Received for publication October 29, 2007.
Revised January 27, 2008.
Accepted for publication January 28, 2008.

Allosteric ligands for the corticotropin releasing factor type 1 receptor modulate conformational states involved in receptor activation

Abstract

Allosteric modulators of G-protein-coupled receptors can regulate conformational states involved in receptor activation (Hall DA (2000) Modeling the functional effects of allosteric modulators at pharmacological receptors: an extension of the two-state model of receptor activation. Mol Pharmacol 58:1412-1423). This hypothesis was investigated for the corticotropin-releasing factor type 1 (CRF₁) receptor, using a novel series of ligands with varying allosteric effect on CRF binding (inhibition to enhancement). For the G-protein-uncoupled receptor, allosteric modulation of CRF binding was correlated with nonpeptide ligand signaling activity; inverse agonists inhibited and agonists enhanced CRF binding. These data were quantitatively consistent with a two-state equilibrium underlying the modulation of CRF binding to the G-protein-uncoupled receptor. We next investigated the allosteric effect on CRF-stimulated G-protein coupling. Ligands inhibited CRF-stimulated cAMP accumulation regardless of their effect on the G-protein-uncoupled state. The modulators reduced CRF E_max, suggesting they reduced the efficacy of a CRF-bound active state to couple to G-protein. Consistent with this hypothesis, the modulators inhibited binding to a guanine nucleotide-sensitive state. Collectively the results are quantitatively consistent with a model in which: 1) The receptor exists in three predominant states, an inactive state, a weakly-active state and a CRF-bound fully-active state. 2) Allosteric inverse agonists stabilize the inactive state and allosteric agonists stabilize the weakly-active state. 3) Antagonism of CRF signaling results from destabilization of the fully-active state. These findings imply nonpeptide ligands differentially modulate conformational states involved in CRF₁ receptor activation and suggest different conformational states can be targeted in designing nonpeptide ligands to inhibit CRF signaling.

Key words: Neuropeptides, CRF, Receptor binding studies, Peptide hormones