Under optimized assay conditions very low concentrations pM

Under optimized assay conditions, very low concentrations (20pM) of [125I]YP20 bound to hCRF1-CHO Idelalisib membranes with high specificity (>95% inhibited by 1μM astressin) and in a membrane protein-dependent manner. Specific binding was selective vs. the hCRF2α subtype because little specific binding was seen with high amounts of hCRF2α-HEK membrane protein. The lack of specific binding to hD1-CHO membranes indicated that specific binding was also selective vs. a class A G protein-coupled receptor and that it was dependent upon transfection of the CHO cells with the hCRF1 receptor. The modest potencies (0.3–0.6μM) of the high affinity (sub nM) CRF2 subtype selective antagonists antisauvagine and K41498 at inhibiting [125I]YP20 binding to the CRF1 receptor provided additional pharmacological evidence for the CRF1-subtype selectivity of this novel radioligand (Higelin et al., 2001, Lawrence et al., 2002). The pharmacology of [125I]YP20 binding indicated that low concentrations (i.e. 20pM) of this radioligand exclusively bound to the ECD of the CRF1 receptor. YP20 and astressin (CRF1receptor-specific peptidic analogs of the C-terminus of CRF) inhibited this binding with Hill coefficients near unity and IC50s comparable in magnitude to those vs. [125I]CRF. In sharp contrast, three J-site specific antagonists (R121929, DMP-696, and CP-154,526) potently inhibited [125I]CRF binding with Hill coefficients near unity, but did not inhibit [125I]YP20 binding even at high concentrations. Consistent with both the J-site and the ECD contributing to affinity of peptidic agonists, agonists CRF and sauvagine exhibited weak potency as [125I]YP20 inhibitors. Thus, the conditions optimized for the high affinity binding of [125I]YP20 are useful for determining the IC50s of test compounds for binding to the ECD of the CRF1 receptor. Saturation binding and kinetic binding experiments indicated that [125I]YP20\'s binding to hCRF1-CHO membranes is complex. A robust saturation binding analysis of high affinity [125I]YP20 binding could not be performed due to a lack of a clear separation of high affinity binding from low affinity/non-saturable binding. However, a Kd of 0.9nM, determined from fitting the low concentrations (≤0.4nM) of the radioligand, was similar in magnitude to the IC50 of unlabeled YP20 at inhibiting [125I]YP20 and [125I]CRF, indicating that this Kd was an accurate estimate of the high affinity site. Kinetic studies using 20pM [125I]YP20 indicated that the majority of binding (61% of the specific binding at 1h) was very rapid (t1/2<1min), but that there was also another component with slow association kinetics (t1/2≈1h). Specific binding was rapidly reversible, but also was best described by two phases: a very rapid (t1/2<1min) and a slower (t1/2≈10min) component. The nature of the complexity of the binding kinetics and saturation properties remains unknown, but may be due to conformational changes in the ligand and/or the ECD upon ligand binding. NMR studies have shown that cyclic CRF30–41 (analog of the same C-terminal amino acids as YP20) is unstructured in solution, but adopts an α-helical conformation when bound to the isolated, soluble ECD of the CRF1 receptor (). NMR studies have also detected large scale changes in the ECD of the CRF2β receptor upon ligand binding (). Consistent with YP20\'s functional antagonism, the receptor/G protein uncoupling agent Gpp(nH)p did not inhibit [125I]YP20 binding or affect its association or dissociation kinetics. Moreover, Gpp(nH)p did not affect the apparent affinity of CRF for this site, which indicates that occupancy of the [125I]YP20 binding site even by agonists is not coupled to receptor activation. Similarly, neither high affinity [125I]YP20 binding itself or the apparent affinity of YP20 for this site was affected by a J-site saturating concentration of R121919, which indicates the absence of an allosteric effect of the J-site on [125I]YP20 binding to the ECD. In contrast, the modest decrease in astressin\'s affinity for the [125I]YP20 binding site in the presence of R121919 is consistent with the reported allosteric inhibition by J-site inhibitors on [125I]astressin binding () while the large decrease in CRF\'s affinity for this site in the presence of R121919 is additional support for the two-step binding model for CRF.