Transmembrane segment five serines of the D4 dopamine receptor uniquely influence the interactions of dopamine, norepinephrine, and Ro10-4548

Abstract

Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with alpha(1)- and alpha(2)-adrenergic, beta(2)-adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional properties for all of them. Dopamine was unable to activate the S5.46A mutant and had reduced potency for the S5.43A mutant and reduced potency and efficacy for the S5.42A mutant. In contrast, Ro10-4548 [RAC-2'-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide], a catechol-like antagonist of the wild-type receptor unexpectedly functions as an agonist of the S5.43A mutant. Other noncatechol ligands had similar properties for mutant and wild-type receptors. This is the first example of a dopamine receptor point mutation selectively changing the receptor's interaction with a specific antagonist to that of an agonist, and together with other data, provides evidence, supported by molecular modeling, that catecholamine-type agonism is induced by different ligand-specific configurations of intermolecular H-bonds with the TM5 conserved serines.

Fig. 1.

Chemical structures of the ligands investigated.

Fig. 2.

All three TM5 serine-to-alanine mutant receptors have significantly reduced affinities for both dopamine and (−)-norepinephrine. These graphs are the cumulative data of three separate experiments with dopamine (A), (−)-norepinephrine (B), the D4-selective ligand Ro10-4548 (C), or (−)-quinpirole (D) in competition with 0.5 nM [³H]methylspiperone at the wild-type D4 (●), D4-S5.42A (□), D4-S5.43A (▵), or D4-S5.46A (♢) receptors. Data points are graphed as the geometric mean ± S.E.M. Corresponding affinity values are listed in Table 2. A much greater reduction (49-fold more) in relative affinity was observed for (−)-norepinephrine (346- versus 7-fold for dopamine) at the D4-S5.43A mutant receptor.

Fig. 3.

Dopamine, but not quinpirole, had drastically reduced function at TM5 serine-to-alanine mutants. Graphed are the cumulative data of three separate experiments that measure the ability of dopamine (A) or (−)-quinpirole (B) to activate wild-type D4 (●), D4-S5.42A (□), D4-S5.43A (▵), and D4-S5.46A (♢) receptors. Data points are graphed as the geometric mean ± S.E.M of accumulated cAMP that was normalized to the maximal cAMP accumulated in the presence of unopposed 6 μM forskolin. Corresponding potency values are listed in Table 2. Note that (−)-quinpirole-mediated D4 receptor inhibition of cAMP accumulation was not significantly affected by the TM5 serine-to-alanine mutations.

Fig. 4.

Screening ligand functional properties at the wild-type and mutant D4 receptors revealed an unexpected gain of function for Ro10-4548 at the D4-S5.43A receptor. Single high concentrations (10 μM) of dopamine, (−)-norepinephrine, (−)-quinpirole, CP226,269, PD168,077, and Ro10-4548 were tested for their ability to mediate cAMP functional responses at wild-type and mutant D4 receptors. At a concentration of 10 μM, (−)-norepinephrine acts as an agonist at the wild-type D4 receptor, but loses agonist activity at all TM5 mutant receptors. Dopamine has a pronounced loss of agonist activity at the S5.42A and S5.46A mutants and reduced activity at the S5.43A mutant. The agonist activity of CP226,269, PD168,077, and (−)-quinpirole for all of the mutant receptors is similar to the wild-type receptor. For all agonist–receptor pairings, the antagonist spiperone was able to inhibit the functional response. No functional response was observed in untransfected CHO10001 cells (data not shown). Data are plotted as the mean ± S.E.M. of three separate experiments normalized to the concentration of accumulated intracellular cAMP generated by unopposed 6 μM forskolin. Significance relative to the wild-type D4 receptor was determined by one-way ANOVA with Dunnett's post hoc (*, p < 0.05).

Fig. 5.

