Characterization of a Putative Receptor for Intestinal Trefoil Factor in Rat Small Intestine: Identification byin SituBinding and Ligand Blotting

Intestinal trefoil factor binds to intestinal epithelial cells and induces nitric oxide production: priming and enhancing effects of mucin

“…Previous observations suggest the presence of ITF-BP on the intestinal epithelial cells [24]. Our recent study further identified this ITF-BP in the crypt cells of rat small intestine [22], and characterized it as a 50 kDa glycosylated membrane protein [22]. The present study demonstrated (1) that the IEC-18 cell expresses the 50 kDa ITF-BP and (2) that the effect of mucin in regulating ITF binding to intestinal epithelial cells could be blocked by inhibitors of transcription and translation, thereby suggesting that the ITF-binding molecule on IEC cells is a protein, most probably ITF-BP.…”

“…Solubilized membrane protein (40 µg) from IEC-18 cells, prepared as described previously [22], was suspended in an equal volume of 2iLaemmli buffer containing 2 % (v\v) 2-mercaptoethanol, then resolved by SDS\PAGE [10 % (w\v) gel] and transferred to a nitrocellulose membrane. After being blocked with PBS containing 5 % (v\v) skimmed milk and bacterial lysates (which expressed Tag protein), the membrane was reacted overnight with PBS containing bTag-ITF peptide (3.4 µg\ml) at 4 mC.…”

“…Initially we examined whether ITF-binding molecules on IEC-18 cell surfaces could be characterized by flow cytometry with bITF, a biotinylated probe [22]. As shown in Figure 4, the histogram of IEC-18 cells incubated with a bITF probe then reacted with PEconjugated goat anti-biotin polyclonal antibody (broken line) shifted to the right, as compared to with that of cells incubated with the antibody only (solid line).…”

“…Previous studies showed that : (1) specific ITF-binding sites exist in the gastric epithelium, crypt cells of small intestine and colon [22][23][24] ; (2) ITF-binding protein (ITF-BP) is a 50 kDa glycosylated intestinal membrane protein [22] ; (3) stimulation by ITF for 24 h (at concentrations as low as 1 nM) leads to cell detachment and to a decreased intracellular adhesion in human carcinoma cells [25] ; (4) ITF (100 nM) leads to the inhibition of extracellular signal-related protein kinase and the mitogenactivated protein kinase pathways in IEC-6 cells through the activation of tyrosine or dual-specific phosphatase [2] ; (5) ITF (10 nM) modulates EGF effects on intestinal epithelial ion transport [26] ; and (6) ITF effect is mediated by signal transduction pathways linked to the protein tyrosine kinase\tyrosine phosphatase pathway [2,18,25]. These observations strongly suggest that a receptor-like membrane protein might mediate the action of ITF.…”

“…Furthermore, we localized ITF-binding molecules on the epithelial cell surface, examined whether mucin regulates ITF-binding molecules on the epithelial cell surface and studied the mechanism of ITF effect on epithelial NO production. and biotinylated Tag control peptide (bTag) were prepared as described previously [22]. To avoid contamination of endotoxin, the rITF was further purified by chromatography through a detoxin gel (Pierce Chemical Co., Rockford, IL, U.S.A.).…”