Effects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in Chinese hamster ovary cells

A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK, receptor to yield a tyrosine/phenylalanine (Tyr/P he) substitution. Chinese hamster ovary cells stably transfected with the w ild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [beta Ala(8)]N KA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinit y as compared to NKA. In a functional assay (production of inositol-1,4,5-t risphosphate, IP3), NKA, GR 64349, and [beta Ala(8)]NKA(4-10) stimulated IP 3 accumulation via the wild-type and mutant receptors with similar potencie s. On the other hand, NKB and SP exhibited a dramatic reduction in their ag onist efficacies at the mutant receptor, NKB acting as a partial agonist (m aximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicat ed that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized th e agonist effect of NKA. The antagonist action of SP at the mutant NK2 rece ptor cannot be ascribed to receptor internalization. The Tyr/Phe replacemen t at position 289 markedly reduced the binding affinity and antagonist pote ncy of the non-peptide ligand, SR 48968, without affecting the binding affi nity and antagonist potency of the bicyclic peptide antagonist MEN 11420. T he results indicate that the hydroxyl radical function of Tyr289 in transme mbrane segment 7 of the human NK2 receptor is, directly or indirectly, invo lved in stimulus transduction when the NK2 receptor is occupied by NKB or S P, but not when using NKA or NK2 receptor-selective agonists. BIOCHEM PHARM ACOL 57;8:899-906, 1999. (C) 1999 Elsevier Science Inc.