The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29-D4). Evidence for its glycoprotein nature.
We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.
D000118:Acetylglucosaminidase; D000230:Adenocarcinoma / Q000032:analysis; D002352:Carrier Proteins / Q000032:analysis; D002460:Cell Line; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D012794:Sialic Acids / Q000032:analysis; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D002352:Carrier Proteins; D006023:Glycoproteins; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D012794:Sialic Acids; D014660:Vasoactive Intestinal Peptide; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase