GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs

Abstract

The orphan G protein-coupled receptor 37 (GPR37) is a substrate of parkin; its insoluble aggregates accumulate in brain samples of Parkinson's disease patients. We report here that GPR37 interacts with the dopamine transporter (DAT) and modulates DAT activity. GPR37 and DAT were found colocalized in mouse striatal presynaptic membranes and in transfected cells and their interaction was confirmed by coimmunoprecipitation assays. Gpr37-null mutant mice showed enhanced DAT-mediated dopamine uptake in striatal membrane samples, with a significant increase in the number of plasma membrane DAT molecules. The null mutant mice also exhibited a decrease in cocaine-induced locomotor activity and in catalepsy induced by dopamine receptor antagonists. These results reveal the specific role of GPR37, a putative peptidergic G protein-coupled receptor, in modulating the functional expression of DAT and the behavioral responses to dopaminergic drugs.

Fig. 1.

GPR37 is enriched in the mouse striatal presynaptic fraction. (A) Western blot analysis of GPR37 and DAT proteins was performed in whole synaptosome (WS, lane 1), plasmalemmal membrane (PM, lane 2), and synaptic vesicle (SV, lane 3) fractions, from the pooled striata of C57BL/6J mice. An aliquot (60 μg) of each fraction was immunoblotted with antibodies specific for GPR37, DAT, and the fraction markers. Both GPR37 and DAT immunoreactive bands are enriched in the plasmalemmal membrane fraction. (B) Western blot analysis of GPR37 and DAT proteins was performed in synaptic membrane fractions, including whole synaptosomes (lane 1), extrasynaptic (lane 2), presynaptic (lane 3), and postsynaptic (lane 4) membrane-enriched fractions, from the pooled striata of C57BL/6J mice. An aliquot (10 μg) of each fraction was immunoblotted by using antibodies specific for the indicated proteins. GPR37 is enriched in the presynaptic fraction. The monoclonal anti-GPR37 antibody labels different N-glycosylation variants of the receptor protein (3).

Fig. 2.

Gpr37^−/− mice exhibit enhanced striatal [³H]DA uptake. (A) [³H]DA (18 nM) uptake by striatal mouse synaptosomes from wild-type (+/+) and Gpr37^−/− (−/−) mice. Data from quadruplicate samples were corrected for unspecific uptake measured in the presence of 10 μM nomifensine and are presented as the mean ± SEM of three separate experiments. (∗, P < 0.05; unpaired t test). (B) Kinetic analysis of [³H]DA uptake in striatal synaptosomes of wild-type (+/+) and Gpr37^−/− male mice. [³H]DA (18 nM) and increasing concentrations of unlabeled DA (from 0.005 to 1 mM) were applied simultaneously. Nonspecific DA uptake was determined in parallel experiments in the presence of 10 μM nomifensine. Data are expressed as [³H]DA uptake in fmol mg⁻¹min⁻¹ and presented as mean ± SEM of three separate experiments, each performed in duplicate. (C) Saturation binding curve of [N-methyl-³H]WIN-35428 to DAT in total striatal extracts from wild-type (+/+) and Gpr37^−/− (−/−) mice. K_d values were 6.55 ± 0.93 × 10⁻⁹ M (+/+) and 5.18 ± 1.10 × 10⁻⁹ M (−/−), whereas B_max values were 2,144 ± 122.3 fmol/mg (+/+) and 1,761 ± 135.3 fmol/mg (−/−, P < 0.05; F test). Data are presented as mean ± SEM of three separate experiments, each performed in duplicate.

Fig. 3.

Gpr37^−/− mice exhibit increased cell-surface levels of DAT. (A) Cell-surface biotinylation experiments were performed in striatal synaptosomes from wild-type (+/+) and Gpr37^−/− (−/−) mice. Equal aliquots of whole striatal synaptosome samples were incubated with sulfo-NHS-biotin. After sonication, biotinylated proteins were isolated with streptavidin beads. One-tenth of the total lysate fractions was used to detect total immunoreactive DAT, and samples were analyzed by SDS/PAGE, followed by Western blot with the anti-DAT antibody. Results shown are representative of four independent experiments. (B) Quantitation of biotinylated immunoreactive bands expressed as percentage of DAT surface density in wild-type (+/+) and Gpr37^−/− (−/−) striatal synaptosomes. Immunoblots from four separate biotinylation experiments were imaged and quantified, after factoring aliquot volumes and sample handling protocol, mean ± SEM values were plotted (+/+, 39.8 ± 2.9%; −/−, 67.9 ± 4.1%). The Gpr37^−/− samples show a significant increase of biotinylated DAT protein (∗∗, P < 0.01; unpaired t test).

Fig. 4.

GPR37 coprecipitates with DAT in transfected HEK293-hDAT cells. HEK293-hDAT cells were transfected with linearized plasmid vector or the GPR37-HA construct. Immunoprecipitations (IP) were performed with anti-HA antibody (Upper) or the anti-DAT monoclonal antibody (Lower), followed by Western blot detection (IB) with anti-DAT or anti-HA, respectively. Total cell lysates were analyzed by Western blotting using specific antibodies to show the expression levels of DAT and GPR37-HA. A representative result from three experiments is shown.

Fig. 5.

Cocaine-induced locomotor activity. Gpr37^−/− mice (−/−) showed a lower response to the administration of 20 mg/kg cocaine compared with their wild-type (+/+) littermates (∗∗, P < 0.01; ∗∗∗, P < 0.001; post hoc Tukey's HSD for unequal n). Symbols represent the mean (±SEM) distance traveled every 10 min after the i.p. injection of either saline (+/+, n = 10; −/−, n = 10), 10 mg/kg cocaine (+/+, n = 10; −/−, n = 10), or 20 mg/kg cocaine (+/+, n = 14; −/−, n = 18). Data are expressed as percentage of baseline locomotor activity measured before the injection (last 10-min interval of habituation phase, data not shown). (Inset) Effect of cocaine treatment on Gpr37^−/− mice locomotor activity. Bars represent the mean (±SEM) distance traveled in 90 min, expressed as percentage of baseline locomotor activity measured before the injection for each genotype and compared across treatment groups. (##, P < 0.01, Gpr37^−/− vs. wild type, 20 mg/kg cocaine; ∗∗, P < 0.01; ∗∗∗, P < 0.001; post hoc Fisher's least-squares difference test).

Fig. 6.

Catalepsy bar test. Response of Gpr37^−/− (−/−) and their wild-type (+/+) littermates to catalepsy induced by the systemic administration of the D1 receptor antagonist SCH23390 (+/+, n = 9; −/−, n = 8) (A) and the D2 receptor antagonist haloperidol (+/+, n = 11; −/−, n = 9) (B). Symbols represent the mean (±SEM) time spent with both front paws resting on the bar; a cut off time was set at 120 s (∗, P < 0.05; ∗∗, P < 0.01; Gpr37^−/− vs. wild type; post hoc Fisher's least-squares difference). Differences between doses are not portrayed. BL, baseline latency to move from the bar before the first injection.