Kinetic Characterization of an Intestinal Trefoil Factor Receptor

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.

Trefoil Factor-3 (TFF3) Stimulates De Novo Angiogenesis in Mammary Carcinoma both Directly and Indirectly via IL-8/CXCR2

“…The functional receptor for TFF3 remains to be identified, albeit much effort has been expended to identify potential binding partners of TFF3. Numerous studies have evident that TFF3 transmits extracellular signals and promotes cell proliferation and migration through EGF-R activation [ 45 , 72 , 73 ] but is likely to be an indirect activation of the EGF-R to stimulate the Ras/MEK/MAPK signaling pathway [ 45 , 74 ] and not that the EGF-R is the specific TFF3 receptor [ 75 ]. TFF3 has also been reported to bind to specific cell surface proteins and stimulate its functional effects independent of the EGF-R signaling pathway [ 49 ].…”

TFF3 is a ligand for LINGO2 that de-represses EGFR to control disease outcome during colitis and gastrointestinal nematode infection

“…Thus, our data demonstrating that TFF3 functions through a previously unrecognized ligandreceptor interaction with LINGO2 to de-repress LINGO2-dependent inhibition of EGFR activation provides a novel conceptual framework explaining how TFF3-mediates mucosal wound healing through enhanced activation of the EGFR pathway. 3 Although Trefoil factor 3 (TFF3) is well known to drive reparative pathways at respiratory, ocular, genitourinary and gastrointestinal mucosa, the identity of a potential TFF3 receptor has remained elusive for several decades [4][5][6][7][8] . In addition to enhancing mucous viscosity 9 , TFF3 induces epithelial cell survival and proliferation, activates EGFR, β-catenin, and STAT3 signaling pathways, and controls tight junction protein expression through ill-defined mechanisms to protect against gastrointestinal (GI) tissue injury and inflammation 3,10 .…”

“…It is likely that this TFF3-LINGO2-EGFR axis has distinct roles in hematopoietic vs. non-hematopoietic cell lineages and may be pertinent to other disease states involving autoimmunity and tumorigenesis. 8…”

LINGO3 interacts with Trefoil factor 2 to enforce mucosal barrier integrity and drive tissue repair during colitis

“…Whether TFFs (TFF1-3) require receptor-mediated signal transduction events or act indirectly through modifying the rheological properties of mucus is an unsolved matter (6, 11, 12). In support of the receptor hypothesis, CXCR4, (the stromal derived factor 1 (SDF-1) receptor), also responds to TFF2 at high concentrations (13).…”