THE LIGAND-BINDING SITE OF THE NEUROKININ-2 RECEPTOR - SITE-DIRECTED MUTAGENESIS AND IDENTIFICATION OF NEUROKININ A BINDING RESIDUES IN THEHUMAN NEUROKININ 2 RECEPTOR

Thirteen residues in the human neurokinin 2 (NK2) receptor were identi fied as potential ligand-binding residues by molecular modeling and am ino acid sequence analysis. Site directed mutagenesis was used to alte r these residues in order to ascertain their importance in binding neu rokinin A (NKA), the physiological peptide ligand for the NK2 receptor , and the non-peptide NK2 receptor selective antagonist SR48968. Four sites appear to be critical for NKA binding (Gln(109), His(198), Ile(2 02), and Gly(273)). The mutant receptors Gln(109) --> His, Ile(202) -- > Val, Gly(273), Pro, and Gly(273), Thr maintain their affinity for SR 48968, despite being unable to bind the peptide ligand. His(198) --> A la and His(198) --> Leu no longer bind NRA or SR48968. We have also id entified a residue (Leu(292)) which appears to play a minor role in th e binding of substance P (SP) and neurokinin B (NKB) to the NK2 recept or. The mutant receptor Leu(292) --> Ser binds NKB and SP with approxi mately a B-fold greater affinity in comparison with the wild type rece ptor while the affinity of NKA remains unaffected. The results suggest that intramembranous residues, as well as residues which lie close to the extracellular side of transmembrane helices 3, 5, and 6, form par t of the NK2 receptor binding site. Binding of SP and NKB to the NK2 r eceptor may also be influenced by residues near the extracellular side of helix 7. These results suggest that some regions of the binding si te for NKA in the NK2 receptor are not used for binding SP in the NH1 receptor. However, it also seems that the NKA binding site includes re gions that are also used by other G-protein-coupled receptors such as rhodopsin and the beta(2)-adrenergic receptors.