is, however, able to reach the cell surface. Despite this, it is unable to bind insulin whether at the cell surface or when solubilized. Insulin-stimulated autophosphorylation is consequently absent. Our experiments using the monoclonal antibodies indicate that the {alpha} -subunit is conformationally altered, but that the ßsubunit appears to remain intact.7^, 百拇医药
Molecular modeling is compatible with these findings. The recently determined quaternary structure of the whole receptor together with bound insulin (10) made it possible to establish the mutual position and interaction of the individual domains and to propose a self-consistent model of IR function (11). Based on these and the known or modeled three-dimensional structure of the homologous IGF-I and IR-related protein (2, 12), we were able to derive a model of the IR, as shown in Fig. 7. Using this model we were able to deduce the putative position of valine 335 (red ribbon) within the L2 domain (blue). Together with its backbone hydrogen-bonded partner valine 311 (pink ribbon) it initiates the first five-stranded parallel ß-sheet. It is thus located just after the flexible hinge that connects the cysteine-rich and L2 domains. The strand containing V335 then crosses to the second parallel ß-sheet, providing the rigid scaffold for the two ß-sheets faces of the L2 domain. Deletion of this residue, which is therefore in such a critical position, would seriously affect the tertiary and quaternary structures of the receptor as a whole by causing the collapse of the initial corner of the ß-sheet in L2, destabilizing cysteines 302 and 323 (yellow) that form one of the disulfide bridges, altering the mutual positions of the two ß-sheets, and lengthening and increasing the flexibility of the hinge between the cysteine-rich domain and L2.
fig.ommitteedfig.ommitteed\0|pzz, 百拇医药
Figure 7. Computer-generated molecular model of the L2 domain. The L2 domain (blue) is connected by a hinge (magenta) to the cysteine-rich domain (gray). The position of the V335 residue and its hydrogen-bonded partner V322 are highlighted as red and pink ribbons, respectively. All disulfide bridges are in yellow; the backbone of the cysteines next to V335 are also colored yellow. Some of the mutations detected in insulin-resistant patients are represented as balls: from top to bottom those affecting insulin binding are red: S323L (21 ), N435S (27 ), and K433E (28 ); those affecting processing are green: F382V (24 ) and W412S (22 ). The figure was produced with Protein Data Bank coordinates of the IGF-I receptor (29 ).\0|pzz, 百拇医药
The putative insulin-binding site is mainly formed by L1, the cysteine-rich domain and the C-terminal tail of the {alpha} -chain. Alanine scanning of the NH2-terminal domain of the {alpha} -subunit (13) and the C-terminal domain (14) have revealed several key residues within these domains that are critical for hormone binding. In the N-terminal domain it appears that mutation of Arg14, Asn15, and Phe64 produced large decreases in binding affinity; the remaining residues tested produced less marked effects. Scanning of the C-terminal domain revealed that the mutation of four residues (Thr704, Phe705, Glu706, and His710) produced large decreases in affinity that were too great to be quantified, and three other amino acids (Tyr708, Leu709, and Phe714) also produced very large decreases in affinity when mutated to alanine.
More recently, studies by Kristensen et al. (15) have attempted to investigate the minimum length of the {alpha} -subunit required for insulin binding. They report that when a fragment consisting of the first 255 amino acids is mixed with the CT (C-terminal) domain in a soluble peptide form, insulin binding is restored. The importance of the C-terminal tail was further demonstrated by the work of Surinya et al. (16). By using IR fragments containing all four of the amino-terminal domains fused to different fragments coded by exon 10, they showed that the precise positioning of this domain critically determines the insulin binding affinity.e, 百拇医药
The importance of the cysteine-rich domain was shown by work carried out by Yip et al. (17). Using receptors labeled with radioactive insulin photoprobes, they isolated a 23-kDa fragment that contained the putative insulin-binding site, and mutational studies revealed this to contain the cysteine-rich domain within it.e, 百拇医药
Despite the fact that the L2 domain is not directly in the insulin binding pocket, it is no less important in helping to confer the receptor’s ability for high affinity ligand binding. L2 anchors the protruding insulin pocket in the main body of the IR and transmits the signal to the interior of the cell by pushing the kinases of each monomer together for mutual activation. Schumacher et al. (18) used IR and IGF-I receptor chimeric receptor constructs to define the sites of high affinity insulin binding. Their work revealed that one of the two major structural determinants required for such binding was indeed located in L2 between amino acids 325 and 524.
