Abstract
ObjectiveTo determine whether intestinal epithelial cells have a receptor for intestinal trefoil factor and characterize receptor-ligand binding kinetics.MethodsRadioligand binding assays were performed to characterize the binding kinetics between [125I]-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.ResultsRadioligand binding assays performed at 4°C showed that the K d 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 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±0.15×108 sites/cell, the very low specific binding between [125I]-ITF and these cells made it impossible to calculate binding kinetic parameters.ConclusionsAn 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.
Highlights
Intestinal trefoil factor (ITF) is a small peptide secreted by intestinal goblet cells that forms a stable gel complex via the association between specific sites in its spatial structure and polysaccharides in mucin to stabilize the intestinal mucus layer [1,2,3]
Much is known about ITF, few studies have focused on ITF receptors and there remains controversy regarding the existence of ITF-specific receptors
Caco2 cells increased with increasing concentrations of [125I]-ITF, whereas non-specific binding (NSB) was observed at low levels and was not dependent on the concentration of [125I]-ITF
Summary
Intestinal trefoil factor (ITF) is a small peptide secreted by intestinal goblet cells that forms a stable gel complex via the association between specific sites in its spatial structure and polysaccharides in mucin to stabilize the intestinal mucus layer [1,2,3]. ITF plays an important role in intestinal self-defense by mitigating gastrointestinal damage that may be caused by a variety of factors [6,7]. Upon stimulation by ITF, EGFR (Epidermal Growth Factor Receptor) is activated and receptor tyrosine protein kinase activity is enhanced; this led to the suggestion that ITF transmits extracellular signals through EGFR to promote cell proliferation and migration [8]. Podolsky claimed the isolation of a specific ITF receptor on the plasma membrane of IEC-6 cells in a U.S patent application, but showed no evidence that this protein is an ITFR [13]. Kalabis et al reported that Vangl plays an important role in the regulation of cell polarity development as an ITF downstream substrate [15], but there is no conclusive evidence to prove Vangl 1 that it is an ITF receptor
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