TABLE 1

Nonpeptide ligand affinity and cooperativity with peptide ligands

Nonpeptide ligand binding activity was measured using two methods: 1) the observed cooperativity between nonpeptide ligand and CRF (C_CRF) and nonpeptide ligand affinity (K_nonpeptide) were measured for the G-protein-uncoupled receptor state (¹²⁵I-astressin vs CRF + GTPγS), as described in Fig. 2. Data are mean ± S.E.M. (n = 3-4). -log K_CRF was not significantly different when measured in assays of the different nonpeptide ligands (P > 0.05, single-factor ANOVA, mean of all values of 7.20 ± 0.04, n = 27, K_CRF = 64 nM). The ¹²⁵I-astressin concentration was 63 to 100 pM and K_d was 31 pM. 2) nonpeptide ligand affinity for the G-protein-uncoupled state was measured more directly by displacement of labeled nonpeptide ligand binding ([³H]NBI 35965 + GTPγS) as described under Materials and Methods. Data are mean ± S.E.M. (n = 3-14). -log K_nonpeptide was significantly different between the two methods (P = 0.0034, two-factor ANOVA), but the Bonferroni post test indicated a significant difference between the methods only for NBI 38242 (P < 0.01) and NBI 30775 (P < 0.05, 3.2- and 2.9-fold differences, respectively). Differences of C_CRF between nonpeptide ligands were tested statistically using single-factor ANOVA (P < 0.0001) with the Newman-Keuls post test indicating all values were significantly different from one another, except for two pairs, NBI 30775 and NBI 34041, and NBI 77173 and NBI 77165.