TABLE 3

Effect of nonpeptide ligand on GTPγS-sensitive and insensitive 125I-sauvagine binding to the CRF1 receptor

The effect of nonpeptide ligand on the G-protein-coupled state of the CRF1 receptor was assessed by measuring GTPγS-sensitive 125I-sauvagine binding, as described in Fig. 6. Ki was determined using the 125I-sauvagine Kd value of 19 pM and the radioligand concentration (56-63 pM). The experiment involved measuring the ligand effect on 125I-sauvagine binding in the presence of GTPγS (GTPγS-insensitive binding), and for these data the IC50 or EC50 value for inhibiting or enhancing 125I-sauvagine binding is given. The 125I-sauvagine bound as [ligand]→ ∞ values were calculated by dividing the specific binding asymptote for infinite ligand by that for no ligand (i.e., the right-hand asymptote by the left-hand asymptote of the curves as presented in Fig. 6). Data are mean ± S.E.M. (n = 3-5).

ligand GTPγS-Sensitive GTPγS-Insensitive
125I-Sauvagine Bound as [ligand]→∞ -log KiKi125I-Sauvagine Bound as [ligand]→∞ -log IC50/EC50 IC50/EC50
% nM % nM
CRF 0 9.83 ± 0.03 0.15 0 8.74 ± 0.23 1.8
NBI 30775 1 ± 1 8.80 ± 0.21 1.6 −10 ± 10 8.29 ± 0.06 5.2
NBI 34041 1 ± 1 8.37 ± 0.10 4.3 −6 ± 15 7.89 ± 0.10 13
NBI 35965 2 ± 1 8.66 ± 0.01 2.2 −2 ± 5 8.30 ± 0.18 5.5
NBI 38242 4 ± 1 7.02 ± 0.11 95 26 ± 1 6.39 ± 0.16 400
NBI 77173 14 ± 3 6.37 ± 0.04 430 220 ± 60 5.44 ± 0.12 3600
NBI 77165 −1 ± 16 7.39 ± 0.02 41 3200 ± 400 6.46 ± 0.07 340
NBI 77178 15 ± 2 6.61 ± 0.18 250 1700 ± 100 5.94 ± 0.05 1100
NBI 77172 20 ± 4 6.79 ± 0.06 160 65 ± 23 5.84 ± 0.06 1500