Pharmacological profile of alanyl-substituted DRY mutant V1aRs Mutant V1aRs were expressed in HEK 293T cells and characterized pharmacologically. Dissociation constants (Ki) were calculated from IC50 values and corrected for radioligand occupancy as described under Materials and Methods. Cell surface expression of each mutant was quantified in parallel by ELISA and expressed as percentage of HA-tagged Wt V1aR. Data shown are mean ± S.E.M. of three individual experiments [unless otherwise stated (n)] with each performed in triplicate. Cell surface expression of 100% corresponds to ∼0.8 pmol/mg of membrane protein.
| Receptor | Binding affinities (Ki) | Cell Surface Expression | |||
|---|---|---|---|---|---|
| Agonist AVP | Antagonists | ||||
| CA | LA | SR 49059 | |||
| nM | % Wt | ||||
| Wt | 1.0 ± 0.1 | 0.5 ± 0.1 | 0.2 ± 0.01 | 0.7 ± 0.1 | 100 |
| D148A | N.D. | N.D. | N.D. | N.D. | 2 ± 2 (7) |
| R149A | 2.6 ± 0.4 | 1.4 ± 0.3 | 1.2 ± 0.1 | 1.9 ± 0.2 | 101 ± 5 (5) |
| Y150A | 1.3 ± 0.3 | 1.0 ± 0.2 | 0.4 ± 0.1 | 1.3 ± 0.2 | 91 ± 4 (6) |
CA, cyclic peptide antagonist; LA, linear peptide antagonist; SR 49059, nonpeptide antagonist; N.D., not detected.