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

The peptide corticotropin-releasing factor (CRF) binds to the CRF1 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 CRF1 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 CRF1 receptor. This ligand, which we named [125I]Yamada peptide 20 ([125I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004). We confirmed its high affinity for the [125I]CRF binding site on the hCRF1 receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF1-CHO cells. Under optimized conditions, 20 pM [125I]YP20 reproducibly bound to hCRF1-CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF1 receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated Kd of ≈ 0.9 nM. The kinetic association of 20 pM [125I]YP20 binding best fit to a rapid component (t1/2 = 0.69 min) and a sluggish component (t1/2 = 42 min). [125I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t1/2 = 0.92 min and t1/2 = 11.7 min). While [125I]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 CRF1 receptor.