Mechanism of Corticotropin-Releasing Factor Type I Receptor Regulation by Nonpeptide Antagonists

• Published in: Molecular pharmacology 2003-03, Vol.63 (3), p.751-765
• Main Authors: Hoare, Sam R J, Sullivan, Sue K, Ling, Nicholas, Crowe, Paul D, Grigoriadis, Dimitri E
• Format: Article
• Language: English
• Subjects: Amphibian Proteins; Animals; Binding Sites; Corticotropin-Releasing Hormone - pharmacology; Humans; Peptide Fragments - pharmacology; Peptide Hormones; Peptides - pharmacology; Pyrazoles - pharmacology; Rats; Receptors, Corticotropin-Releasing Hormone - antagonists & inhibitors; Receptors, Corticotropin-Releasing Hormone - metabolism; Triazines - pharmacology; Tritium
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.63.3.751

Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF 1 ) receptor regulation by nonpeptide antagonists. The antagonist mechanism was investigated at the G protein-coupled (RG) and uncoupled (R) states of the receptor in membranes from Ltk − cells expressing the cloned human CRF 1 receptor. R was detected with the antagonist 125 I-astressin with 30 μM guanosine 5′- O -(3-thiotriphosphate present, and RG detected using 125 I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited 125 I-astressin binding (22–32% maximal inhibition). NBI 35965 accelerated 125 I-astressin dissociation and only partially increased the IC 50 value of unlabeled sauvagine, CRF, and urocortin for displacing 125 I-astressin binding (by 4.0–7.1-fold). Reciprocal effects at the R state were demonstrated using [ 3 H]NBI 35965: agonist peptides only partially inhibited binding (by 13–40%) and accelerated [ 3 H]NBI 35965 dissociation. These data are quantitatively consistent with nonpeptide antagonist and peptide ligand binding spatially distinct sites, with mutual, weak negative cooperativity (allosteric inhibition) between their binding. At the RG state the compounds near fully inhibited 125 I-sauvagine binding at low radioligand concentrations (79–94 pM). NBI 35965 did not completely inhibit 125 I-sauvagine binding at high radioligand concentrations (82 ± 1%, 1.3–2.1 nM) and slowed dissociation of 125 I-sauvagine and 125 I-CRF. The antagonist effect at RG is consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites. These findings demonstrate a novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG. Strong inhibition at RG explains the antagonist properties of the compounds.