Homozygous deletion of the human insulin receptor gene results in leprechaunism.

Homozygous inactivation of a gene, as is frequently performed to generate mouse models, provides an opportunity to elucidate the role that the gene plays in normal physiology. However, studies of human disease provide direct insight into the effect of inactivating mutations in man. In this investigation, we have identified a one year-old boy from a consanguineous pedigree who is homozygous for deletion of the insulin receptor gene resulting in leprechaunism. Contrary to previous predictions, the complete deletion of the insulin receptor gene is compatible with life.

Immunoprecipitation of insulin receptors from cultured human lymphocytes (IM-9 cells) by antibodies to pp60src.

The family of tyrosine-specific protein kinases includes proteins encoded by retroviral oncogenes as well as receptors for insulin and several growth factors. Antibodies to pp60src, the protein encoded by the src oncogene of Rous sarcoma virus (RSV), can specifically immunoprecipitate affinity-labeled insulin receptors from cultured human lymphocytes (IM-9 cells). This precipitation is specifically inhibited by the src gene product purified from RSV-transformed rat cells. These observations provide evidence that there is structural homology between the insulin receptors and pp60src.

Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

Defect in cooperativity in insulin receptors from a patient with a congenital form of extreme insulin resistance.

Previously, we have described a novel qualitative defect in insulin receptors from a patient with a genetic form of extreme insulin resistance (leprechaunism). Receptors from this insulin-resistant child are characterized by two abnormalities: (a) an abnormally high binding affinity for insulin, and (b) a markedly reduced sensitivity of 125I-insulin binding to alterations in pH and temperature. In this paper, we have investigated the kinetic mechanism of this abnormality in steady-state binding. The increased binding affinity for 125I-insulin results from a decrease in the dissociation rate of the hormone-receptor complex. In addition, the cooperative interactions among insulin binding sites are defective with insulin receptors from this child with leprechaunism. With insulin receptors on cultured lymphocytes from normal subjects, both negative and positive cooperativity may be observed. Porcine insulin accelerates the dissociation of the hormone-receptor complex (negative cooperativity). In contrast, certain insulin analogs such as desoctapeptide-insulin and desalanine-desasparagine-insulin retard the dissociation of the hormone-receptor complex (positive cooperativity). With insulin receptors from the leprechaun child, positive cooperativity could not be demonstrated, although negative cooperativity appeared to be normal. It seems likely that the same genetic defect may be responsible for the abnormalities in both insulin sensitivity and positive cooperativity.

Insulin receptor biosynthesis in cultured lymphocytes from insulin-resistant patients.

In some patients with genetic forms of extreme insulin resistance, the cause of insulin resistance is a marked (greater than or equal to 90%) reduction in the number of insulin receptors on the cell surface. In the present work, we describe studies of insulin receptor biosynthesis in Epstein-Barr virus (EBV)-transformed lymphocytes from three patients (A-1, A-5, and A-8) with type A extreme insulin resistance. Insulin receptors are composed of two major glycoprotein subunits (apparent molecular weight [Mr] of 135 and 95 kD), which are both derived from a common precursor molecule with Mr of 190 kD. In one patient (A-1), there was a marked reduction in the biosynthesis of both the 190-kD precursor and the mature receptor. Thus, in this patient, the defect appears to occur early in the biosynthetic pathway (i.e., before the synthesis of the 190-kD precursor). In contrast, in two sisters (A-5 and A-8) with type A extreme insulin resistance, biosynthesis of the 190-kD precursor proceeds at a normal rate. However, there appears to be a defect subsequent to the biosynthesis of the 190-kD precursor, but before the insertion of the mature receptor in the plasma membrane. Our observations suggest the existence of at least two distinct types of biosynthetic defects which may give rise to a marked reduction in the number of insulin receptors on the cell surface. In addition, for comparison, we have studied receptor biosynthesis in cultured EBV lymphocytes from a fourth patient (A-7) with type A extreme insulin resistance. Although the cells of patient A-7 have a normal number of insulin receptors, we have detected subtle abnormalities in the posttranslational processing of the insulin receptor precursor, which may be a biochemical marker for a postbinding defect that causes insulin resistance in this patient.

Insulin receptor degradation is accelerated in cultured lymphocytes from patients with genetic syndromes of extreme insulin resistance.

