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Titolo:
Photoaffinity labeling of mutant neurokinin-1 receptors reveals additionalstructural features of the substance P/NK-1 receptor complex
Autore:
Macdonald, D; Mierke, DF; Li, HZ; Pellegrini, M; Sachais, B; Krause, JE; Leeman, SE; Boyd, ND;
Indirizzi:
Boston Univ, Sch Med, Dept Pharmacol & Expt Therapeut, Boston, MA 02118 USA Boston Univ Boston MA USA 02118 ol & Expt Therapeut, Boston, MA 02118 USA Brown Univ, Dept Mol Pharmacol, Div Biol & Med, Providence, RI 02912 USA Brown Univ Providence RI USA 02912 v Biol & Med, Providence, RI 02912 USA Brown Univ, Dept Chem, Providence, RI 02912 USA Brown Univ Providence RI USA 02912 v, Dept Chem, Providence, RI 02912 USA Univ Penn, Dept Pathol & Lab Med, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 & Lab Med, Philadelphia, PA 19104 USA Neurogen Corp, Branford, CT 06405 USA Neurogen Corp Branford CT USA 06405Neurogen Corp, Branford, CT 06405 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 8, volume: 40, anno: 2001,
pagine: 2530 - 2539
SICI:
0006-2960(20010227)40:8<2530:PLOMNR>2.0.ZU;2-3
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR-MAGNETIC-RESONANCE; DISTANCE GEOMETRY CALCULATIONS; FIRST EXTRACELLULAR LOOP; 3RD CYTOPLASMIC LOOP; P NK-1 RECEPTOR; NMR-SPECTROSCOPY; SECONDARY STRUCTURE; 3-DIMENSIONAL STRUCTURE; SPECIES SELECTIVITY; HORMONE-RECEPTOR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
50
Recensione:
Indirizzi per estratti:
Indirizzo: Boyd, ND Boston Univ, Sch Med, Dept Pharmacol & Expt Therapeut, Room L611,80 E Concord St, Boston, MA 02118 USA Boston Univ Room L611,80 E Concord StBoston MA USA 02118 118 USA
Citazione:
D. Macdonald et al., "Photoaffinity labeling of mutant neurokinin-1 receptors reveals additionalstructural features of the substance P/NK-1 receptor complex", BIOCHEM, 40(8), 2001, pp. 2530-2539

Abstract

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/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 Bpa(8)-SP resulted ina marked reduction in photoincorporation efficiency compared to the wild-type receptor, In contrast, photoaffinity labeling of the M174A mutant usingradioiodinated Bpa(4)-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2)loop sequence, that is not the primary sire of attachment in the wild-typereceptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembranehelices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha -helix, extending from Pro175 to Glu183, as well as smaller alpha -helices at thetermini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha -helix, approximately 11 Angstrom apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 14/07/20 alle ore 12:55:59