Binding specificity and intramolecular signal transmission of uncleaved insulin proreceptor in transformed lymphocytes from a patient with extreme insulin resistance

Abstract

An alteration of an amino acid sequence in the processing site of the insulin proreceptor by a point mutation of the insulin receptor gene produced extreme insulin resistance. We characterized functional properties of the unprocessed insulin receptor in transformed lymphocytes from a patient. Insulin binding to intact cells and to a partially purified insulin receptor preparation was radically decreased to 20% and 18% of the control values, respectively. In competitive insulin binding to intact cells, [LeuA3]-, [LeuB24]-, [SerB24]-insulin, and mini-proinsulin ([ B(1-29)-Ala-Ala-Lys-A(1-21)]-insulin) had the same relative binding activity in both the patient's and the control cells, but proinsulin and IGF-I were markedly less able to displace 125I-insulin in the patient's cells. In contrast to the study in intact cells, proinsulin and IGF-I as well as other insulin analogues had the same relative binding activity to bind to the partially lectin-purified insulin receptor preparations from both the patient's and the control cells. As regards the signal transduction after receptor binding, insulin-stimulated autophosphorylation of the unprocessed insulin proreceptor occurred proportionally to the amount of decreased insulin binding. With 0.025% trypsin treatment, the abnormal binding characteristics and autophosphorylation were normalized through conversion to functionally normal receptors. In spite of the abnormal processing, self-association of receptors into oligomeric structures was observed in the proreceptor. These results suggest that the unprocessed insulin proreceptor in the plasma membranes has an altered conformation which affects its binding characteristics but not its intramolecular signal transmission.

Long-term follow up in type A insulin resistant syndrome treated by insulin-like growth factor I.

“…The method for the 125I-insulin binding to erythrocytes, transformed lymphocytes, and cultured fibroblasts has been described by Sasaoka et al 5 6 Surface labelling of Epstein-Barr virus transformed lymphocytes was determined by the method described previously. 7 We performed subsequent DNA analysis of insulin receptor gene by a modification of the method described by Kusukawa et al8 and examined transfection of insulin receptor c-DNA to COS7 cells according to the method of Kobayashi et al 9 Recombinant human IGF-I was provided by the Fujisawa pharmaceutical industry (Osaka, Japan).…”

A mutation (Trp1193?Leu1193) in the tyrosine kinase domain of the insulin receptor associated with type A syndrome of insulin resistance

“…Insulin binding to transformed lymphocytes. The method of 125I-insulin binding to transformed lymphocytes has been described elsewhere [5]. The binding study was carried out at 15 ~ for 3.5 h.…”

“…Preparation of partially purified insulin receptor. The method of purifying insulin receptors, using WGA agarose column has been described previously [5].…”

“…Autophosphorylation of insulin receptor. The lectin-purified extracts were preincubated with various concentrations of insulin at 4 ~ for 16 h. The phosphorylation was initiated by adding a solution composed of 20 mmol/1 Mn acetate, 5 mmol/1 CTP, 20 gmol/1 ATP, and 30 gCi [y_32p] ATR After incubation at 4 ~ for 10 min, the reaction was terminated by adding stopping solution containing 0.2 % Triton X-100, 10 mmol/1 EDTA, 100 mmol/1 NaF, 20 mmol/l sodium pyrophosphate, 20 mmol/1 ATP, and 20 mmol/1 Hepes (pH 7.6) as previously described [5].…”

Loss of Tumorigenicity and Metastatic Potential in Carcinoma Cells Expressing the Extracellular Domain of the Type 1 Insulin-Like Growth Factor Receptor

“…Interestingly, it was recently shown that the expression of a deletion mutant of IGF-IR lacking the 36 amino acid residues immediately NH 2 -terminal to the transmembrane domain of IGF-IR (IGF-IR ⌬870 -905 ) in BALB/c 3T3 cells resulted in the intracellular accumulation of a high-molecular-mass (ϳ200 kDa) proreceptor (13). Another study showed that the insulin proreceptor can bind insulin (14); it is therefore possible that the structurally similar IGF-IR proreceptors can also retain a ligandbinding capacity.…”