Signalling and agonist-selective antagonism at corticotropin-releasing factor receptors

抄録

Background. Corticotropin-releasing factor (CRF), urocortin 1, urocortin 2 and urocortin 3 are potentially involved in physiological responses including anxiety stress and depression. These peptides bind two distinct receptors: CRF1 and CRF2. CRF and urocortin 1 bind non-selectively to both CRF1 and CRF2 receptors, whereas urocortin 2 and urocortin 3 are selective for the CRF2 receptor. To understand (patho)physiology and potentially treat anxiety, stress and depression, several CRF receptor antagonists have been developed. However, for many antagonists blockade of CRF has only been examined in binding studies or at a single signalling pathway. This study aimed to address this by investigating signalling and antagonist action at CRF1 and CRF2 receptors.

Methods. Cos7 cells were transiently transfected with human CRF1 or CRF2 receptors. Cells were stimulated with CRF or urocortin 1 and cAMP, IP1 or phosphorylated ERK1/2 measured. The action of CP-376,395, a CRF1 receptor antagonist, was quantified by its ability to antagonise CRF- and urocortin 1-stimulated cAMP and IP1 using global Schild analysis.

Results. CRF and urocortin 1 stimulated cAMP responses, ERK1/2 phosphorylation and IP1 responses at the CRF1 receptor. However, at the CRF2 receptor, although CRF and urocortin 1 activated cAMP responses and ERK1/2 phosphorylation, IP1 responses were only seen with urocortin 1. This suggests that CRF displays apparent bias for cAMP and ERK1/2 phosphorylation over IP1 signalling at the CRF2 receptor. The CRF1 receptor antagonist, CP-376,395 potently antagonised CRF-stimulated cAMP responces at the CRF1 receptor. However, CP-376,395 was approximately 50-fold less potent antagonist of urocortin 1. Interestingly, when IP1 responses were examined CP-376,395 caused a concentration-dependent reduction in maximal signalling.

Conclusions. At the CRF receptors, the agonist signalling profiles and antagonist behaviour was dependent upon which receptor and signalling pathways were examined. Therefore, multiple signalling pathways should be taken into consideration when determining the physiological roles of CRF and urocortin 1. Biased signalling and agonist-selective antagonism should also be considered when developing antagonists that target the CRF1 and CRF2 receptors.