Estimates of the NBI 35965 affinity and cooperativity for allosteric modulation of peptide ligand binding to the R state of the CRF1 receptor
| Peptide | pKN(1/KN) | α | pKL(1/KL) |
|---|---|---|---|
| nM | nM | ||
| Sauvagine | 9.25 ± 0.142-a | 0.33 ± 0.082-b 2-c 2-d | 6.36 ± 0.04e |
| (0.56) | (430) | ||
| CRF | 8.71 ± 0.072-a | 0.14 ± 0.032-b | 6.52 ± 0.032-f |
| (1.9) | (300) | ||
| UCN I | 8.94 ± 0.172-a | 0.11 ± 0.032-b | 8.57 ± 0.072-g |
| (1.1) | (2.7) | ||
| Astressin | 8.87 ± 0.332-a | 0.65 ± 0.032-c 2-d | N.A. |
| (1.4) |
The fitted parameters are those for the R state of the CRF1receptor in L-CRF1 cell membranes. (30 μM GTPγS was included in the assays.) To determine the cooperativity (α) between the binding of NBI 35965 and astressin, data for inhibition of125I-astressin binding (Fig. 1A) were fitted to the allosteric ternary complex model (eq. 1, see ). For the peptide agonists (sauvagine, CRF, and UCN I) K N and α were estimated by fitting the data of Fig. 2 to the allosteric ternary complex model (eq. 3). Data are the mean ± S.E.M (n = 3). In all analyses the affinity constant of125I-astressin was fixed at the value measured in saturation experiments (1.43 × 1010M−1).
N.A., not applicable.
↵2-a The pK N values of NBI 35965 for modulating binding of the four different ligands were not significantly different (p = 0.27, single-factor ANOVA). The α values for cooperativity between NBI 35965 and the four different ligands were significantly different (p = 0.0001). Post hoc analysis (Newman-Keuls test) was used to identify significant differences of a between pairs of peptide ligands.
↵2-b Significantly different from the astressin α value.
↵2-c Significantly different from the CRF α value.
↵2-d Significantly different from the UCN I α value. Equation 3 also provides estimates of unlabeled agonist affinity for the R state (pK L). To test the validity of the fit to eq. 3, these values were compared with those measured in the absence of a second, unlabeled ligand. The fitted value of agonist affinity (pK L) from eq. 3 (Fig. 2) was not significantly different from the K i value obtained from competitive inhibition of 125I-astressin binding in the absence of NBI 35965 (e p = 0.11, sauvagine pK i = 6.55 ± 0.07;
↵2-f p = 0.27, CRF pK i = 6.68 ± 0.09;
↵2-g p = 0.33, UCN I pK i = 8.72 ± 0.11; two-tailed Student's t test, pK i values from manuscript in preparation). Equation 3 also provides estimates of cooperativity between NBI 35965 and 125I-astressin (β). The fitted value of β for analysis of NBI 35965 / agonist interaction was 0.75 ± 0.05, 0.73 ± 0.05, and 0.68 ± 0.05 for sauvagine, CRF, and UCN I, respectively. These values were not significantly different from each other or from the value for NBI 35965 and 125I-astressin measured in the absence of unlabeled agonist (α from eq. 1, 0.65 ± 0.03; Fig. 1A) (p= 0.40, single-factor ANOVA).