Evidence for the Proximity of the Extreme N-Terminus of the Neurokinin-2 (NK2) Tachykinin Receptor to Cys167 in the Putative Fourth Transmembrane Helix

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• 作者：Nirmala Bhogal ; Frank E. Blaney ; Paul M. Ingley ; James Rees ; and John B. C. Findlay
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：March 23, 2004
• 年：2004
• 卷：43
• 期：11
• 页码：3027 - 3038
• 全文大小：911K
• 年卷期：v.43,no.11(March 23, 2004)
• ISSN：1520-4995

文摘

Neurokinin-2 receptor (NK₂R) binding of [³H]-SR48968, a piperidinyl antagonist, is inhibitedby methanethiosulfonate ethylammonium (MTSEA) in a time- and concentration-dependent manner. Bythe systematic alanine replacement of putative loop and transmembrane region cysteine residues (Cys⁴,Cys⁸¹, Cys¹⁶⁷, Cys²⁶², Cys²⁸¹, Cys³⁰⁸, and Cys³⁰⁹), we have determined that MTSEA perturbs [³H]-SR48968binding by modifying Cys¹⁶⁷ in transmembrane helix 4. Data were substantiated using glycine, serine,and threonine substitutions of Cys¹⁶⁷. MTSEA preferentially modifies cysteine residues that are in proximityto a negatively charged environment. Hence, aspartate and glutamate residues were systematically substitutedwith leucine or valine, respectively, and the inhibitory effects of MTSEA on [³H]-SR48968 binding werereevaluated to determine those acidic residues close to the MTSEA binding crevice. Most significantly,substitution of Asp⁵ in the receptor's extreme N-terminus abolished the effects of MTSEA on [³H]-SR48968 binding. Therefore, our data would suggest close association of the extreme N-terminus withthe extracellular surfaces of helices 4 and 3 in the NK₂R in forming a binding crevice for MTSEA. Theinhibition of SR48968 binding appears to result from loss of the SR48968 binding conformation of Gln¹⁶⁶induced by MTSEA when it is coupled to Cys¹⁶⁷. Hence, it is proposed that there is mutually exclusivehydrogen bonding of SR48968 and MTSEA to Gln¹⁶⁶.