Previous studies of the insulin receptor in disease states have utilized primarily techniques of equilibrium binding and, to a limited extent structural, analysis. Though techniques have been developed to study receptor degradation in normal cells, they have not been applied to disease states. In the present study we have examined insulin receptor degradation rate in B lymphocytes that were obtained from peripheral blood of normal subjects and patients with several syndromes of extreme insulin resistance. B lymphocytes were established in culture from each patient's peripheral cells by transformation with Epstein-Barr virus. The insulin receptors were surface labeled using Na125I/lactoperoxidase and the cells were returned to incubate in growth media. After varying periods of incubation, aliquots of cells were solubilized and the cell content of labeled receptor subunits were measured by immunoprecipitation with anti-receptor antibodies and NaDodSO4/polyacrylamide gel electrophoresis. The fall in 125I-insulin receptor content approximated a single exponential and was quantitated as receptor subunit half-life (t1/2). In cell lines from four patients in whom the number of insulin receptors was reduced by greater than 90%, the rate of receptor loss was greater than normal (t1/2 equals 3.8 +/- 0.9 h vs. 6.5 +/- 1.2 h; mean +/- SD, P less than 0.01). However, a similar acceleration in receptor degradation was seen in cells from five patients with extreme insulin resistance but low-normal insulin receptor concentration (t1/2 equals 4.4 +/- 0.9 h). This group included cells from one patient with a qualitatively abnormal receptor. Thus, all the patients with genetic syndromes of insulin resistance had accelerated receptor degradation, regardless of their receptor concentration. By contrast, insulin receptors on cultured lymphocytes that were obtained from patients with extreme insulin resistance secondary to autoantibodies to the insulin receptor had normal receptor degradation (t1/2 equals 6.1 +/- 1.9 h). We conclude that (a) accelerated insulin receptor degradation is an additional feature of cells from patients with genetic forms of insulin resistance; (b) that accelerated insulin receptor degradation may explain the low-normal receptor concentrations that were seen in some patients with extreme insulin resistance; and (c) that accelerated degradation does not explain the decreased receptor concentration in patients with very low insulin receptor binding and, therefore, by inference, a defect in receptor synthesis must be present in this subgroup.

Clinical disorders associated with autoantibodies to the insulin receptor. Simulation by passive transfer of immunoglobulins to rats.

Patients with autoantibodies to the insulin receptor (Anti-R) may exhibit either fasting hypoglycemia or hyperglycemia and extreme insulin resistance. Occasionally, both these phenomena are observed in the same patient at different times in the clinical course. In an effort to understand what determines the patient's response to Anti-R, we developed an animal model of these clinical disorders by passive transfer of Anti-R IgG to rats. IgG fractions from the plasma of Anti-R patients and control subjects were prepared by affinity chromatography with staphylococcal protein A-Sepharose. Anti-R IgG, injected into fasting rats, induced severe and persistent hypoglycemia (plasma glucose 30-60 mg/dl). Rats injected with control IgG maintained a plasma glucose within the range of 75 (fasting) to 165 mg/dl (feeding). In comparison with the effects of insulin, the hypoglycemic response to Anti-R IgG had a slower onset (2-4 h) and lasted longer (8-24 h). Similar, dose-dependent hypoglycemic responses were observed in rats whether the Anti-R IgG was derived from an insulin-resistant or hypoglycemic patient. When Anti-R IgG was administered in sufficiently high doses for several days to fed rats, persistent hyperglycemia (plasma glucose 200-400 mg/dl) developed. Based on these in vivo and previous in vitro studies, we attribute the hypoglycemic response to an insulin-like effect of Anti-R, and the hyperglycemic response to a desensitization of host tissues to the effects of insulin, with more prolonged exposure to higher levels of Anti-R.

Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance.

The nucleotide sequence was determined for all 22 exons of the insulin receptor gene from three patients with genetic syndromes associated with extreme insulin resistance. In all three patients, insulin resistance was caused by decreased insulin binding to the cell surface. The patient with leprechaunism (leprechaun/Winnipeg) came from a consanguineous pedigree and was homozygous for a missense mutation substituting arginine for His209 in the alpha-subunit of the insulin receptor. The other two patients were both compound heterozygotes with a nonsense mutation in one allele of the insulin receptor gene, and a missense mutation in the other allele. In the patient with the Rabson-Mendenhall syndrome (patient RM-1), the missense mutation substituted lysine for Asn15 in the alpha-subunit. In the patient with type A extreme insulin resistance (patient A-1), the missense mutation substituted serine for Asn462 in the alpha-subunit. Both nonsense mutations markedly reduced the levels of insulin receptor mRNA transcribed from the alleles with the nonsense mutation as compared to the transcripts from the other allele. The reduction in the level of mRNA would be predicted to greatly reduce the rate at which the truncated receptors would be synthesized. Furthermore, the truncated receptors would be severely impaired in their ability to mediate insulin action.

Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance.

Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.

Identification of a family of sorting nexin molecules and characterization of their association with receptors.

Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008-1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of approximately 100 amino acids, which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms. Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes.

Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.

Deletion of C-terminal 113 amino acids impairs processing and internalization of human insulin receptor: comparison of receptors expressed in CHO and NIH-3T3 cells.

