Electrostatic and aromatic microdomains within the binding-site crevice of the D2 receptor: Contributions of the second membrane-spanning segment

被引:64
作者
Javitch, JA
Ballesteros, JA
Chen, JY
Chiappa, V
Simpson, MM
机构
[1] Columbia Univ, Coll Phys & Surg, Ctr Mol Recognit, New York, NY 10032 USA
[2] Columbia Univ, Coll Phys & Surg, Dept Psychiat, New York, NY 10032 USA
[3] Columbia Univ, Coll Phys & Surg, Dept Pharmacol, New York, NY 10032 USA
[4] CUNY Mt Sinai Sch Med, Dept Physiol & Biophys, New York, NY 10029 USA
关键词
D O I
10.1021/bi9905314
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an cl-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7.
引用
收藏
页码:7961 / 7968
页数:8
相关论文
共 40 条
[1]   IDENTIFICATION OF ACETYLCHOLINE-RECEPTOR CHANNEL-LINING RESIDUES IN THE ENTIRE M2 SEGMENT OF THE ALPHA-SUBUNIT [J].
AKABAS, MH ;
KAUFMANN, C ;
ARCHDEACON, P ;
KARLIN, A .
NEURON, 1994, 13 (04) :919-927
[2]   ACETYLCHOLINE-RECEPTOR CHANNEL STRUCTURE PROBED IN CYSTEINE-SUBSTITUTION MUTANTS [J].
AKABAS, MH ;
STAUFFER, DA ;
XU, M ;
KARLIN, A .
SCIENCE, 1992, 258 (5080) :307-310
[3]   THE PROBABLE ARRANGEMENT OF THE HELICES IN G-PROTEIN-COUPLED RECEPTORS [J].
BALDWIN, JM .
EMBO JOURNAL, 1993, 12 (04) :1693-1703
[4]   Functional microdomains in g-protein-coupled receptors - The conserved Arginine-cage motif in the gonadotropin-releasing hormone receptor [J].
Ballesteros, J ;
Kitanovic, S ;
Guarnieri, F ;
Davies, P ;
Fromme, BJ ;
Konvicka, K ;
Chi, L ;
Millar, RP ;
Davidson, JS ;
Weinstein, H ;
Sealfon, SC .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (17) :10445-10453
[5]   ANALYSIS AND REFINEMENT OF CRITERIA FOR PREDICTING THE STRUCTURE AND RELATIVE ORIENTATIONS OF TRANSMEMBRANAL HELICAL DOMAINS [J].
BALLESTEROS, JA ;
WEINSTEIN, H .
BIOPHYSICAL JOURNAL, 1992, 62 (01) :107-109
[6]  
Ballesteros JA., 1995, METH NEUROSCI, V25, P366, DOI DOI 10.1016/S1043-9471(05)80049-7
[7]   HELIX GEOMETRY IN PROTEINS [J].
BARLOW, DJ ;
THORNTON, JM .
JOURNAL OF MOLECULAR BIOLOGY, 1988, 201 (03) :601-619
[8]  
CHENG Y, 1973, BIOCHEM PHARMACOL, V22, P3099
[9]   MOLECULAR-BIOLOGY OF THE DOPAMINE-RECEPTORS [J].
CIVELLI, O ;
BUNZOW, JR ;
GRANDY, DK ;
ZHOU, QY ;
VANTOL, HHM .
EUROPEAN JOURNAL OF PHARMACOLOGY-MOLECULAR PHARMACOLOGY SECTION, 1991, 207 (04) :277-286
[10]   IDENTIFICATION AND SEQUENCE OF A BINDING-SITE PEPTIDE OF THE BETA-2-ADRENERGIC RECEPTOR [J].
DOHLMAN, HG ;
CARON, MG ;
STRADER, CD ;
AMLAIKY, N ;
LEFKOWITZ, RJ .
BIOCHEMISTRY, 1988, 27 (06) :1813-1817