Improper expression of insulin receptors on fibroblasts from a leprechaun patient

Abstract

Leprechaunism is an inherited human disorder characterized by severe insulin resistance. We have examined the properties of the insulin receptor in fibroblasts from a leprechaun patient. In vitro, severe insulin resistance is reflected by a low level of insulin binding to the patients fibroblasts and impaired insulin-mediated uptake of 2-deoxyglucose. Quantification of the receptor in detergent-solubilized total glycoprotein indicates a normal receptor number, in agreement with the observed normal level of insulin receptor mRNA on northern blots. The insulinstimulated autophosphorylation of the patient's receptor shows a normal profile. The insulin receptor is present on the plasma membrane as indicated by cell-surface iodination experiments. No abnormalities in the molecular masses of the receptor's M and p chains were observed. The results indicate that an apparently normal receptor is synthesized in sufficient amounts but functional expression of the receptor on the plasma membrane is impaired.

Coupling of insulin‐responsive glucose transport to receptors for insulin‐like growth factor 1 in primary human fibroblasts

“…After washing the precipitate with 1 ml ethanol, autophosphorylation was quantified by SDS/polyacrylamide gel electrophoresis and autoradiography as described for insulin receptor autophosphorylation. Insulin receptor autophosphorylation was as described [12]. In brief, 10 pg glycoprotein was incubated for 1 h at 22'C with the indicated concentration of hormone in 50 mM Hepes pH 7.4, 10 mM MgCI2, 5 mM MnCI2, 1 mM 2-mercaptoethanol, 0.3 M N-acetylglucosamine, 1 pg/ml leupeptin, 0.1 mM phenylmethylsulphonyl fluoride and 1 mg/ml bovine serum albumin.…”

“…The reaction was terminated by addition of 1 ml 10% trichloroacetic acid. The resulting protein precipitate was dissolved in 75 pl 50 mM sodium bicarbonate, 0.1% SDS; 750 p1 immune precipitation buffer (50 mM Tris/HCl pH 7.50, 30 mM EDTA, 200 mM NaC1, 10 mM NaF, 10 mM 4-nitrophenyl phosphate, 10 mM sodium orthovanadate, 1 pg/ml leupeptin, 1 pg/ml trasylol, 1 mM phenylmethylsulphonyl fluoride, 0.1% NP40) and 5 pl anti-(insulin receptor) antiserum [12] was added. After incubation on ice for 16 h, the immune complex was isolated using formalin-treated Staphylococcus aureus.…”

“…Experiments were in triplicate, standard deviations are indicated. Incorporation of radioactivity was determined by densitometry of autoradiograms as described [12].…”

“…Receptor autophosphorylution IGF-1 receptor autophosphorylation was performed on the glycoprotein fraction of fibroblasts as described for insulin receptor autophosphorylation [12] ; 5 pg glycoprotein was used in each assay. The autophosphorylation reaction was terminated by addition of 1 ml 10% trichloroacetic acid and the protein precipitate was collected by centrifugation.…”

An in-frame insertion in exon 3 and a nonsense mutation in exon 2 of the insulin receptor gene associated with severe insulin resistance in a patient with Rabson-Mendenhall syndrome

“…Preparation of partially purified insulin receptors by chromatography on wheat germ agglutinin sepharose and insulin-stimulated autophosphorylation was performed as previously described [20]. Insulin was added to 10 ~tl glycoprotein fraction (1 mg protein/ml) and incubation was continued for 1 h at 22 ~ Phosphorylation was initiated by addition of 6 mmol/1 MnC12 and 32p-ATP (25 gmol/l; 200 Ci/mmol).…”