Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK2 receptor

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK2 receptor, either wild-type or mutated, at four aro matic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmem brane segments V to VII, to assess the role of these residues in the bindin g of natural tachykinins and peptide and nonpeptide antagonists. Three radi oligands, the agonist [I-125]neurokinin A (NKA), the peptide antagonist [H- 3]MEN 11420, and the nonpeptide antagonist [H-3]SR 48968 bound to the wild- type receptor with high affinity (K-d = 2.4 nM, 0.3 nM, and 4.0 nM, respect ively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K-d = 4.8 and 11.5 nM, respecti vely); (YF)-F-266 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); F(270)A mutatio n abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K-d = 1.6 nM); (YF)-F-289 mutation abrogated the binding of SR 48968 but n ot that of NKA and MEN 11420 (K-d = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at varianc e with SR 48968, resulted heavily compromised by H(198)A and (YF)-F-266 mut ations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for th e (YF)-F-289 mutant receptor. Taken together, these results indicate that d ifferent, partially overlapping sets of sites may be involved in the bindin g of agonists and diverse antagonists to the human tachykinin NK2 receptor.