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Biomedical subjects

S I Taylor

Publications and source records attributed to S I Taylor.

At least 19 recordsLinked to original sources
PubMed1992

Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation.

Some patients with extreme insulin resistance have mutations in their insulin receptor gene. We previously identified five such mutations located in the extracellular domain of the insulin receptor (Asn-->Lys15, His-->Arg209, Phe-->Val382, Lys-->Glu460, and Asn-->Ser462) and studied the effects of these mutations upon posttranslational processing, insulin binding, and tyrosine autophosphorylation. We now characterize the ability of these mutant receptors to mediate biological actions of insulin in transfected NIH-3T3 fibroblasts. All cell lines expressing mutant receptors showed marked impairment in insulin-stimulated c-jun expression and thymidine incorporation when compared with cells expressing wild-type human insulin receptors. The most severe impairment was seen in cells expressing the Val382 mutant (a mutation which causes an intrinsic defect in receptor autophosphorylation). These cells had insulin responses similar to the untransfected cells (used as a negative control). In contrast, cells expressing the Lys15 mutant have the ability to achieve a normal level of maximal autophosphorylation but require an abnormally high concentration of insulin to do so (as the result of decreased insulin binding affinity). These cells show a higher basal rate and much lower insulin stimulation of both c-jun expression and thymidine incorporation when compared with the cells expressing the wild-type human insulin receptors. This pattern is also seen in the cells expressing the other mutants with normal autophosphorylation (Arg209, Glu460, and Ser462). Although the most severe defects in insulin action are seen with the mutation which has an intrinsic defect in receptor autophosphorylation, the ability to undergo normal autophosphorylation does not seem to preclude mutations from impairing the ability of receptors to mediate some of the actions of insulin.

Cell Line↗
PubMed1992

Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.

3T3 Cells↗
PubMed1992

Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain.

Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain-binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane.

3T3 Cells↗
PubMed1992

Mutations in the insulin receptor gene in patients with genetic syndromes of insulin resistance and acanthosis nigricans.

Mutations of the insulin receptor gene have been identified in patients with genetic syndromes of insulin resistance associated with acanthosis nigricans. These mutations impair insulin responses by reducing the number of insulin receptors on the surface of target cells, or by reducing the receptor's ability to bind insulin or to undergo insulin-stimulated autophosphorylation, an important step in insulin action. Studies of mutant receptors expressed in transfection systems have contributed to our understanding of the structure-function relationships of the insulin receptor.

Acanthosis Nigricans↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Detection of mutations in insulin receptor gene by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5' end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied. Two mutant alleles were identified in this patient. The allele inherited from the father had a mutation substituting alanine for Val-28; in the allele inherited from the mother, arginine was substituted for Gly-366.

Alanine↗
PubMed1991

Substitution of arginine for histidine at position 209 in the alpha-subunit of the human insulin receptor. A mutation that impairs receptor dimerization and transport of receptors to the cell surface.

Receptors for insulin and epidermal growth factor contain cysteine-rich domains in the extracellular portion of the molecule. His209 (insulin receptor numbering system) is 1 of 2 amino acid residues that are identically conserved in the cysteine-rich domains of insulin receptors, epidermal growth factor receptors, and other homologous receptors. Previously, we have identified a patient with leprechaunism who is homozygous for a mutation substituting Arg for His209 in the insulin receptor gene (Kadowaki, T., Kadowaki, H., Rechler, M. M., Serrano-Rios, M., Roth, J., Gorden, P., and Taylor, S. I. (1990) J. Clin. Invest. 86, 254-264). In this investigation, the Arg209 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs several steps in the post-translational processing of the insulin receptor:dimerization of 190-kDa proreceptors into a disulfide linked species, proteolytic cleavage of the proreceptor into alpha- and beta-subunits, and terminal processing of the high mannose form of N-linked oligosaccharide into complex carbohydrate. In addition, the defects in post-translational processing within the endoplasmic reticulum and Golgi apparatus are associated with a marked inhibition in transport of receptors to the plasma membrane. Nevertheless, a small number (approximately 10%) of the receptors are transported to the cell surface. These receptors on the cell surface bind insulin with normal affinity and have normal tyrosine kinase activity.

