Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells.
The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.
el Battari
A
A
Institut de Chimie Biologique, Centre National de la Recherche Scientifique Unité Associée n.202, Université de Provence, Marseille, France.
Martin
J M
JM
Luis
J
J
Pouzol
O
O
Secchi
J
J
Marvaldi
J
J
Pichon
J
J
D000230:Adenocarcinoma / Q000378:metabolism*; D002460:Cell Line; D003110:Colonic Neoplasms / Q000378:metabolism*; D003902:Detergents; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification* / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide; D012995:Solubility; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D003902:Detergents; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide