Cells and culture conditions

Wild type (WT) human IR-A, IR-B and mutated IRs were inserted in pZem vector and stably over-expressed in Baby Hamster Kidney (BHK) cells to achieve the desired receptor expressions. In brief, 1 x 10⁶ cells were transfected with 10 μg plasmid by liposome transfer using Lipofectamine (Life Technologies, Tåstrup, Denmark) and allowed to recover three days before applying selection pressure in the form of 1 μM methotrexate. After approximately three weeks single cell clones from each transfection appeared and 12 clones from each transfection were picked and subjected to IR expression analysis by Western Blotting. Based on the highest expression level one clone for each transfection was selected and used in the subsequent experiments. Cells were cultured in Dulbecco’s modified Eagle medium containing high glucose, 10% fetal calf serum and 2 mM L-glutamine, 10 mg/ml streptomycin, and 100 units/ml penicillin (Invitrogen, Nærum, Denmark) at 37°C in a 5% CO₂ enriched, humidified atmosphere.

Midi receptor mutants of human IR were made in the soluble high affinity midi receptor construct [31], but with an intact functional furine processing site followed by a small part of the β-subunit giving rise to a genuine IR C-terminal alpha subunit sequence without tags. Two unique restriction sites (AvrII and BstBI) were made to frame exon 11 without changing the amino acid sequence. Oligos with the desired mutations were designed to include the AvrII site (upstream of exon 11). An oligo downstream of exon 11 and the BstB1 was used to obtain a PCR product which was subcloned, hereby replacing the AvrII/BstBI fragment in human IR-A or human IR-B. The point mutation at position 718 in full-length receptors included IR-A P718A, P718E and P718K and IR-B K718A, K718E and K718P.