Abstract
The characteristics of a high-affinity antagonist radioligand are compared with those a high-affinity agonist in binding to the cloned corticotropin-releasing factor receptor type 1 (CRF-R1) and type 2 (CRF-R2) and to the native receptors that exist in rat cerebellum and brain stem. The relative potencies of CRF antagonists and agonists to the two types of cloned CRF receptors overexpressed stably in Chinese hamster ovary cells are determined using the antagonist radioligand 125I- [DTyr1]astressin (Ast*), and the agonist radioligand, 125I -[Tyr0]rat urocortin (Ucn*). The inhibitory binding constants (Ki) of astressin and urocortin are 1 to 2 nM for all receptors and are independent of which radioligand is employed. Astressin binds with high affinity to the native cerebellar/brain stem receptor and relative potencies of selected CRF analogs determined with Ast* on the native receptor are 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 contrast to another CRF agonist, the binding of Ucn* to the cloned receptors is relatively insensitive to guanyl nucleotides at both 20°C and 37°C; however, its binding to the native receptor is displaced by guanyl nucleotides at 37°C and, to a lesser degree, at 20°C. As expected, the binding of the antagonist Ast* is not affected by guanyl nucleotides. Because it is a high-affinity, specific CRF antagonist, astressin is eminently suitable as a ligand for detection and characterization of both endogenous and cloned CRF receptors.
Footnotes
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Send reprint requests to: Marilyn Perrin, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Rd., La Jolla, CA 92037. E-mail:perrin{at}salk.edu
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↵1 This work was supported by National Institutes of Health Grant DK-26741 and in part by The Foundation for Research (to M.P. and W.V.). W.V. is a Foundation for Research Senior Investigator.
- Abbreviations:
- CRF
- corticotropin-releasing factor
- CRF-R1
- corticotropin-releasing factor receptor type 1
- CRF-R2α
- corticotropin-releasing factor receptor type 2α
- CRF-R2β
- corticotropin-releasing factor receptor type 2β
- G protein
- GTP-binding proteins
- CHO
- Chinese hamster ovary
- HPLC
- high-performance liquid chromatography
- Ucn
- urocortin
- Ast
- astressin
- Received July 9, 1998.
- Accepted August 28, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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