title: Evidence for the proximity of the extreme N-terminus of the neurokinin-2 (NK2) tachykinin receptor to Cys167 in the putative fourth transmembrane helix.
abstract: Neurokinin-2 receptor (NK(2)R) binding of [(3)H]-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 (Cys(4), Cys(81), Cys(167), Cys(262), Cys(281), Cys(308), and Cys(309)), we have determined that MTSEA perturbs [(3)H]-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 [(3)H]-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 [(3)H]-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 NK(2)R 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 Cys(167). Hence, it is proposed that there is mutually exclusive hydrogen bonding of SR48968 and MTSEA to Gln(166).
authors.affiliation: School of Biochemistry and Molecular Biology, University of Leeds, UK. N.Bhogal@Leeds.ac.uk
authors.forename: Nirmala
authors.initials: N
authors.lastname: Bhogal
authors.forename: Frank E
authors.initials: FE
authors.lastname: Blaney
authors.forename: Paul M
authors.initials: PM
authors.lastname: Ingley
authors.forename: James
authors.initials: J
authors.lastname: Rees
authors.forename: John B C
authors.initials: JB
authors.lastname: Findlay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001549:Benzamides / Q000037:antagonists & inhibitors / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000737:chemistry; D005110:Extracellular Space / Q000737:chemistry*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010880:Piperidines / Q000037:antagonists & inhibitors / Q000737:chemistry; D017433:Protein Structure, Secondary; D018041:Receptors, Neurokinin-2 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D055672:Static Electricity; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014316:Tritium
chemical_list: D001549:Benzamides; D010446:Peptide Fragments; D010880:Piperidines; D018041:Receptors, Neurokinin-2; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid; C073839:SR 48968; D005020:Ethyl Methanesulfonate; D003545:Cysteine