E R van der Vorm

Leiden University, Leyden, South Holland, Netherlands

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Publications (8)32.45 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) alpha subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to alpha and beta subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I-labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant alpha subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR alpha and beta subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, 125I-insulin was efficiently cross-linked to the receptor alpha subunit by disuccinimidyl suberate. These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the alpha subunit carboxyl terminus to insulin binding.
    Journal of Biological Chemistry 09/1996; 271(31):18719-24. · 4.65 Impact Factor
  • E R van der Vorm, J A Maassen
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    ABSTRACT: Leprechaunism is a syndrome of severe insulin resistance. In general, mutations in both alleles of the insulin receptor are required for developing the leprechaun phenotype. Recently, we described a leprechaun patient having an Arg for Gly substitution in one allele of the insulin receptor whereas the other allele has the normal sequence. To explain the leprechaun phenotype, we searched for additional defects at the receptor level. The insulin receptor exists as two splice variants, either with (B form) or without exon 11 (A form). It has been suggested that a decrease in the relative amount of the A isoform can contribute to the development of an insulin resistant state. Fibroblasts from the leprechaun patient show levels of the A isoform of less than 20% whereas in control fibroblasts approximately 50% of the A isoform is present. However, in fibroblasts from two other patients with severe insulin resistance where the disease has been demonstrated to result from two mutated insulin receptor alleles, the same decline of the A isoform was seen. We conclude that the decrease in the relative amount of the A isoform in these patients is probably an epiphenomenon due to impaired insulin action.
    Hormone and Metabolic Research 01/1995; 26(12):599-601. · 2.15 Impact Factor
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    ABSTRACT: Here we report the identification of a new mutation in the alpha-chain of the insulin receptor, changing Trp412 into Ser using DNA from consanguineous parents who gave birth to a child with leprechaunism. The mutant receptor was expressed stably in CHO and transiently in COS-1 cells. It was found that the Ser412 mutant is not cleaved into alpha- and beta-subunits and remains as a 210-kDa proreceptor at an intracellular site. This property of the mutant receptor is in line with the observed decreased insulin binding to the parental fibroblasts. Cross-linking experiments show that the Ser412 proreceptor is able to bind insulin with an affinity comparable to that of the wild-type alpha-chain. Despite its capacity to bind insulin, the mutant receptor is not autophosphorylated. We postulate that the patient was homozygous for the Trp412-->Ser mutation and that the mutation was responsible for the leprechaun phenotype. This is the first description of a transport-defective receptor with the mutation outside the tetrabasic processing site and a functional insulin binding domain. The ability of the Ser412 mutant to bind insulin in cross-linking experiments suggests that the impaired transport of the proreceptor to the cell surface is the primary cause for the binding defect to intact cells.
    Journal of Biological Chemistry 05/1994; 269(19):14297-302. · 4.65 Impact Factor
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    ABSTRACT: We have studied the structure and function of the insulin receptor in a patient (PK) with severe insulin resistance and Rabson-Mendenhall syndrome. Insulin binding to cultured fibroblasts from PK was almost not detectable and insulin-induced insulin receptor autophosphorylation and glucose uptake was abolished. The structure of the receptor gene was analysed by sequencing amplified products of the 22 exons with the flanking intron regions directly as well as after subcloning in pUCBM20 plasmids. Two mutant alleles of the insulin receptor gene were detected. One allele contains in-frame 12 additional base pairs in exon 3 coding for the amino acids Leu-His-Leu-Val located between Asp-261 and Leu-262 in the receptor's extracellular domain, being the first report of an insertion mutation of the insulin receptor gene. In the other allele Arg-86 in exon 2 is changed into a stop codon. Therefore, PK is compound heterozygous at the insulin receptor locus. Direct cDNA sequencing indicates that both mutant alleles are expressed in the patient's fibroblasts. Studies of the parents' fibroblasts revealed that PK inherited the insertion mutation from the father and the nonsense mutation from the mother. Insulin binding to fibroblasts of the mother was reduced (63% of control cells) and hormone binding to the father's cells shows a larger reduction (37% of control cells), but less severe than the patient's cells (11% of control). This investigation provides further evidence that the Rabson-Mendenhall syndrome is causally related to mutations in the insulin receptor gene.
    Diabetologia 12/1993; 36(11):1168-74. · 6.49 Impact Factor
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    ABSTRACT: Lipodystrophic diabetes mellitus of the Seip-Berardinelli type is a syndrome associated with insulin resistance and recessive inheritance. We have examined whether mutations in the insulin receptor are pathogenetic factors in this syndrome. Fibroblasts from three different patients with Seip-Berardinelli's lipodystrophy were tested for insulin binding, and insulin-stimulated receptor autophosphorylation. In addition, the coding region of both alleles of the iinsulin receptor gene was sequenced. No abnormalities in the number of high affinity insulin binding sites, and insulin-stimulated receptor autophosphorylation were detected. The insulin receptor related insulin-like growth factor I receptor also showed no functional changes. DNA sequence analysis of the amplified exons of the insulin receptor gene showed a silent mutation in patient 1 at codon Ser339, changing AGT to AGC. In patient 2 a heterozygous Met for Val substitution at position 985 was detected, which is a rare polymorphism. In patient 3 no mutations, other than described polymorphisms, were observed. These findings demonstrate that the primary genetic lesion in Seip-Berardinelli's lipodystrophy is outside the insulin receptor gene and that an involvement of the insulin-like growth factor I receptor is also unlikely.
    Diabetologia 03/1993; 36(2):172-4. · 6.49 Impact Factor
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    ABSTRACT: In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited.
    Journal of Biological Chemistry 02/1992; 267(1):66-71. · 4.65 Impact Factor
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    ABSTRACT: We have previously shown that a homozygous mutation encoding a substitution of proline for leucine at position 233 in the insulin receptor is linked with the syndrome of leprechaunism, being a lethal form of insulin resistance in newborn children. Specific binding of insulin and insulin-stimulated autophosphorylation of the insulin receptor are nearly absent in fibroblasts from the leprechaun patient. To examine the molecular basis of the observed insulin receptor abnormalities, CHO cell lines overexpressing mutant insulin receptors were made by transfection. The results show that the mutation inhibits cleavage and transport of the proreceptor from intracellular sites to the cell surface. As the mutant receptor is poorly precipitated by two different monoclonal antibodies recognizing epitopes on undenatured wild-type alpha-subunits, the mutation probably affects overall folding of the alpha-subunit. The mutant proreceptor is unable to bind insulin and exhibits no insulin-stimulated autophosphorylation. These data explain the abnormalities seen in the patient's fibroblasts. Pulse-chase labeling experiments on transfected cells show that the mutant precursor has an extended half-life (approximately 5 h) compared to the precursor of wild-type insulin receptors (approximately 2 h). This mutation is the first example of a naturally occurring mutation in the insulin receptor which completely blocks cleavage of the proreceptor and transport to the cell surface.
    Biochemistry 12/1991; 30(44):10778-83. · 3.38 Impact Factor
  • Nederlands tijdschrift voor geneeskunde 07/1991; 135(26):1165-70.

Publication Stats

78 Citations
32.45 Total Impact Points

Institutions

  • 1996
    • Leiden University
      Leyden, South Holland, Netherlands
  • 1993–1994
    • CSU Mentor
      Long Beach, California, United States
    • University of Cologne
      • Division of Cardiology, Pneumology, Angiology and Intensive Care
      Köln, North Rhine-Westphalia, Germany