We have studied the structure and the function of a truncated human insulin receptor in which 113 amino acids (aa 1231-1343) at the C-terminus of the beta-subunit were deleted. In this study, wild-type and truncated insulin receptors were expressed by stable transfection in NIH-3T3 cells and CHO cells. The mutation impairs post-translational processing of the insulin receptor; proteolytic cleavage is retarded, and degradation of the truncated receptor is accelerated. Furthermore, insulin-stimulated autophosphorylation of the mutant insulin receptor is impaired. This is associated with a defect in insulin-stimulated endocytosis. Finally, in NIH-3T3 cells, the mutant insulin receptor failed to mediate the mitogenic effects of insulin. In CHO cells, transfection of insulin receptor cDNA (either wild-type or mutant) did not alter mitogenic response to insulin. It has previously been shown that deletion of 43 amino acids at the C-terminus of the beta-subunit did not affect insulin receptor tyrosine kinase activity. Our data suggest that the structural domain located 43-113 amino acids from the C-terminus appears to have several functional roles. First, the domain appears to promote folding of receptor into the optimal conformation for post-translational processing. Second, the presence of this domain appears to promote the stability of the receptor beta-subunit in intact cells. Finally, perhaps as a consequence of the effects upon the stability of the receptor, this domain is required in intact cells for insulin-stimulated autophosphorylation and signal transmission.

Lilly Lecture: molecular mechanisms of insulin resistance. Lessons from patients with mutations in the insulin-receptor gene.

Insulin resistance contributes to the pathogenesis of NIDDM. We have investigated the molecular mechanisms of insulin resistance in patients with genetic syndromes caused by mutations in the insulin-receptor gene. In general, patients with two mutant alleles of the insulin-receptor gene are more severely insulin-resistant than are patients who are heterozygous for a single mutant allele. These mutations can be put into five classes, depending upon the mechanisms by which they impair receptor function. Some mutations lead to a decrease in the number of insulin receptors on the cell surface. For example, some mutations decrease the level of insulin receptor mRNA or impair receptor biosynthesis by introducing a premature chain termination codon (class 1). Class 2 mutations impair the transport of receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane. Mutations that accelerate the rate of receptor degradation (class 5) also decrease the number of receptors on the cell surface. Other mutations cause insulin resistance by impairing receptor function--either by decreasing the affinity to bind insulin (class 3) or by impairing receptor tyrosine kinase activity (class 4). The prevalence of mutations in the insulin receptor gene is not known. However, theoretical calculations suggest that approximately 0.1-1% of the general population are heterozygous for a mutation in the insulin-receptor gene; the prevalence is likely to be higher among people with NIDDM. Accordingly, it is likely that mutations in the insulin-receptor gene may be a contributory cause of insulin resistance in a subpopulation with NIDDM.

Immunological abnormalities in insulin receptors on cultured EBV-transformed lymphocytes from insulin-resistant patient with leprechaunism.

Defects in insulin-receptor structure can impair insulin-receptor function. We have previously identified qualitative abnormalities in insulin binding to insulin receptors from an insulin-resistant patient (Lep/Ark-1). The defects in insulin binding are probably caused by a defect in receptor structure. In this study, we used immunological probes to investigate the structural defect(s) responsible for the abnormal function. Several anti-receptor antibodies were impaired in their abilities to bind to the insulin receptor of Lep/Ark-1. For example, monoclonal antibody MoAb-51 was much less effective in inhibiting binding to insulin receptors from Lep/Ark-1 (ID50 70 nM) than to those of normal subjects (ID50 8 nM). In addition, there was a 10-fold reduction of the avidity with which human polyclonal antibody B-d immunoprecipitated the patient's insulin receptors. The avidity of antibody B-10 was also reduced, although to a lesser extent. In contrast, several site-specific antibodies against epitopes on the beta-subunit of the receptor bound to receptors from Lep/Ark-1 with normal avidity. The data with monoclonal and polyclonal antibodies are consistent with the hypothesis that the structural defect resides in the extracellular domain of this patient's insulin receptor. The normal immunoreactivity of two putative phosphorylation sites on the beta-subunit with site-specific antibodies gives further support to the conclusion that this patient's receptors have normal tyrosine kinase activity.

Proximal enhancer of the human insulin receptor gene binds the transcription factor Sp1.

