Abstract
Recently we reported the deletion of Lys-121 in one allele of the insulin receptor gene from a child with severe insulin resistance. In the present work, this mutant receptor (M121) was shown to have an abnormal sensitivity to temperature and an alteration in "negative cooperativity." In contrast to the wild-type receptor (HIRC), insulin binding by the M121 receptor was rapidly and irreversibly lost at temperatures above 30 degrees C with the phosphorylated form of the receptor being more temperature-sensitive than the nonphosphorylated form. Although insulin binding activity was lost, Western analysis and other studies showed that the mutant receptor remained intact. Measurements of 125I-insulin dissociation at 21 degrees C in the presence of native insulin (an estimate of negative cooperativity) demonstrated a difference between the mutant and wild-type receptor. Insulin dissociation from the mutant receptor was not as pronounced as that found with the wild-type receptor. Thus, an abnormality in insulin binding by the mutation was evident at lower "permissive" temperatures. The results of these and other studies argue that Lys-121 occupies an important position for the regulation of insulin receptor conformation. This regulation apparently influences negative cooperative interactions with insulin and modulates signal transduction.
Highlights
To prepare autophosphorylated insulin receptor, 100,...g of Wheat germ agglutinin (WGA) extract was autophosphorylated under conditions described previously [2, 16] and incubated with against phosphotyrosine (a-PY) absorbed to protein G-agarose
The results indicate that each cell line is generally homogeneous for the concentration of insulin receptors, i.e. no significant subpopulation of cells exists which express significantly more or less receptors than the general cell population
The alteration in M121 cell-associated insulin correlated with the reduction in the amount of 125I_insulin internalized, which was 20-30% the amount found in CHOHIRC (Fig. 2A)
Summary
Materials-The CHO cell lines CHO-HIRC and CHO·MI21 have been described in detail previously [16]. Both lines were derived from stable transfections with cytomegalovirus- driven expression plasmids that expressed wild-type (HIRC) insulin receptor or the Lys-121 deletion mutation (MI2l) prepared by site-directed mutagenesis. Following selection by G418, cells were selected by fluorescence-activated cell sorting using an antibody against the a-subunit of the human insulin receptor [2]. An enriched population of cells was obtained which expressed similar amounts of wild-type insulin receptor or mutant receptor as determined by fluorescence-activated cell sorting. The costs of publication of this article were defrayed in part by the payment of page charges.
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