Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method.
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. We have developed a method to map systematically all the residues forming the surface of this binding-site crevice, and we have applied this method to the third membrane-spanning segment (M3). We mutated, one at a time, 23 residues in and flanking M3 to cysteine and expressed the mutant receptors heterologously. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist. Thus, the side chains of these residues are exposed in the binding-site crevice, which like M3 extends from the extracellular to the intracellular side of the membrane. The pattern of exposure is consistent with a short loop followed by six turns of an alpha helix.
Javitch
J A
JA
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Fu
D
D
Chen
J
J
Karlin
A
A
D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004622:Embryo, Mammalian; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney; D008698:Mesylates / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping / Q000379:methods*; D017433:Protein Structure, Secondary; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D007202:Indicators and Reagents; D008698:Mesylates; D000644:Quaternary Ammonium Compounds; D017448:Receptors, Dopamine D2; C093171:methanethiosulfonate ethylammonium; C039795:trimethylethylammonium; C074502:methanethiosulfonate; D005020:Ethyl Methanesulfonate; D003545:Cysteine