RECEPTOR-BINDING AND TYROSINE KINASE ACTIVATION BY INSULIN ANALOGS WITH EXTREME AFFINITIES STUDIED IN HUMAN HEPATOMA HEPG2 CELLS

The insulin-receptor affinity of five human insulin analogues with one to four amino acid substitutions was measured with human hepatoma cells (HepG2). The binding affinities ranged from 0.05% for AspB25 insulin, 18% for AspB9,GluB27 insulin, 80% for AspB26 insulin, and 327% for AspB10 insulin to 687% for HisA8,HisB4,GluB10,HisB27 insulin relative to human insulin. Binding constants obtained by competition experiments at steady state with [I-125]TyrA14-labeled insulin and unlabeled analogues and by kinetic studies with [I-125]TyrA14-labeled analogues and insulin gave essentially the same values. The kinetic studies showed that differences in affinity between analogues were due to differences in both dissociation and association rate constants. The affinity for insulinlike growth factor I receptor was low, ranging from < 0.005% for AspB25 insulin to 0.6% for HisA8,HisB4,GluB10,HisB27 insulin. The potencies of insulin analogues in activation of the tyrosine kinase of solubilized and partially purified insulin receptors from HepG2 cells, measured with the exogenous substrate poly(Glu80-Tyr20), ranked in the same order as the binding affinities, the actual values being somewhat elevated for the high-affinity analogues, however. We conclude that these human insulin analogues are active in insulin-receptor binding and tyrosine kinase stimulation but show wide variation in affinity.