guanosine-triphosphate and dithiobis(succinimidylpropionate)

High-performance liquid chromatography-purified 125I-vasoactive intestinal peptide (VIP) bound to T-47D human breast cancer cells in a specific, saturable, and reversible manner. Scatchard plots were compatible with the presence of one class of VIP receptors with high affinity (Kd = 4.5 X 10(-10) M VIP, and Bmax = 293 fmol/mg protein). The neuropeptide and its natural analogues inhibited the binding of 125I-VIP and stimulated cyclic AMP (cAMP) generation in T-47D cells 96-fold (EC50 = 7 X 10(-10) M VIP), in the following order of potency: VIP greater than helodermin greater than human peptide with N-terminal histidine and C-terminal methionine greater than human pancreatic growth hormone-releasing factor greater than human secretin. In contrast, 125I-VIP binding was not displaced by pancreatic glucagon, human oxyntomodulin, truncated glucagon-like peptide-1, glucagon-like peptide-2, the somatostatin analogue SMS 201-995, gastric inhibitory peptide, and a series of steroid hormones or peptides unrelated to VIP. VIP also increased cAMP generation in seven other human breast cancer cell lines: H4-66B, HSL 53, HSL 78, MCF 7, MDA-MB231, T-47D2, and ZR75-1. Adenylate cyclase activity rose from 72.2 +/- 14 to 1069 +/- 66 pmol cAMP/min mg protein after the addition of 10(-7) M VIP to T-47D plasma membranes. In agreement with our pharmacological results and the Scatchard analysis of the binding data, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized receptor in the T-47D membranes permitted identification of one autoradiographic band with a molecular weight of 69,000. The sensitivity of the Mr 69,000 binding site to GTP and low doses of VIP implies that in T-47D cells, this component constitutes the membrane domain involved in the functional regulation of adenylate cyclase by VIP receptors. Our results indicate a role for the VIP receptor-cAMP system in human breast cancer cells.

Topics: Adenylyl Cyclases; Breast Neoplasms; Cell Line; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Female; Guanosine Triphosphate; Humans; Molecular Weight; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Succinimides; Vasoactive Intestinal Peptide

The structure of the secretin receptor in purified plasma membranes isolated from the antral and fundic parts of the rat gastric mucosa was probed, using the cross linking reagent dithiobis succinimidyl propionate (DSP) and HPLC-purified [125I] secretin. [125I] secretin binding sites were preferentially located in rat antrum and displayed the pharmacological properties expected for specific secretin receptors: secretin greater than helodermin greater than rhGRF greater than rPHI. SDS gel electrophoresis of the solubilized receptor allowed identification of two radiolabeled peptides of 62 and 33 KDa connected by disulfide bonds. According to the sensitivity of the 62 KDa component to low doses of secretin and to GTP, it constitutes the membrane domain involved in the physiological regulation of adenylate cyclase by secretin in rat gastric glands.

Topics: Animals; Carrier Proteins; Gastric Mucosa; Guanosine Triphosphate; In Vitro Techniques; Iodine Radioisotopes; Male; Molecular Weight; Rats; Rats, Inbred Strains; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Secretin; Succinimides

To characterize the molecular components of the vasoactive intestinal peptide (VIP) receptor in human intestine, [125I]VIP was covalently bound to human colonic epithelial membranes using dithio-bis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel autoradiographic studies of affinity labeled membranes revealed three major bands corresponding to [125I]VIP-protein complexes of 66,000, 33,000, and 16,000 mol wt. Labeling of the 66,000 and 33,000 mol wt complexes was specific, since it was abolished by VIP, while labeling of the 16,000 mol wt complex was not. Densitometric scanning of autoradiographs indicated that labeling of the 66,000 mol wt complex was inhibited by low VIP concentrations in the 10(-10)-10(-8) M range, but was unaffected by glucagon or octa-cholecystokinin. It was also reduced by VIP-(2-28), with a potency 1/100th that of VIP, and by GTP in the concentration range of 10(-7)-10(-3) M. The 33,000 mol wt complex behaved similarly to the 66,000 mol wt complex with respect to specificity and GTP sensitivity, but differed in one major feature, its affinity for VIP. Its labeling was inhibited by native VIP concentrations in the 10(-9)-10(-7) M range. Assuming one molecule of [125I]VIP bound per molecule of protein, two proteins of 63,000 and 30,000 mol wt were identified as VIP-binding sites. The 63,000 mol wt protein had the properties expected for the VIP receptor coupled to adenylate cyclase in human colon, while the 30,000 mol wt protein was a low affinity binding site. Treatment of human colonic membranes with the sulfhydryl reducing agent dithiothreitol before [125I]VIP binding strongly reduced the labeling of the two proteins. This finding does not support the hypothesis that the low affinity 30,000 mol wt binding site may be a monomer of the high affinity binding site.

Topics: Animals; Cell Membrane; Colon; Cross-Linking Reagents; Dithiothreitol; Epithelium; Guanosine Triphosphate; Humans; Molecular Weight; Rats; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Succinimides; Vasoactive Intestinal Peptide