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 (IGF1) 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 KD of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with KD of 0.14 nM and for the high-affinity insulin binding sites with KD 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.

Fluorescence correlation spectroscopy reveals details of ligand binding to insulin receptors and IGF1 receptors on single cells. (A) Equilibrium binding of FITC-insulin to the surface of live cells demonstrates two distinct classes of ligand binding sites. (B) Complete dissociation of FITC-insulin from ligand binding sites on live cells occurs over approximately two hours.Highlights► FCS permits examination of interactions between biological molecules on individual live cells. ► 2H3-RBL cells express IR and IGF1R homodimers, as well as hybrid IR-IGF1R heterodimers. ► Insulin and IGF1 compete with differing affinities for available binding sites on these cells. ► Hybrid IR-IGF1R heterodimer formation is likely due to random-association of IR and IGF1R monomers.