Kinetic Characterization of an Intestinal Trefoil Factor Receptor

Abstract

Objective:To determine whether intestinal epithelial cells have a receptor for intestinal trefoil factor and characterize receptor-ligand binding kinetics.Methods:Radioligand binding assays were performed to characterize the binding kinetics between [125I]-labeled ITF and IEC-6, HT-29, Caco2 and HaCaT cells. The Kd, Bmax and other kinetic variables describing the interaction between ITF and its potential receptors were determined.Results:Radioligand binding assays performed at 4°C showed that the Kd value for the association between [125I]-ITF and IEC-6, HT-29, and Caco2 cells were 1.99±0.12×10-9 M, 3.89±0.42×10-9 M, and 2.04±0.17×10-9 M, respectively. Bmax values were 1.17±0.04×1011, 3.97±0.29×1011, and 2.03±0.08×1011 sites/cell, respectively. The Ki values for the interaction between IEC-6, HT-29, and Caco2 cells and non-labeled ITF were 20.98±0.57 nM, 36.87±3.35 nM, and 21.38±0.93 nM, respectively, and the IC50 values were 25.21±0.39 nM, 40.68±0.27 nM, and 23.61±0.25 nM, respectively. Radioligand binding kinetic results showed the association rate constants (k+1) for IEC-6, HT-29, and Caco2 cells were 0.22±0.04 min-1, 0.29±0.04 min-1, and 0.26±0.05 min-1, respectively, and the dissociation rate constants (k-1) were 0.06±0.02 min-1, 0.03±0.01 min-1, and 0.04±0.01 min-1, respectively. For the HaCaT cells, the Kd was 4.86±0.28×10-8 M and Bmax was 5.81±0.15×108 sites/cell, the very low specific binding between [125I]-ITF and these cells made it impossible to calculate binding kinetic parameters.Conclusions:An ITF-specific receptor appears to be present on the three types of intestinal epithelial cells (IEC-6, HT-29, and Caco-2), and there may be no ITF receptor on epidermal cells. © 2013 Yong et al.