RESIDUES IN THE 5TH MEMBRANE-SPANNING SEGMENT OF THE DOPAMINE D2 RECEPTOR EXPOSED 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 in the third membrane-spanning segment (M3) that are exposed in the binding-site crevice [Javitch et al. (1995) Neuron 14, 825]. W e have now mutated, one at a time, 24 consecutive residues in and flan king the fifth membrane-spanning segment (M5) to cysteine and expresse d the mutant receptors in HEK 293 cells. Thirteen of these mutants rea cted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagen ts, added extracellularly, and were protected from reaction by another reversible dopamine antagonist, sulpiride. Thus, the side chains of t hese residues are exposed in the binding-site crevice. Of the 13 expos ed residues, 10 are consecutive, from Phe189 to Phe198. This pattern o f exposure is inconsistent with the expectation that M5, like M3, form s a fixed alpha-helix, one side of which is exposed in the binding-sit e crevice. The exposed region of M5, which contains the serines likely to bind agonist [Strader et al. (1989) J. Biol. Chem. 264, 13752], mi ght loop out into the lumen of the binding-site crevice and be complet ely accessible to water and thus to MTSEA. Alternatively, the exposed region of M5 might be embedded in the membrane and also in contact wit h other membrane-spanning segments. At any instant, only a limited set of residues might be exposed in the binding-site crevice; however, M5 might move rapidly to expose different sets of residues.