Kinetic characterization of an intestinal trefoil factor receptor
Zhang Yong1, Wang Lin, Sun Yong
1State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burns of PLA, Southwest Hospital, Third Military Medical University, Chongqing, PR China.
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.
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