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 |
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Authors: | C L Wood M S O'Dorisio L M Vassalo W B Malarkey T M O'Dorisio |
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Affiliation: | 1. Department of Medicine, The Ohio State University College of Medicine, Columbus, OH, U.S.A.;2. Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, U.S.A. |
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Abstract: | 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. |
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Keywords: | DSS disuccinimidyl suberate EGS ethylene glycol bis(succinimidyl succinate) GHRF human pancreatic (1–44) growth hormone releasing factor stimulatory guanine nucleotide binding protein HBSS Hank's balanced salt solution HSAB PHI peptide histidine isoleucine RPMI 1640 Roswell Park Memorial Institute Medium 1640 SANAH SBSS Seligman's balanced salt solution vasoactive intestinal peptide adenylate cyclase affinity binding affinity labeling |
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