Relationship of insulin binding to amino acid transport by cultured 14-day embryonic chick heart cells

Abstract

The characteristics of insulin receptors were studied in cultured embryonic chick heart cells which demonstrated insulin-responsive amino acid transport. Binding of [125I]iodoinsulin was time dependent, reversible, saturable, species specific, and proportional to cell number. Optimum binding occurred at pH 7.8 in the presence of 0.1% bovine serum albumin. Curvilinear Scatchard plots were found for chicken and bovine insulin binding at 15 and 30 C. Equilibrium association constants (Kas) and maximum capacities were calculated based on a two-receptor model. When studied at 15 C, chicken insulin was bound with Kas of 5.0 and 0.026 nM-1 for the high and low affinity receptors, respectively. Bovine insulin bound with Kas of 2.1 and 0.03 nM-1. The binding capacities of 600 and 9000 molecules/cell for the high and low affinity receptors, respectively, were the same for both species of ligand. At 30 C, the Ka of the high affinity chicken insulin receptor interaction decreased to 1.6 nM-1, whereas the low affinity Ka was not changed. In competitive binding assays, chicken insulin was 4 and 250 times more potent than bovine and guinea pig insulin, respectively. Human GH and mouse epidermal growth factor did not compete with chicken insulin. Although mouse epidermal growth factor did not compete with insulin, it did stimulate 2-aminoisobutyric acid accumulation. The maximal stimulation by this hormone was less than and additive to that produced by the maximal stimulation of insulin. These data indicate that insulin and epidermal growth factor acted through different receptors to stimulate amino acid transport. When insulin-stimulated functional response was compared to calculated receptor occupancy in this two-receptor model, stimulation of amino acid transport paralleled occupancy of the low affinity receptor such that at half-maximal transport stimulation, approximately 50% of these receptors were occupied. Half-maximal stimulation of 2-aminoisobutyric acid transport occurred at 18- and 20-nM concentrations of bovine and chicken insulins, respectively. Guinea pig insulin failed to stimulate 2-aminoisobutyric acid uptake even at 350 nM. Both the affinity of binding in the low affinity class and the insulin concentration for half-maximal stimulation of 2-aminoisobutyric acid transport were independent of temperature in the temperature ranges studied. We conclude that 14-day embryonic chick heart cells possess at least two classes of receptors which bind insulin. Occupancy of the lower affinity class of insulin receptors correlates quantitatively with insulin stimulation of 2-aminoisobutyric acid transport.