Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice.
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, we previously mapped the residues that form the surface of the binding-site crevice in the third and fifth membrane-spanning segments (M3 and M5). We have now mutated to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant receptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of these residues are in the water-accessible surface of the binding-site crevice. The pattern of accessibility of the cysteine-substitution mutants is consistent with M7 being a kinked alpha-helix.
Fu
D
D
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Ballesteros
J A
JA
Weinstein
H
H
Chen
J
J
Javitch
J A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000235:genetics; D018492:Dopamine Antagonists / Q000494:pharmacology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013134:Spiperone / Q000031:analogs & derivatives / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism / Q000494:pharmacology; D013469:Sulpiride / Q000494:pharmacology
D018492:Dopamine Antagonists; D008565:Membrane Proteins; D017448:Receptors, Dopamine D2; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; D013134:Spiperone; D013469:Sulpiride; C039167:3-N-methylspiperone; D005020:Ethyl Methanesulfonate; D019204:GTP-Binding Proteins; D003545:Cysteine