Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

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• 作者：Douglas Macdonald ; Dale F. Mierke ; Hanzhong Li ; Maria Pellegrini ; Bruce Sachais ; James E. Krause ; Susan E. Leeman ; and Norman D. Boyd
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：February 27, 2001
• 年：2001
• 卷：40
• 期：8
• 页码：2530 - 2539
• 全文大小：215K
• 年卷期：v.40,no.8(February 27, 2001)
• ISSN：1520-4995

文摘

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopyhave been combined to further define the molecular details of the binding of substance P (SP) to the ratneurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated,photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8,respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa⁸-SP resulted in a markedreduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinitylabeling of the M174A mutant using radioiodinated Bpa⁴-SP gave the unexpected result of an increase inthe efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemicalfragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachmentwere not the substituted alanine, but rather the other methionine on the second extracellular (E2) loopsequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest aclose spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structuraldisposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to theentire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipidsused to mimic a membrane. The structural features of the E2 loop include a centrally located -helix,extending from Pro175 to Glu183, as well as smaller -helices at the termini, corresponding to thetransmembrane regions. The two methionine residues are located on the same face of the central -helix,approximately 11 Å apart from each other, and are therefore consistent with the conclusions of thephotoaffinity labeling results.