Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation: comparison with peptide histidine isoleucine and growth hormone-releasing factor

• Published in: Regulatory peptides Vol. 12; no. 3; pp. 237 - 248
• Main Authors: WOOD, C. L, O'DORISIO, M. S, VASSALO, L. M, MALARKEY, W. B, O'DORISIO, T. M
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier 07.11.1985; Amsterdam
• Subjects: Adenylyl Cyclases - metabolism; Animal tumors. Experimental tumors; Animals; Binding, Competitive; Biological and medical sciences; Cyclic AMP - metabolism; Enzyme Activation - drug effects; Experimental endocrine tumors; Growth Hormone-Releasing Hormone - pharmacology; Medical sciences; Peptide PHI; Peptides - pharmacology; Pituitary Gland - metabolism; Rats; Receptors, Cell Surface - metabolism; Receptors, Vasoactive Intestinal Peptide; Tumors; Vasoactive Intestinal Peptide - metabolism; Vasoactive Intestinal Peptide - pharmacology; Rat; Rodentia; Peptide hormone; Adenylate cyclase; Vertebrata; Mammalia; Fixation; Established cell line; Pituitary gland; Experimental tumor; VIP; Peptide PHI; STH-RH
• ISSN: 0167-0115; 1873-1686
• DOI: 10.1016/0167-0115(85)90065-5

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.