title: 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.
abstract: 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.
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D004848:Epithelium / Q000378:metabolism; D007082:Ileum / Q000378:metabolism*; D007583:Jejunum / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D000262:Adenylyl Cyclases