Although L2 has not been directly probed for insulin binding by alanine scanning mutagenesis, the naturally occurring mutations described to date clearly indicate its importance in this function as well as receptor processing and its transport to the cell surface. Figure 7 shows the mutations found in the L2 domain only. V335 is crucial for both functions, whereas the other mutations described to date are implicated in one or other of them. Apart from V335, all other mutations in L2 are located in the connecting loops outside the rigid secondary structure elements. These will therefore only affect local side-chain interactions, as opposed to the global structural change that the deletion of V335 has been shown to cause with such drastic clinical consequences.cu, 百拇医药
Although multiple nonsense and missense mutations in the {alpha} -subunit have been found in patients with Donohue’s and Rabson-Mendenhall syndromes, deletions of single amino acids have rarely been reported. Jospe et al. (19) described a 3-bp deletion in exon 2 leading to a deletion of the lysine residue in position 121. This was found in a female patient with Donohue’s syndrome (patient Rochester). The mutation was present in heterozygous form in this subject; the presumed second mutated allele was not found. Initially, functional studies of the 121 suggested that, unlike V335, cell surface expression and insulin binding were normal. Later investigations (20) revealed that this receptor did, in fact, have altered insulin binding, but that this was temperature sensitive. When measured at 21 C, binding was similar to that in the WT, but at 37 C, insulin binding was 50% WT binding.
There have also been several other descriptions of naturally occurring substitution mutations in the extracellular domain. The nearest of these to V335 is the substitution of serine for leucine at position 323. This was found in a patient with Rabson-Mendenhall syndrome. Functional studies by Krook et al. (21) showed that unlike V335, processing was normal. However, insulin binding was abolished, as was subsequent ligand-stimulated autophosphorylation.{e5+, 百拇医药
Another mutation described by Van de Vorm (22) resulted in a substitution of tryptophan at position 412 into serine. This mutation in the L2 domain was found in a patient with Donohue’s syndrome in a homozygous form. When studied in COS1 cells, this was found to be retained intracellularly as the 210-kDa precursor. However, it retained its insulinbinding activity in cross-linking experiments and was able to undergo autophosphorylation.{e5+, 百拇医药
Maasen et al. (23) investigated a substitution mutation at position 233 of the receptor, which was transfected into CHO cell lines. This leucine to proline mutation was found in a homozygous form in a patient with Donohue’s syndrome. Here, receptor processing was defective; the mutation prevented proreceptor cleavage. In a similar manner to {Delta} V335, the mutant receptor was also poorly precipitated by two antibodies targeted to the extracellular domain, indicating that it, too, caused a conformational change, such as to alter the epitopes for these antibodies. However, unlike {Delta} V335, this receptor though was not transported to the cell surface. When solubilized, it was also unable to bind insulin or undergo autophosphorylation.
Accili et al. (24) described another mutation in L2 that was found in two sisters who were both homozygous for the same mutation. Experiments in transfected NIH-3T3 cells revealed that this substitution of phenylalanine to valine at position 382 affected posttranslational processing and transport to the cell surface.qv+*n$7, http://www.100md.com
There have been several other naturally occurring mutations within the L2 domain (25, 26, 27), but unlike these and the ones described above, {Delta} V335 is the only single residue homozygous deletion within the extracellular domain of the IR to have been implicated in causing severe insulin resistance. Its position within the L2 domain appears to be critical in keeping the structure of the extracellular domain intact. Therefore, in its absence, proreceptor processing is impaired and insulin binding is abolished, and clinically it results in the most severe phenotype of the insulin resistance spectrum.qv+*n$7, http://www.100md.com
Acknowledgmentsqv+*n$7, http://www.100md.com
Recombinant human IGF was kindly provided by Chiron Corp. (Emeryville, CA).
