Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method
Javitch, J.A.; Fu, D.; Chen, J.; Karlin, A.
Neuron 14(4): 825-831
ISSN/ISBN: 0896-6273 PMID: 7718244 DOI: 10.1016/0896-6273(95)90226-0
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.