The conserved serine microdomain of TM5 is critical for (−)-norepinephrine-mediated activation of the D4 receptor. Functional assays with a concentration of (−)-norepinephrine (250 μM) that should saturate or nearly saturate even the mutant receptors, which have reduced affinity for (−)-norepinephrine, revealed that none of the mutant D4 receptors can be activated by (−)-norepinephrine. Surprisingly, (−)-norepinephrine was also unable to prevent 100 nM (−)-quinpirole from functionally activating the mutant receptors. Individual data sets were normalized to the concentration of accumulated intracellular cAMP generated by unopposed 6 μM forskolin and graphed as the geometric mean ± S.E.M. of three separate experiments. Significance relative to the wild-type D4 receptor was determined by one-way ANOVA with Dunnett's post hoc (*, p < 0.05).

Fig. 6.

ABT-724, PNU-101,387G, L-750,667, and Ro-10-5824 exhibit antagonist properties at wild-type and mutant D4 receptors. Saturating concentrations of L-750,667, ABT-724, Ro10-5824, and PNU101,387G were tested, alone or in competition with the agonist (−)-quinpirole, for D4 receptor-mediated inhibition of forskolin-induced intracellular cAMP accumulation. In all cases, L-750,667, ABT-724, Ro10-5824, and PNU101,387G failed to elicit a change in forskolin-stimulated cAMP responses, but were able to block 100 nM (−)-quinpirole from activating the receptors. Individual data sets were normalized to the concentration of accumulated intracellular cAMP generated by unopposed 6 μM forskolin and graphed as the geometric mean ± S.E.M. of three separate experiments. Significance relative to the wild-type D4 receptor was determined by one-way ANOVA with Dunnett's post hoc (*, p < 0.05).

Fig. 7.

Mean intermolecular contact maps for catechol/catechol-like ligand poses at dopamine receptor constructs. Mean contact maps were computed from pose subsets that pass the filter for each ligand–receptor system examined by docking. The fingerprints of intermolecular contacts are shown as colored matrices for mean H-bonding frequency (A, B, C, and E) with color indicating the relative frequency of observing an intermolecular H bond at a particular cleft residue site from red (high) to yellow (moderate) to green (low). Alternatively, mean van der Waals contacts are displayed in matrix D using an alternative color spectrum from red (high) to yellow (moderate) to blue (low). Residue positions near the cleft are indexed on the left for D4 wild type. The homologous positions for rat D1–4 sequences are listed on the right with gray highlights to emphasize the regions of sequence heterogeneity. A, the mean H-bond map for dopamine at the D4 receptor constructs. B, the mean H-bond map for epinephrine at the D4 receptor constructs. C, the mean H-bond contact map for Ro10-4548 at the D4 receptor constructs. D, the mean van der Waals contact maps for dopamine at wild-type receptor subtypes D1–4. E, the mean H-bond map for dopamine at wild-type receptor subtypes D1–4. At the bottom of each column, N indicates the number of poses in the subset.

Fig. 8.

Filtered pose distributions for catechol and catechol-like ligands at the D4 receptor constructs. Shown are the positions of the catechol and catechol-like moieties from all poses that pass the filter in the following systems: dopamine at D4-WT (A), S5.42A (B), S5.43A (C), and S546A (D); norepinephrine at D4-WT (E), S5.42A (F), S5.43A (G), and S546A (H); and Ro10-4548 at D4-WT (I), S5.42A (J), S5.43A (K), and S5.46A (L). For wild type (A, E, and I), the aromatic microdomain residues 5.47, 6.48, 6.51, 6.52, and 6.55 are shown as dark spheres in addition to the critical D3.32 residue on TM3 that makes an H-bond-reinforced ionic interaction with the amine ligands. The light spheres represent the general positions of the conserved TM5 serines viewing from the bilayer. Only the initial backbone conformation is depicted as a ribbon for reference, although backbone flexibility was allowed in the docking procedure. Only ligands' catechol and catechol-like groups are displayed as dark sticks with white oxygen atoms; the remaining ligand atoms were removed for clarity. Note in J the two poses are almost superimposed.