SPECIFIC RESIDUES AT THE TOP OF TRANSMEMBRANE SEGMENT-V AND SEGMENT-VI OF THE NEUROKININ-1 RECEPTOR INVOLVED IN BINDING OF THE NONPEPTIDE ANTAGONIST CP-96,345

Previously we have found that binding of the nonpeptide substance P an tagonists, CP 96,345, to the neurokinin-1 (NK-1) receptor was critical ly dependent on two short segments adjacent to the top of transmembran e segments (TM) V and VI, called segments A (residues 183-195) and D ( residues 271-276), respectively. In the present study we have systemat ically performed substitutions of nonconserved residues within these t wo segments with residues from the homologous NK-3 and/or NK-2 recepto r. In segment A, deletion of residues Glu(193) and Lys(194), which are not present in the NK-3 receptor, or substituting them with leucines as in the NK-2 receptor, decreased the affinity of CP 96,345 10- and 2 2-fold, respectively. Surprisingly, switching the position of Glu(193) and Lys(194) did not affect the affinity of CP 96,345, suggesting tha t, rather than interacting directly with CP 96,345, an interaction of these residues with one another is important for CP 96,345 binding. In segment D substitution of Tyr(272) with threonine as in the NK-2 rece ptor and with alanine as in the NK-3 receptor decreased the affinity o f CP 96,345 7- and 24-fold, respectively. Mutation of the preceding Pr o(271) to, glycine alone did not affect CP 96,345 binding, but, combin ed with the mutation of Tyr(272) to threonine, the affinity decreased 28-fold. A series of CP 96,345 analogues with modifications of the maj or chemical moieties exhibited equally reduced affinity as that of CP 96,345 for the Tyr(272)- and Lys(193)-Glu(194)- substituted constructs , except CP 95,555, which lacks one of the phenyl rings in the benzhyd ryl group and which was almost unaffected by these mutations, In concl usion, our data indicate a direct interaction between CP 96,345 and Ty r(272), which are located at the top of TM VI likely in close spatial proximity to the previously identified interaction point, His(197), at the top of the adjacent TM V. Furthermore, the data demonstrated a cr itical involvement in CP 96,345 binding of Lys(193) and Glu(194) locat ed one alpha-helical turn above His(197).