Interaction of Met(297) in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disu lfide interactions of Cys-substituted NK2 receptor mutants with analogues o f neurokinin A (NKA) containing single cysteine substitutions. The NKA anal ogues were N-biotinylated to facilitate the rapid detection of covalent ana logue-receptor interactions utilizing streptavidin reactivity. N-biotinyl[T yr(1),Cys(9)]NKA, N-biotinyl-[Tyr(1),Cys(10)]NKA were both found to reversi bly disulfide bond to the NK2 receptor mutant Met(297) --> Cys. This is con sistent with the improved affinities of these particular analogues for the Met(297) --> Cys receptor as compared with those for the wild-type and Met( 297) --> Leu receptors. In our three-dimensional model, Met(297) occupies t he equivalent position in helix 7 to the retinal binding Lys(296) in rhodop sin. Binding of the NK2 receptor antagonist [H-3]SR 48968 and of I-125-NKA was used to characterize additional receptor mutants. It seems that the aro matic residues Trp(99) (helix 3), His(198) (helix 5), Tyr(266), His(267), a nd Phe(270) play an important role in NKA binding as structural determinant s. The existence of overlapping SR 48968 and NKA binding sites is also evid ent. These data suggest that the peptide binding site of the NK2R is at lea st in part formed by residues buried deep within the transmembrane bundle a nd that this intramembranous binding domain may correspond to the binding s ites for substantially smaller endogenous GPCR ligands.