A CLUSTER OF AROMATIC RESIDUES IN THE 6TH MEMBRANE-SPANNING SEGMENT OF THE DOPAMINE D2 RECEPTOR IS 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 in the third, fifth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have n ow mutated to cysteine, one at a time, 22 consecutive residues in the sixth membrane-spanning segment (M6) and expressed the mutant receptor s in HEK 293 cells. Ten of these mutants reacted with charged, hydroph ilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and all but one were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of the res idues at the reactive loci (V378, F382, W386, P388, F389, F390, T392, H393, I394, and I397) are on tile water-accessible surface of the bind ing-site crevice. The pattern of accessibility is consistent with an a lpha-helical conformation with a wide angle of accessibility near the binding site itself and a narrower stripe continuing toward the cytopl asmic portion of the binding-site crevice, This pattern of accessibili ty is consistent with the presence of a proline kink which could bend the extracellular portion of M6 into the binding-site crevice where it would be more broadly accessible than the cytoplasmic portion of the membrane-spanning segment. Four highly conserved aromatic residues and a histidine are clustered together un the water-accessible surface of the binding-site crevice. They define an interconnected ''aromatic cl uster'' that may be involved in ligand binding and receptor activation .