Received August 5, 2002.3!]z2o, 百拇医药
Accepted for publication November 4, 2002.3!]z2o, 百拇医药
References3!]z2o, 百拇医药
Longo N, Wang Y, Smith SA, Langley SD, DiMeglio LA, Giannella-Neto D 2002 Genotype-phenotype correlation in inherited severe insulin resistance. Hum Mol Genet 11:1465–14753!]z2o, 百拇医药
Ward CW, Garrett TP, McKern NM, Lou M, Cosgrove LJ, Sparrow LG, Frenkel MJ, Hoyne PA, Elleman TC, Adams TE, Lovrecz GO, Lawrence LJ, Tulloch PA 2001 The three dimensional structure of the type I insulin-like growth factor receptor. Mol Pathol 54:125–1323!]z2o, 百拇医药
Soos MA, Siddle K, Baron MD, Heward JM, Luzio JP, Bellatin J, Lennox ES 1986 Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. Biochem J 235:199–2083!]z2o, 百拇医药
Prigent SA, Stanley KK, Siddle K 1990 Identification of epitopes on the human insulin receptor reacting with rabbit polyclonal antisera and mouse monoclonal antibodies. J Biol Chem 265:9970–99773!]z2o, 百拇医药
Rau H, Kocova M, O’Rahilly S, Whitehead JP 2000 Naturally occurring amino acid substitutions at Arg1174 in the human insulin receptor result in differential effects on receptor biosynthesis and hybrid formation, leading to discordant clinical phenotypes. Diabetes 49:1264–1268
Sambrook J FE, Maniatis T 1989 Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Pressu:v}$, 百拇医药
Hart LM, Lindhout D, Van der Zon GC, Kayserilli H, Apak MY, Kleijer WJ, Van der Vorm ER, Maassen JA 1996 An insulin receptor mutant (Asp707 –> Ala), involved in leprechaunism, is processed and transported to the cell surface but unable to bind insulin. J Biol Chem 271:18719–18724u:v}$, 百拇医药
Zhang B, Roth RA 1991 A region of the insulin receptor important for ligand binding (residues 450–601) is recognized by patients’ autoimmune antibodies and inhibitory monoclonal antibodies. Proc Natl Acad Sci USA 88:9858–9862u:v}$, 百拇医药
Elleman TC, Frenkel MJ, Hoyne PA, McKern NM, Cosgrove L, Hewish DR, Jachno KM, Bentley JD, Sankovich SE, Ward CW 2000 Mutational analysis of the N-linked glycosylation sites of the human insulin receptor. Biochem J 347:771–779u:v}$, 百拇医药
Luo RZ, Beniac DR, Fernandes A, Yip CC, Ottensmeyer FP 1999 Quaternary structure of the insulin-insulin receptor complex. Science 285:1077–1080
Ottensmeyer FP, Beniac DR, Luo RZ, Yip CC 2000 Mechanism of transmembrane signaling: insulin binding and the insulin receptor. Biochemistry 39:12103–121127r(;.5x, 百拇医药
Marino-Buslje C, Martin-Martinez M, Mizuguchi K, Siddle K, Blundell TL 1999 The insulin receptor: from protein sequence to structure. Biochem Soc Trans 27:715–7267r(;.5x, 百拇医药
Williams PF, Mynarcik DC, Yu GQ, Whittaker J 1995 Mapping of an NH2-terminal ligand binding site of the insulin receptor by alanine scanning mutagenesis. J Biol Chem 270:3012–30167r(;.5x, 百拇医药
Mynarcik DC, Yu GQ, Whittaker J 1996 Alanine-scanning mutagenesis of a C-terminal ligand binding domain of the insulin receptor {alpha} subunit. J Biol Chem 271:2439–24427r(;.5x, 百拇医药
