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 (Kd = 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 (Kd = 4.8 and 11.5 nM, respectively); Y266F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (Kd = 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 (Kd = 1.6 nM); Y289F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (Kd = 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.
Footnotes
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Send reprint requests to: Dr. Anna Rita Renzetti, Pharmacology Department, Menarini Ricerche S.p. A., Via Rismondo 12A, I-50131 Florence, Italy.
- Abbreviations:
- SP
- substance P
- CHO
- Chinese hamster ovary
- NKA
- neurokinin A
- NKB
- neurokinin B
- TM
- transmembrane segment
- α-MEM
- minimum essential medium α-modification
- DHFR
- dihydrofolate reductase
- hNK2R
- human tachykinin NK2receptor
- FCS
- fetal calf serum
- Received January 29, 1999.
- Accepted April 6, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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