Fluorescence Correlation Spectroscopic Examination of Insulin and IGF1 Binding to Receptors on 2H3 Rat Basophilic Leukemia Cells

Abstract

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are tyrosine kinase receptors with significant structural similarity. Additionally, both IR and IGF1R have pleotropic but differential effects on cell growth and energy metabolism. The interactions of insulin and insulin-like growth factor (IGF1) with IR and IGF1R are well-characterized in vitro. Each ligand binds both classes of receptors with different apparent affinities. However, the kinetics of binding of these ligands at physiological concentrations in vivo remain elusive. We have examined, using fluorescence correlation spectroscopy, the binding of insulin as well as IGF1 to both IR and IGF1R on individual live 2H3 rat basophilic leukemia (RBL-2H3) cells. Ligand saturation experiments with fluorescein isothiocyanate-conjugated insulin (FITC-insulin) indicated the presence of two classes of low-capacity binding sites with surface densities of 49 sites/µm2 and 110 sites/µm2 and KD's of 106pM and 75nM, respectively. Pre-labeling of cells with IGF1 showed that 81 sites/µm2 of the FITC-insulin binding sites bind IGF1 with KD of 82pM. Competitive binding experiments indicated that FITC-insulin and insulin bind with similar KD of 106pM and 181pM and that IGF1 binds high insulin-affinity receptors with KD of 22nM. These experiments also indicate that the relative affinities of insulin and IGF1 for these receptors remained constant when probed with 100pM to10nM FITC-insulin. Finally, we evaluated the off-rates (Koff) of IGF1 and FITC-insulin as 0.013min−1 and 0.016min−1 respectively. The on-rates Kon of insulin and IGF1 for both IR and IGF1R were then estimated using values determined for KD and Koff. This project was supported in part by the NIH (RR023156), NSF (CHE-0628260) and American Heart Association (AHA0650081Z).