Binding kinetics of mutated insulin receptors in transfected cells grown in suspension culture: Application to the Tyr → Phe 960 insulin receptor mutant

Abstract

Site-directed mutagenesis of the insulin receptor cDNA is now widely used to elucidate the role of various domains and residues of the receptor, particularly in order to examine the functional importance of the β chain - associated tyrosine kinase. However, little has been done to correlate the functional repercussions of such mutations with alterations in the complex insulin binding kinetics. This is due in part to the difficulty of conducting large scale experiments using transfected cells on culture dishes. In an effort to overcome this problem, we have developed a method for culturing Chinese hamster ovary (CHO) cells in suspension culture, which provides a large number of cells and obviates the need for enzymatic or mechanical detachment of cells. The feasibility of this approach is demonstrated in a detailed study of the kinetics of insulin binding to the Tyr → Phe 960 insulin receptor mutant.