Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors

Abstract

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,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 receptorselective 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. Dopamine modulates diverse biological functions, including movement, endocrine function, and memory formation, through activation of five distinct dopamine receptor subtypes that belong to the G protein-coupled receptor (GPCR) superfamily and are grouped into two subfamilies, D 1 -like dopamine receptors and D 2 -like dopamine receptors, based on their structure, pharmacology, and transduction pathways. The D 1 and D 2 receptors are the most abundant dopamine receptor subtypes and are most similar to the traditional pharmacologically defined D 1 and D 2 receptors (Kebabian and Calne, 1979). The D 1 receptor has a long carboxyl terminus and a short third intracellular loop, couples to the adenylate cyclase stimulatory G proteins G␣ s/olf , and stimulates cyclic AMP accumulation. In contrast, the D 2 receptor has a short carboxyl terminus and a long third intracellular loop, couples to the pertussis toxin-sensitive G proteins G␣ i/o , inhibits cyclic AMP accumulation, and also modulates a variety of G␤␥-regulated effectors such as calcium and potassium ion channels, mitogen-activated protein kinases, and phospholipases (Neve et al., 2004). D 1 and D 2 receptor-selective agonists and antagonists are current or potential therapeutic drugs for treatment o...

Ligand Selectivity of D2Dopamine Receptors Is Modulated by Changes in Local Dynamics Produced by Sodium Binding

“…Although a few docking poses were observed in closed conformers (Ϫ7 to Ϫ1) with the signature H-bond-reinforced ionic interaction between the ligand's pyridyl ammonium group and the carboxylate side chain of D3.32(114), the benzamide ring remained extracellular of the presumed primary binding site, without deeper penetration into the primary cleft. In contrast, the frames corresponding to the opened phase of the harmonic motion of M 19 Naϩ (frames ϩ1, ϩ2) accommodate (Ϫ)-raclopride in orientations that satisfy the experimentally suggested interactions, such as the H-bond-reinforced ionic interaction, an H-bond interaction between the ligand's hydroxyl group and the side chain of Y7.43(388) (not shown), and the deeper access of the benzamide ring into the primary pocket (Lan et al, 2006). B, histograms of docking poses for (Ϫ)-raclopride in wild-type D 2 receptor.…”

“…In the D 2 receptor, the allosteric site is likely coupled to insta- Naϩ trajectory. A, initial (0) and closed frames (Ϫ1, Ϫ2) restrict (Ϫ)-raclopride occupancy in the primary binding pocket (situated between TMs 4 and 6) that is expected to accommodate the substituted benzamide ring moiety of (Ϫ)-raclopride (Lan et al, 2006). Although a few docking poses were observed in closed conformers (Ϫ7 to Ϫ1) with the signature H-bond-reinforced ionic interaction between the ligand's pyridyl ammonium group and the carboxylate side chain of D3.32(114), the benzamide ring remained extracellular of the presumed primary binding site, without deeper penetration into the primary cleft.…”

Addressing Selective Polypharmacology of Antipsychotic Drugs Targeting the Bioaminergic Receptors through Receptor Dynamic Conformational Ensembles

“…The importance of these residues for selectivity to the D 2 1 receptor has been shown in the mutagenesis studies. 47,50 In addition, we have identified four partially distinct sites for the receptors of group 1, i.e. aromatic (4), hydrophobic (5), acceptor (12), and donor (13) The available mutagenesis and structure−activity relationships studies in the literature suggest the importance of several residues, identified above, in the ligand selectivity to the bioaminergic receptors.…”

Dopaminergic isoquinolines with hexahydrocyclopenta[ ij ]-isoquinolines as D 2 -like selective ligands

“…13 illustrates the most relevant hydrophobic interactions that contribute to the stabilization process. Since the active site at D 1 DR was greater than at D 2 DR [40,41], it is likely that compound interactions were favored to D 2 DR vs. D 1 (Fig. 12).…”