Receptor binding and tyrosine kinase activation by insulin analogues with extreme affinities studied in human hepatoma HepG2 cells

• Published in: Diabetes (New York, N.Y.) Vol. 40; no. 11; pp. 1488 - 1495
• Main Authors: Drejer, K., Kruse, V., Larsen, U. D., Hougaard, P., Bjorn, S., Gammeltoft, S.
• Format: Journal Article
• Language: English
• Published: American Diabetes Association 01-11-1991
• ISSN: 0012-1797; 1939-327X; 0012-1797
• DOI: 10.2337/diabetes.40.11.1488

Receptor binding and tyrosine kinase activation by insulin analogues with extreme affinities studied in human hepatoma HepG2
cells.
K Drejer ,
V Kruse ,
U D Larsen ,
P Hougaard ,
S Bjørn and
S Gammeltoft
Novo Research Institute, Bagsvaerd, Denmark.
Abstract
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 AspB28 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 [125I]TyrA14-labeled insulin and unlabeled analogues
and by kinetic studies with [125I]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 less than 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.