Nonconserved residues in the second transmembrane-spanning domain of the D(4) dopamine receptor are molecular determinants of D(4)-selective pharmacology.

The molecular determinants that govern selective ligand binding to the rat D(4) dopamine receptor were investigated by substituting D(2) dopamine receptor sequences into a D(4) dopamine receptor background. The resulting mutant D(4) dopamine receptors were then screened with a panel of 10 selective and nonselective ligands, which included two allosteric modulators as sensitive measures of protein conformational changes. Mutation of a phenylalanine at position 88 in the second transmembrane-spanning domain (TMS2) of the D(4) receptor to the corresponding valine in the D(2) receptor D(4)-F88V resulted in an approximately 100-fold decrease in the affinity of the highly D(4)-selective drug 3-([4-(4-iodophenyl) piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine (L-750,667) without substantially affecting the binding of the other ligands. Mutations at the extracellular side of D(4)-TMS3 produced moderate decreases in L-750,667 binding affinities with concomitant increases in binding affinity for the D(2)/D(3)-selective antagonist (-)-raclopride. However, the binding affinities of these same D(4)-TMS3 mutants for the allosteric modulator isomethylbutylamiloride also were an anomalous 6- to 20-fold higher than either wild-type receptor. In the combined D(4)-F88V/TMS3 mutants, L-750,667 binding affinity was further decreased, but this decrease was not additive. More importantly, the combined D(4)-F88V/TMS3 mutants had (-)-raclopride and isomethylbutylamiloride binding properties that reverted back to those of the wild-type D(4)-receptor. In contrast to the D(4)-F88V mutant, the adjacent D(4)-L87W mutant had an increased affinity for ligands with extended structures, but had essentially no effect on ligands with compact structures. These findings demonstrate that two residues near the extracellular side of D(4)-TMS2 are critical molecular determinants for the selective binding of L-750,667 and ligands with extended structures.