[125I]YP20: A novel radioligand specific for the extracellular domain of the CRF1 receptor

Abstract The peptide corticotropin-releasing factor (CRF) binds to the CRF 1 receptor via a two-domain mechanism such that the extracellular domain (ECD) of the receptor captures the CRF's C-terminus to facilitate the binding of CRF's N-terminus to the juxta-membrane or “J”-site. Known small molecule antagonists bind to the J-site while known CRF 1 receptor peptide radioligands bind to both sites. We report here the in vitro binding properties of the first radioligand that binds exclusively to the ECD of the CRF 1 receptor. This ligand, which we named [ 125 I]Yamada peptide 20 ([ 125 I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004) . We confirmed its high affinity for the [ 125 I]CRF binding site on the hCRF 1 receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF 1 -CHO cells. Under optimized conditions, 20 pM [ 125 I]YP20 reproducibly bound to hCRF 1 -CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF 1 receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated K d of ≈ 0.9 nM. The kinetic association of 20 pM [ 125 I]YP20 binding best fit to a rapid component (t 1/2  = 0.69 min) and a sluggish component (t 1/2  = 42 min). [ 125 I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t 1/2  = 0.92 min and t 1/2  = 11.7 min). While [ 125 I]YP20's binding kinetics are complex, its high affinity and pharmacological specificity indicate that it is an excellent radioligand for probing the ECD site of the CRF 1 receptor.