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.