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 homol ogous G protein-coupled receptors, is contained within a water-accessi ble 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 tim e, 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 extracell ularly, and were protected from reaction by a reversible dopamine anta gonist. Thus, the side chains of these residues are exposed in the bin ding-site crevice, which like M3 extends from the extracellular to the intracellular side of the membrane. The pattern of exposure is consis tent with a short loop followed by six turns of an alpha helix.