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Titolo:
Determination of amino acid residues that are accessible from the ligand binding crevice in the seventh transmembrane-spanning region of the human A(1) adenosine receptor
Autore:
Dawson, ES; Wells, JN;
Indirizzi:
Vanderbilt Univ, Sch Med, Dept Pharmacol, Nashville, TN 37232 USA Vanderbilt Univ Nashville TN USA 37232 Pharmacol, Nashville, TN 37232 USA
Titolo Testata:
MOLECULAR PHARMACOLOGY
fascicolo: 5, volume: 59, anno: 2001,
pagine: 1187 - 1195
SICI:
0026-895X(200105)59:5<1187:DOAART>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
DOPAMINE D2 RECEPTOR; SITE-DIRECTED MUTAGENESIS; SEGMENT; CHANNEL; A(1)-ADENOSINE; EXPRESSION; CLONING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
27
Recensione:
Indirizzi per estratti:
Indirizzo: Wells, JN Vanderbilt Univ, Sch Med, Dept Pharmacol, 221 Kirkland Hall, Nashville, TN37232 USA Vanderbilt Univ 221 Kirkland Hall Nashville TN USA 37232 232 USA
Citazione:
E.S. Dawson e J.N. Wells, "Determination of amino acid residues that are accessible from the ligand binding crevice in the seventh transmembrane-spanning region of the human A(1) adenosine receptor", MOLEC PHARM, 59(5), 2001, pp. 1187-1195

Abstract

The substituted-cysteine accessibility method (SCAM) was applied to transmembrane span seven of the human A(1) adenosine receptor (hA(1)AR) to reveala subset of amino acids that are exposed to the ligand-binding crevice. The SCAM approach involved a systematic probe of receptor structure by individual substitutions of residues K265 (7.30) to R296 (7.61) with cysteine. Inmost cases, hA(1)AR substituted-cysteine mutant membranes displayed antagonist dissociation binding constants that did not differ significantly from wild-type (WT). Radioligand binding assays were used to compare cell membranes that were treated with hydrophilic, sulfhydryl-specific methanethiosulfonate derivatives with control cell membranes. Position H278 was previouslyreported to be required for A(1)AR ligand binding; however, that report did not establish that H278 represents a contact point for ligands. Cysteine-substitution at H278 yields membrane preparations with greatly decreased receptor density compared with WT membranes from cells in the same transfection experiment. However, H278C membranes retain a measurable fraction of antagonist binding. This observation allows for the investigation of binding-crevice accessibility at position 278 and suggests that H278 may not be required for binding of antagonist ligands. Our data reveal the binding-creviceaccessibility of residues T270 (7.35), A273 (7.38), I274 (7.39), T277 (7.42), H278 (7.43), N284 (7.49), and Y288 (7.53) in the hA(1)AR. These data are consistent with the high-resolution structure of bovine rhodopsin that features three alpha -helical turns in this region that are interrupted by anelongated, nonhelical structure from positions 7.43 to 7.48 in the primaryamino acid sequence.

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Documento generato il 23/01/20 alle ore 03:27:44