3T3 Cells↗
PubMed1991

Targeted inactivation of the insulin receptor gene in mouse 3T3-L1 fibroblasts via homologous recombination.

To study the role of the insulin receptor in determining adipocyte differentiation of the mouse cell line 3T3-L1, we have introduced a mutation that inactivates the insulin receptor gene by homologous recombination. In two independent clones, inactivation of one allele of the insulin receptor gene was associated with a 50-70% reduction in the number of insulin receptors. In addition, both clones were markedly impaired in their ability to differentiate into adipocytes. The defect in adipocyte-specific differentiation was corrected by expression of transfected human insulin receptor cDNA. These data suggest that the insulin receptor may play an important role in promoting differentiation of 3T3-L1 cells into adipocytes in vitro.

Animals↗
PubMed1991

A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation.

Mutations of the insulin receptor gene have been shown to cause insulin-resistant diabetes in patients with genetic forms of insulin resistance. We have previously reported that a mutation substituting valine for Phe382 in the alpha-subunit of the insulin receptor is associated with impaired transport of the mutant receptor to the plasma membrane (Accili, D., Frapier, C., Mosthaf, L., McKeon, C., Elbein, S. C., Permutt, M. A., Ramos, E., Lander, E. S., Ullrich, A., and Taylor, S. I. (1989) EMBO J. 8, 2509-2517). In this study, we demonstrate that the Val382 mutation impairs the ability of insulin to activate receptor autophosphorylation. Furthermore, the Val382 receptor has reduced activity to phosphorylate other peptide substrates in the presence of insulin. Nevertheless, when the Val382 mutant and wild-type receptors are mixed together, the wild-type human insulin receptor is able to phosphorylate the Val382 mutant receptor, thereby activating the tyrosine kinase activity of the mutant receptor. Thus, the conformational change caused by the Val382 mutation compromises the ability of the receptor to transmit a signal across the plasma membrane. Furthermore, our observations suggest that receptor phosphorylation by an intermolecular mechanism (i.e. transphosphorylation) may play a role in mediating the action of insulin upon the target cell.

Animals↗
PubMed1991

Unusual forms of insulin resistance.

Insulin resistance is an important feature in the pathogenesis of non-insulin-dependent diabetes mellitus. Some rare patients have syndromes associated with a severe form of insulin resistance, usually involving acanthosis nigricans and, in premenopausal females, hyperandrogenism. This review discusses two of the causes of insulin resistance: mutations in the insulin receptor gene and autoantibodies directed against the insulin receptor.

Acanthosis Nigricans↗
PubMed1991

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.

Amino Acids↗
PubMed1991

A mutation in the tyrosine kinase domain of the insulin receptor associated with insulin resistance in an obese woman.

Insulin resistance is frequently associated with acanthosis nigricans and hyperandrogenism. In patients with type A insulin resistance, this has been shown to be due to genetic defects in insulin receptor function. However, other patients with a similar clinical syndrome have been reported to have a variant of this syndrome, in which assays of insulin receptor function were normal. We have sequenced a portion of the insulin receptor gene in one such patient, a 29-yr-old woman with obesity and insulin resistance. The patient is heterozygous for a mutation substituting isoleucine for methionine at position 1153. Met1153 is located in the intracellular domain of the receptor near the cluster of tyrosine phosphorylation sites at positions 1158, 1162, and 1163. Studies of the mutant receptor expressed in NIH-3T3 cells demonstrated that the Ile1153-mutation impairs the ability of insulin to stimulate autophosphorylation of solubilized insulin receptors. In addition, the mutation impairs the ability of insulin to stimulate receptor tyrosine kinase activity to phosphorylate an artificial substrate [poly(Glu-Tyr)]. It seems likely that this defect in receptor tyrosine kinase activity explains the defect in the ability of the patient's insulin receptors to mediate insulin action in vivo. Furthermore, this patient provides a paradigm in which genetic factors act in concert with other risk factors, such as obesity, to cause clinically important insulin resistance.

Adult↗
PubMed1991

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes.

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.

Adult↗