Abstract

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK₂ receptor, either wild-type or mutated, at four aromatic residues (His¹⁹⁸, Tyr²⁶⁶, Phe²⁷⁰, Tyr²⁸⁹) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [¹²⁵I]neurokinin A (NKA), the peptide antagonist [³H]MEN 11420, and the nonpeptide antagonist [³H]SR 48968 bound to the wild-type receptor with high affinity (K_d = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H¹⁹⁸A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K_d = 4.8 and 11.5 nM, respectively); Y²⁶⁶F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K_d = 2.8 nM and 1.2 nM, respectively); F²⁷⁰A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K_d = 1.6 nM); Y²⁸⁹F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K_d = 2.0 and 2.9 nM, respectively). Y²⁶⁶A and Y²⁸⁹A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H¹⁹⁸A and Y²⁶⁶F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y²⁸⁹F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK₂ receptor.