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 [(125)I]-labeled ITF and IEC-6, HT-29, Caco2 and HaCaT cells. The K d, 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 K d value for the association between [(125)I]-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 × 10(11), 3.97 ± 0.29 × 10(11), and 2.03 ± 0.08 × 10(11) sites/cell, respectively. The K i 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 K d was 4.86 ± 0.28 × 10(-8) M and B max was 5.81 ± 0.15 × 10(8) sites/cell, the very low specific binding between [(125)I]-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.