Abstract

Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF₁) 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 CRF₁ receptor. R was detected with the antagonist¹²⁵I-astressin with 30 μM guanosine 5′-O-(3-thiotriphosphate present, and RG detected using ¹²⁵I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited ¹²⁵I-astressin binding (22–32% maximal inhibition). NBI 35965 accelerated ¹²⁵I-astressin dissociation and only partially increased the IC₅₀ value of unlabeled sauvagine, CRF, and urocortin for displacing¹²⁵I-astressin binding (by 4.0–7.1-fold). Reciprocal effects at the R state were demonstrated using [³H]NBI 35965: agonist peptides only partially inhibited binding (by 13–40%) and accelerated [³H]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 ¹²⁵I-sauvagine binding at low radioligand concentrations (79–94 pM). NBI 35965 did not completely inhibit ¹²⁵I-sauvagine binding at high radioligand concentrations (82 ± 1%, 1.3–2.1 nM) and slowed dissociation of ¹²⁵I-sauvagine and ¹²⁵I-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.