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  • indocid br Acknowledgments This research was supported by gr


    Acknowledgments This research was supported by grants from the Ministerio de Ciencia e Innovación (Grants SAF/FEDER 2010-17907; 2013-49076-P), Spain, Red de trastornos adictivos (RETICS RD12/0028//0003, Instituto de Salud Carlos III, Spain) and Fundación Séneca (15405/PI/10, Región de Murcia, Spain). García-Carmona JA was supported by a predoctoral fellowship from Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia (ref. 15519/FPI/10). Conflict of interest statement
    Introduction Corticotrophin (CRF) is a 41 amino indocid peptide regulating the stress response of the hypothalamic-pituitary-adrenal axis (HPA axis). CRF's actions are mediated by two pharmacologically distinct receptor subtypes, CRF1 and CRF2, which both stimulate adenylate cyclase (Wietfeld et al., 2004). CRF receptors are class B G protein-coupled receptors, a class activated by peptidic agonists and characterized by a large extracellular domain (ECD). The general mechanism for activation of class B G protein-coupled receptors has been termed the two-domain model (Hoare, 2005, Grace et al., 2004). In this model, the carboxyl terminus of CRF first binds to the ECD with moderate affinity which then facilitates binding of the amino terminus of CRF to a second site near the membrane (the juxta-membrane or “J”-site, a region of the receptor composed of the transmembrane domain) which leads to receptor coupling to the G protein (Hoare et al., 2004, Beyermann et al., 2000). Consequently, functional inhibition of CRF's in vivo activity can be potentially achieved by antagonists binding to either the ECD or the J-site. Because a convergence of clinical and rodent behavioral studies have suggested that overstimulation of CRF1 receptors results in an increase in anxiety-like behaviors, there has been considerable effort in the pharmaceutical industry over the last decade to develop orally active antagonists of the CRF1 receptor for the treatment of anxiety and depression (Valdez, 2009, Holsboer and Marcus, 2008). All of these compounds are non-peptidic small molecule antagonists that allosterically antagonize the CRF1 receptor by specifically binding to the J-site with high affinity (). However, the drugability of known J-site antagonists has been limited by their lipophilicity (i.e. high logD), due to the hydrophobicity of the J-site itself. One approach for identifying CRF1 receptor antagonists with more CNS-friendly physical properties is to target the ECD site since it is a more hydrophilic environment. Although at least part of the binding of [125I]Tyr0-CRF (), [125I]Tyr0-sauvagine (Grigoriadis et al., 1996) and [125I]Tyr0-astressin () is to the ECD of the CRF1 receptor, the binding of these ligands is either robustly (agonist radioligands), or partially ([125I]Tyr0-astressin) inhibited by J-site antagonists via negative allosteric interactions (Hoare et al., 2003). Thus, searching for CRF1 receptor ECD-specific antagonists has been confounded by the lack of a high affinity, competitive ECD-specific radioligand. Yamada et al. (2004) reported a biologically stabilized, small C-terminal peptidic CRF analog (analog number 20, which we named Yamada peptide 20 or YP20) that retained high affinity (3.1nM) for the human CRF1 receptor and antagonized CRF inhibition of ACTH secretion in rats. To develop a specific probe of the CRF binding domain on the ECD of the CRF1 receptor, we radiolabeled YP20 with 125I using the Bolton–Hunter reagent (Fig. 1). We describe here the in vitro characterization of this radioligand's binding to membranes expressing the hCRF1 receptor and its potential for screening for small molecule CRFR1 receptor ECD antagonists.
    Materials and methods
    Discussion We confirmed the high affinity of Yamada peptide #20 () at inhibiting [125I]CRF binding to the hCRF1R and demonstrated that this peptide potently inhibited CRF-stimulated cAMP production in hCRF1-CHO cells. Subsequently, we synthesized [125I]YP20 and developed a radioligand binding assay useful for determining the IC50s of ligands that compete for [125I]YP20 binding to the ECD of the CRF1 receptor.