RESIDUES IN THE 7TH 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 homol ogous G-protein-coupled receptors, is contained within a water-accessi ble crevice formed among its seven membrane-spanning segments. Using t he substituted-cysteine accessibility method, we previously mapped the residues that form the surface of the binding-site crevice in the thi rd and fifth membrane-spanning segments (M3 and M5). We have now mutat ed to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant r eceptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracel lularly, and were protected from reaction by a reversible dopamine ant agonist, sulpiride. Thus, we infer that the side chains of these resid ues are in the water-accessible surface of the binding-site crevice. T he pattern of accessibility of the cysteine-substitution mutants is co nsistent with M7 being a kinked alpha-helix.