Dopamine D4/D2 receptor selectivity is determined by a divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments

Conserved features of the sequences of dopamine receptors and of homologous G-protein-coupled receptors point to regions, and amino acid residues with in these regions, that contribute to their ligand binding sites. Difference s in binding specificities among the catecholamine receptors, however, must stem from their nonconserved residues. Using the substituted-cysteine acce ssibility method, we have identified the residues that form the surface of the water-accessible binding-site crevice in the dopamine D2 receptor. Of a pproximately 80 membrane-spanning residues that differ between the D2 and D 4 receptors, only 20 were found to be accessible, and 6 of these 20 are con servative aliphatic substitutions. In a D2 receptor background, we mutated the 14 accessible, nonconserved residues, individually or in combinations, to the aligned residues in the D4 receptor. We also made the reciprocal mut ations in a D4 receptor background. The combined substitution of four to si x of these residues was sufficient to switch the affinity of the receptors for several chemically distinct D4-selective antagonists by three orders of magnitude in both directions (D2- to D4-like and D4- to D2-like). The muta ted residues are in the second, third, and seventh membrane-spanning segmen ts (M2, M3, M7) and form a cluster in the binding-site crevice. Mutation of a single residue in this cluster in M2 was sufficient to increase the affi nity for clozapine to D4- like levels. We can rationalize the data in terms of a set of chemical moieties in the ligands interacting with a divergent aromatic microdomain in M2-M3-M7 of the D2 and D4 receptors.