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Endocrinology, Vol 120, 978-985, Copyright © 1987 by Endocrine Society

ARTICLES

Selective photolabeling of high and low affinity binding sites for vasoactive intestinal peptide (VIP): evidence for two classes of covalent VIP-receptor complexes in intestinal cell membranes

A Robichon and JC Marie

The biological activities of VIP derivatives as photoaffinity labels are described. The derivatives were obtained by VIP modification with azido phenyl glyoxal at arginyl residues 12 and 14 ([Az Bz Arg12- 14]VIP) or only at position 14 ([Az Bz Arg14]VIP). The first derivative exhibited pronounced hydrophobic properties. The level of cAMP produced in HT 29 cells by this derivative represented 15% of that obtained by VIP at equimolar concentration (10(-10) M). The second derivative ([Az Bz Arg14]VIP) was synthesized by a new procedure, in which amino acids of VIP buried in the active site of the receptor were protected from azido phenyl glyoxal. This analog retained a high binding affinity for receptor (Kd, 0.5 nM for [125I-Tyr,Az Bz Arg14]VIP vs. 0.1 nM for [125I] VIP) and was found to be biologically active, as judged by stimulation of adenylate cyclase activity (production of cAMP was 120 pmol/10(6) cells vs. 140 pmol for VIP at 10(-10) M). Photolysis of this analog in the presence of HT 29 cell membranes resulted in a stimulation of adenylate cyclase which persisted in spite of repeated washings. Our findings indicate that [125I-Tyr,Az Bz Arg14] VIP binds covalently to active sites to form an active peptide-receptor complex. When low affinity binding sites were specifically photolabeled using a selective protection of high affinity sites by incubating membranes with 10(-10) M VIP for 5 min, the derivative did not stimulate adenylate cyclase activity. This suggests that VIP acts through a high affinity site to produce the biological activity and that the functional relevance of low affinity sites is unclear.




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Copyright © 1987 by The Endocrine Society