The insulin receptor is a growth regulator present on the surface of most cells that transmits a mitogenic signal in response to insulin. Thus, the gene for the insulin receptor is constitutively expressed at low levels in all cells. We characterize a constitutive enhancer element that is present in the proximal promoter of the human insulin receptor gene. We have localized the enhancer to a 26-base-pair (26-bp) sequence from -528 to -503. When this sequence is inserted into the proximal promoter, a three- to fourfold increase in promoter activity is observed, and when two copies are inserted, a five- to sixfold increase is seen. Electrophoretic mobility shift analysis demonstrates that nuclear factors binding to this sequence are found in many different cell types. At least two proteins with different specificities bind within this 26-bp sequence. The identity of the predominant binding protein is Sp1, because an oligonucleotide composed of an Sp1 consensus binding sequence can compete for several of the DNA-protein complexes. In addition, we demonstrate that purified Sp1 can bind to the 26-bp oligonucleotide and that this complex comigrates with a DNA-protein complex formed with a HeLa nuclear extract. Finally, an antibody to human Sp1 protein is able to bind to the enhancer DNA/HeLa protein complex and supershift this complex. These findings suggest that this sequence corresponds to a general element that may contribute to the ubiquitous expression of the human insulin receptor gene.

Non-insulin-mediated glucose disappearance in subjects with IDDM. Discordance between experimental results and minimal model analysis.

Both insulin and glucose contribute to the regulation of glucose metabolism in vivo. We directly measured the ability of glucose per se to promote glucose disposal in subjects with insulin-dependent diabetes mellitus (IDDM). We compared our results with predictions of the minimal model of glucose metabolism. To identify minimal model parameters, a frequently sampled intravenous glucose tolerance test (FSIVGTT) was administered to each subject while they were connected to a Biostator (a device that monitors blood glucose and gives insulin to mimic normal insulin secretion). Data from this test reflected normal glucose tolerance and were in excellent agreement with minimal model predictions. The FSIVGTT was then repeated without the Biostator in the same diabetic subjects in order to directly measure the effect of glucose per se to promote glucose disposal in the absence of an incremental insulin effect (a basal insulin drip was maintained). To compare these results with minimal model predictions, the equations describing glucose disappearance in the absence of an incremental insulin effect were solved using parameters identified from the Biostator experiment. The glucose disappearance measured in the absence of an incremental insulin response was much slower than the minimal model predictions. Thus, the minimal model appears to overestimate the effect of glucose per se on glucose uptake and underestimate the contribution of incremental insulin.

Insulin-receptor biosynthesis in cultured lymphocytes from an insulin-resistant patient (Rabson-Mendenhall syndrome). Evidence for defect before insertion of receptor into plasma membrane.

In some patients with genetic forms of extreme insulin resistance, there is a marked decrease in the number of insulin receptors on the cell surface. We studied an insulin-resistant patient (RM-1) with the Rabson-Mendenhall syndrome. As judged by insulin-binding studies, Epstein-Barr virus-transformed lymphocytes from patient RM-1 exhibit a 90% decrease in the number of insulin receptors. Similarly, with either lactoperoxidase-catalyzed radioiodination of cell surface receptors or biosynthetic labeling of receptors with [3H]glucosamine, we demonstrated an 80-90% decrease in the number of insulin receptors in cells from patient RM-1. Previous studies have shown that the marked decrease in insulin receptors of the Rabson-Mendenhall patient is not due to accelerated receptor degradation. Therefore, we investigated the possibility that a slow rate of receptor biosynthesis might account for the 90% reduction of insulin receptors in cells from this patient. Insulin-receptor biosynthesis proceeds through a glycoprotein precursor with an apparent Mr of 190,000. It undergoes endopeptidase cleavage and further posttranslational processing to yield the mature 135,000- and 95,000-Mr glycoprotein subunits. We studied the biosynthesis of the 190,000-Mr precursor and mature receptor subunits by a pulse-chase labeling technique with [2-3H]mannose. The time course of insulin-receptor biosynthesis appeared normal in cells from patient RM-1, despite a 10-fold reduction in the number of receptors on the cell surface. Parallel pulse-chase experiments with either [2-3H]mannose or [35S]methionine yielded the same results regardless of which label was employed. Thus, the receptor precursor in the Rabson-Mendenhall patient seems to be synthesized at a normal rat.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel human insulin receptor gene mutation uniquely inhibits insulin binding without impairing posttranslational processing.

The precise nature of the insulin-binding site of the insulin receptor (IR) has not been determined, although the importance of several regions of the alpha-subunit in insulin binding has been demonstrated. A naturally occurring mutation in a patient with severe insulin resistance that changes the Ser323 codon in the alpha-subunit of the IR to a leucine codon is associated with markedly impaired insulin binding to cells from the patient and to transfected cells expressing the mutant receptor. However, unlike other IR alpha-subunit mutations associated with decreased insulin binding, this mutation does not lead to a defect in posttranslational processing or cell-surface expression of IRs. Thus, the defect in insulin binding associated with the Leu323 mutant IR is a direct result of an alteration in the insulin-binding site. No natural IR mutation described thus far is associated with both decreased insulin binding and normal cell-surface expression of the mutant receptor. This study demonstrates the critical role that Ser323 of the IR alpha-subunit plays in insulin binding, either by forming part of the binding site or by stabilizing its conformation.