Kristensen C, Andersen AS, Ostergaard S, Hansen PH, Brandt J 2002 Functional reconstitution of insulin receptor binding site from non-binding receptor fragments. J Biol Chem 277:18340–183457r(;.5x, 百拇医药
Surinya KH, Molina L, Soos MA, Brandt J, Kristensen C, Siddle K 2002 Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies. J Biol Chem 277:16718–16725
Yip CC, Hsu H, Patel RG, Hawley DM, Maddux BA, Goldfine ID 1988 Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor {alpha} -subunit. Biochem Biophys Res Commun 157:321–329%, 百拇医药
Schumacher R, Soos MA, Schlessinger J, Brandenburg D, Siddle K, Ullrich A 1993 Signaling-competent receptor chimeras allow mapping of major insulin receptor binding domain determinants. J Biol Chem 268:1087–1094%, 百拇医药
Jospe N, Zhu J, Liu R, Livingston JN, Furlanetto RW 1994 Deletion of 3 basepairs resulting in the loss of lysine-121 in the insulin receptor {alpha} -subunit in a patient with leprechaunism: binding, phosphorylation, and biological activity. J Clin Endocrinol Metab 79:1294–1302%, 百拇医药
Liu R, Zhu J, Jospe N, Furlanetto RW, Bastian W, Livingston JN 1995 Deletion of lysine 121 creates a temperature-sensitive alteration in insulin binding by the insulin receptor. J Biol Chem 270:476–482%, 百拇医药
Krook A, Soos MA, Kumar S, Siddle K, O’Rahilly S 1996 Functional activation of mutant human insulin receptor by monoclonal antibody. Lancet 347:1586–1590
van der Vorm ER, Kuipers A, Kielkopf-Renner S, Krans HM, Moller W, Maassen JA 1994 A mutation in the insulin receptor that impairs proreceptor processing but not insulin binding. J Biol Chem 269:14297–143026*#, 百拇医药
Maassen JA, Van der Vorm ER, Van der Zon GC, Klinkhamer MP, Krans HM, Moller W 1991 A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface. Biochemistry 30:10778–107836*#, 百拇医药
Accili D, Frapier C, Mosthaf L, McKeon C, Elbein SC, Permutt MA, Ramos E, Lander E, Ullrich A, Taylor SI 1989 A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes. EMBO J 8:2509–25176*#, 百拇医药
Barbetti F, Gejman PV, Taylor SI, Raben N, Cama A, Bonora E, Pizzo P, Moghetti P, Muggeo M, Roth J 1992 Detection of in insulin receptor gene by denaturing gradient gel electrophoresis. Diabetes 41:408–4156*#, 百拇医药
Longo N, Langley SD, Griffin LD, Elsas LJ 1995 Two mutations in the insulin receptor gene of a patient with leprechaunism: application to prenatal diagnosis. J Clin Endocrinol Metab 80:1496–15016*#, 百拇医药
Kadowaki T, Kadowaki H, Rechler MM, Serrano-Rios M, Roth J, Gorden P, Taylor SI 1990 Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. J Clin Invest 86:254–2646*#, 百拇医药
Kadowaki T, Bevins CL, Cama A, Ojamaa K, Marcus-Samuels B, Kadowaki H, Beitz L, McKeon C, Taylor SI 1988 Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science 240:787–7906*#, 百拇医药
Garrett TP, McKern NM, Lou M, Frenkel MJ, Bentley JD, Lovrecz GO, Elleman TC, Cosgrove LJ, Ward CW 1998 Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor. Nature 394: 395–399(S. George A. Johansen M. A. Soos H. Mortensen S. Gammeltoft V. Saudek K. Siddle L. Hansen and S. O’)