[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 K(d) of ≈0.9 nM. The kinetic association of 20 pM [¹²⁵I]YP20 binding best fit to a rapid component (t(1/2)=0.69 min) and a sluggish component (t(1/2)=42 min). [¹²⁵I]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 [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.