Insulin receptor and insulin sensitivity in a chicken hepatoma cell line

Abstract

Insulin receptors have been characterized in a cell line recently isolated from a chicken hepatoma (LMH). The binding of 125I-insulin to LMH cells or membranes displayed the expected criteria for insulin receptors: affinity, temperature dependency, curvilinearity of Scatchard plot, rank order of potency for insulin analogs and insulin induced down-regulation. The α-subunit of LMH cell insulin receptors exhibited a normal size of 135 kDa. Following autophosphorylation, LMH WGA-purified receptors revealed a 95 kDa β-subunit and a 72 kDa protein (pp72). Both proteins were phosphorylated in a time-, insulin- (and insulin-like growth factor 1; IGF-1) and manganese-dependent manner, and were precipitated by antiphosphotyrosine and two anti-insulin receptor antibodies. The 72 kDa protein was not present under non-reducing condition PAGE or in normal chicken liver. These results strongly suggest that pp72 is either a truncated form of the insulin receptor β-subunit specific to LMH cells or a degradation product. Lectin-purified insulin receptors from LMH cells or chicken liver membranes exhibited similar tyrosine kinase activity, using artificial substrate poly(Glu-Tyr) 4:1. Finally, amino acid uptake by LMH cells was insulin stimulatable.