Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

Abstract

Recently we reported the association of severe insulin resistance and congenital fibre type disproportion myopathy (CFTDM) in two brothers from a Danish family [1]. It has now been shown that the insulin resistance and possibly the myopathy in the two brothers is associated with compound heterozygous mutations of the insulin receptor gene [2]. The allele inherited from their father, who is less insulin resistant and has no CFTDM [1], has a missense mutation that changes Arg 1174 ®Gln. The allele inherited from their mother, who is less insulin resistant than the patients and her husband [1], carries a point mutation at the last base pair in exon 17 (position 3396). This G to A change affects the ±1 donor splice site of exon 17, resulting in a mixture of two mRNAs from that allele. The abnormal skipping of exon 17 (exon 17± variant) shifts the amino acid reading frame and creates a stop codon 36 amino acids after the end of the sequence encoded by exon 16 and this leads to a truncated receptor missing the entire tyrosine kinase domain. In the exon 17 + variant, the point mutation is silent and should result in a normally transcribed insulin receptor. Diabetologia (1999) Summary We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg 1174 ®Gln mutation, in situ activation of the receptor kinase in skeletal muscle was reduced about 70 %. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70 % of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother's skeletal muscle, suggesting that virtually no truncated receptor was expressed. Receptor kinase activity was, however, reduced by 95 and 91 % in the compound heterozygous brothers. This suggests that the mother's mutated allele contributes little to the generation of functional receptor protein and that the receptors in the mother's skeletal muscle are transcribed almost exclusively from the non-mutated allele. The mutation in exon 17 could lead to reduced transcription or rapid degradation of a predominantly transcribed truncated gene product or both. [Diabetologia (1999) Abbreviations: CFTDM, congenital fibre type disproportion myopathy; aPY, anti-phosphotyrosine antibody; aIR, anti-insulin receptor antibody; IRS-1, insulin receptor substrate-1; GLU4:TYR1, Polymer with a 4:1 ratio of glutamic acid and tyrosine, used as insulin receptor substrate; IGF-1, insulin-like growth factor-1.

Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

“…All ten family members affected by hypoglycaemia carried a heterozygous mutation in the IRTK domain of INSR (Arg1174Gln). This mutation has previously been found in three females with the type A syndrome of insulin resistance, and in an apparently healthy male [4][5][6][7]. Complete cosegregation (logarithm of the odds score 3.21) with the disease phenotype supported the proposition that the Arg1174Gln mutation was the cause of hypoglycaemia [3].…”

“…Studies of the functional properties of the Arg1174Gln mutation have shown normal insulin binding and affinity to the receptor, but a 70-75% reduction in insulin-stimulated IRTK activity [5][6][7]. Furthermore, in CHO cell lines homozygous for the mutant INSR, stimulation with insulin in the physiological range (1,000 pmol/l) showed a pronounced inability to increase tyrosine phosphorylation of IRS-1, PI3K activity, SLC2A4 translocation and glycogen synthesis [7,19].…”

Insulin signaling and action in cultured skeletal muscle cells from lean healthy humans with high and low insulin sensitivity.

“…For the IR kinase assays, 40 µl lysates (3.5 mg/ml) were added to microwells coated with anti-IR antibody (28) at 4°C. After overnight incubation, the wells were washed, and the kinase activity of immobilized IR was measured in the presence of 0.3 µmol/l ATP containing 3 µCi/well [␥-32 P]ATP and 2.4 µg/ml recombinant IRS-1 as previously described (29). Wells were then washed again, and insulin binding activity (defined as the amount of insulin specifically bound at 8.7 nmol/l) was analyzed as described earlier (28).…”

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms β2 and θ

“…After stimulation with insulin, whole-cell lysates of HEK 293 fibroblasts were added to microwells coated with anti-insulin receptor (aIR) antibody [28]. After the receptor had bound to the antibody, wells were washed and kinase activity was measured in the presence of 0.3 mmol/l [g-32 P]-ATP (3.7±7.4 TBq/mmol) and 2.4 mg/ml recombinant insulin receptor substrate-1 (IRS-1) (Upstate Biotechnology, New York, N. Y., USA) as described previously [29]. Wells were then washed again and insulin binding activity (defined as the amount of insulin specifically bound at 8.7 nmol/l) was analysed as described previously [28] to be sure of similar amounts of insulin receptor.…”