Evidence for the proximity of the extreme N-terminus of the neurokinin-2 (NK2) tachykinin receptor to Cys167 in the putative fourth transmembrane helix

Neurokinin-2 receptor (NK2R) binding of [H-3]-SR48968, a piperidinyl antagonist, is inhibited by methanethiosulfonate ethylammonium (MTSEA) in a time- and concentration-dependent manner. By the systematic alanine replacement of putative loop and transmembrane region cysteine residues (CyS4, Cys(81) Cys(167), Cys(262), Cys(281), Cys(301), and Cys(309)), we have determined that MTSEA perturbs [H-3]-SR48968 binding by modifying Cys(167) in transmembrane helix 4. Data were substantiated using glycine, serine, and threonine substitutions of Cys(167). MTSEA preferentially modifies cysteine residues that are in proximity to a negatively charged environment. Hence, aspartate and glutamate residues were systematically substituted with leucine or valine, respectively, and the inhibitory effects of MTSEA on [H-3]-SR48968 binding were reevaluated to determine those acidic residues close to the MTSEA binding crevice. Most significantly, substitution of Asp(5) in the receptor's extreme N-terminus abolished the effects of MTSEA on [H-3]SR48968 binding. Therefore, our data would suggest close association of the extreme N-terminus with the extracellular surfaces of helices 4 and 3 in the NK2R in forming a binding crevice for MTSEA. The inhibition of SR48968 binding appears to result from loss of the SR48968 binding conformation of Gln(166) induced by MTSEA when it is coupled to Cys167. Hence, it is proposed that there is mutually exclusive hydrogen bonding of SR48968 and MTSEA to Gln(166).