Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (ICF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K-D of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K-D of 0.14 nM and for the high-affinity insulin binding sites with K-D of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min(-1) and 0.013 min(-1), respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells. (C) 2011 Elsevier B.V. All rights reserved.