Pharmacology, molecular identification and functional characteristics of vasoactive intestinal peptide receptors in human breast cancer cells.
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.
Gespach
C
C
INSERM U. 55, Unité de recherches sur les neuropeptides digestifs et le diabète, Hôpital Saint-Antoine, Paris, France.
Bawab
W
W
de Cremoux
P
P
Calvo
F
F
D000262:Adenylyl Cyclases / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D003432:Cross-Linking Reagents; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides; D014660:Vasoactive Intestinal Peptide; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C011240:dithiobis(succinimidylpropionate)