Identification of a Zn2+-binding site on the dopamine D2 receptor.

Zinc (II) modulates the function of many integral membrane proteins. To identify the Zn(2+)-binding site responsible for allosteric modulation of the D(2) dopamine receptor, we first demonstrated that the binding site is likely located in extracellular loops or in transmembrane regions that are accessible from the extracellular milieu. We mutated every histidine in these regions to alanine; two mutants, H394A and H399A, exhibited a reduced response to Zn(2+). Combined mutation of H394 and H399 caused a larger effect of zinc than did either single mutation. Mutation of other potential Zn(2+)-binding residues predicted to be in proximity to H394 or H399 did not substantially alter the potency of Zn(2+). The double mutant H394A/H399A was similar to D(2) in affinity for [(3)H]spiperone and ability to inhibit cyclic AMP accumulation. We conclude that binding of Zn(2+) to H394 and H399 on the dopamine D(2) receptor contributes to allosteric regulation of antagonist binding.

Structural determinants of pharmacological specificity between D(1) and D(2) dopamine receptors.

To test the hypothesis that pharmacological differentiation between D(1) and D(2) dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D(2) receptor to the corresponding aligned residues in the D(1) receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D(2) receptors and 11 mutant D(1) receptors for D(1)- and D(2)-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D(1) receptor mutant W99(3.28)F showed dramatically increased affinity for several D(2)-selective antagonists, particularly spiperone (225-fold), whereas the D(2) receptor mutant Y417(7.43)W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D(1)-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V(5.39) caused modest but consistent and reciprocal changes in affinity of the receptors for D(1) and D(2)-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D(1)/D(2) receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.