2 NONPEPTIDE TACHYKININ ANTAGONISTS ACT THROUGH EPITOPES ON CORRESPONDING SEGMENTS OF THE NK1 AND NK2 RECEPTORS

The molecular mechanism of action for two chemically distinct and high ly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was stu died by development of a series of chimeric constructs between their r espective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, s ubstance P and neurokinin A, were not affected by exchanging almost th e entire C-terminal half of the NK1 receptor with the corresponding se gment of the NK2 receptor. In contrast, it was found that transfer fro m the NK2 to the NK1 receptor of a segment corresponding to transmembr ane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the suscep tibility for the nonpeptide antagonists. This chimeric exchange, corre sponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK 1 receptor-i.e., the K(i) value for inhibition of binding of I-125-lab eled substance P decreased from >10,000 to 0.97 nM. At the same time t he affinity for the NK1-selective compound CP-96,345 decreased >30-fol d. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [H-3]SR-48,968, which did not bind to th e NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor s egment incorporated around transmembrane segments VI and VII, K(d) = 1 .5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act th rough epitopes located around transmembrane segment VI on their respec tive target receptors and that at least the nonconserved residues in t hese epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.