Comparison of an agonist, urocortin, and an antagonist, astressin, as radioligands for characterization of corticotropin-releasing factor receptors

The characteristics of a high-affinity antagonist radioligand are compared with those a high-affinity agonist in binding to the cloned corticotropin-r eleasing factor receptor type 1 (CRF-RI) and type 2 (CRF-R2) and to the nat ive receptors that exist in rat cerebellum and brain stem. The relative pot encies of CRF antagonists and agonists to the two types of cloned CRF recep tors overexpressed stably in Chinese hamster ovary cells are determined usi ng the antagonist radioligand I-125-[DTyr(1)]astressin (Ast*), and the agon ist radioligand, I-125-[Tyr]rat urocortin (Ucn*). The inhibitory binding co nstants (K-i) of astressin and urocortin are 1 to 2 nM for all receptors an d are independent of which radioligand is employed. Astressin binds with hi gh affinity to the native cerebellar/brain stem receptor and relative poten cies of selected CRF analogs determined with Ast* on the native receptor ar e similar to those obtained for the cloned CRF-R1. The specific binding of Ast* to endogenous brain receptors is greater than that of Ucn*, resulting in more sites being detected by the antagonist than by the agonist. In cont rast to another CRF agonist, the binding of Ucn* to the cloned receptors is relatively insensitive to guanyl nucleotides at both 20 degrees C and 37 d egrees C; however, its binding to the native receptor is displaced by guany l nucleotides at 37 degrees C and, to a lesser degree, at 20 degrees C. As expected, the binding of the antagonist Ast* is not affected by guanyl nucl eotides. Because it is a high-affinity, specific CRF antagonist, astressin is eminently suitable as a ligand for detection and characterization of bot h endogenous and cloned CRF receptors.