Kinetic Characterization of an Intestinal Trefoil Factor Receptor

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), B(max) 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. B(max) 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 IC(50) 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.