Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A

Abstract

Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.

Fig. 1.

Identification of SERT/PP2Ac association in transfected cells. A, OA-sensitive phosphatase activity is present in SERT immunoprecipitates. Phosphatase activity was measured in SERT (#48) immune complexes (n = 3), as described in Materials and Methods, and compared with activity found with nonimmune sera (*p < 0.05, Student's ttest). B, PP2Ac coimmunoprecipitates with hSERT in stably transfected HEK-293 cells. Total cell extracts from 293 hSERT cells (250 μg) were immunoprecipitated overnight with SERT immune sera (#48) or preimmune sera (PI), and the resulting blot was probed with PP2Ac monoclonal antibody as described in Materials and Methods. C, Multiple SERT antisera immunoprecipitate PP2Ac. Top panel, SERT/PP2Ac complexes immunoprecipitated using multiple SERT antisera. In addition to Ab #48, PP2Ac immunoreactivity was detected in immunoprecipitates using either SERT sera #50 orCT2B (lanes 2, 3) but was significantly reduced in immunoprecipitations using nonimmune sera (NI) or #50 sera preabsorbed with 100 μg/ml peptide. Bottom panel, PP2Ac immunoreactivity is enriched in 293-hSERT cells over parental HEK-293 cells. Levels of PP2Ac in parental HEK-293 cell immunoprecipitates were equivalent to that seen with preimmune serum. D, SERT/PP2Ac immune complexes (Ab #48) are formed in COS-7 cells transiently transfected with hSERT cDNA as compared with pcDNA3 vector. E, SERT/PP2Ac complexes are detected in plasma membrane-enriched preparations. 293-hSERT cells were biotinylated using membrane-impermeant NHS-biotin and surface proteins isolated on monomeric streptavidin beads as described in Materials and Methods. Associated proteins were eluted with 2 mm free biotin and immunoprecipitated overnight. Blots of immunoprecipitates reveal SERT/PP2Ac complexes that were diminished in immunoprecipitations using preimmune sera (PI). The bottom panels show representative blots of proteins from the eluted fractions, the last wash (LW), or total extracts probed with antibodies against PP2Ac, SERT, Na⁺/K⁺ ATPase (as a plasma membrane marker), or calnexin (as an intracellular marker) used to determine plasma membrane-enriched fractions. Quantitation of protein immunoreactivities reveals a fivefold enrichment of Na⁺/K⁺ ATPase over calnexin, consistent with predominant recovery of cell-surface proteins in biotinylated fractions.

Fig. 2.

Identification of SERT/PP2Ac associations in native tissues. A, Midbrain synaptosomes (100 μg) were preincubated with vehicle (DMSO, veh), OA(1 μm), or 1-norokadone (NO, 1 μm) for 30 min at 37°C and then assayed for 5-HT (2 μm final, 10 min) uptake as described in Materials and Methods. Data are the average ± SEM of three separate experiments. Nonspecific uptake was defined as the uptake in the presence of 0.1 μm paroxetine and subtracted from the total accumulation to yield specific uptake. *p < 0.05, Student's t test. B, SERT/PP2Ac complexes identified in mouse midbrain synaptosomes. Adult mouse midbrain synaptosomal protein (500 μg) was immunoprecipitated overnight with preimmune sera (lane 1) or SERT CT2B sera (lane 2), and the resulting Western blot was probed for PP2Ac. Right panel, Immunoblot of total mouse midbrain synaptosomal protein probed with the PP2Ac monoclonal antibody.C, Coimmunoprecipitation of PP2Ac from rat tissues. Representative blot showing PP2Ac immunoreactivity recovered from immunoprecipitations from 500 μg rat midbrain (mb) (lanes 1, 2), cerebellum (cb) (lanes 3, 4), skeletal muscle (mus) (lanes 3,4), or cortex (ctx) (lanes 5, 6) using SERT immune sera CT2B (I) or preimmune sera (PI). Bottom panel shows similar levels of PP2Ac in all tissue extracts.

Fig. 3.

PP2A inhibitors regulate PP2Ac/SERT associations in intact cells. A, 293-hSERT cells treated with various phosphatase inhibitors display inhibitor-specific effects on SERT/PP2Ac associations. 293-hSERT cells were incubated for 30 min at 37°C in the presence or absence of phosphatase inhibitors: OA (1 μm), norokadone (NO, 1 μm), calyculin A (CA, 1 μm), tautomycin (TA, 1 μm), or cyclosporine A (CY, 5 μm). Detergent extracts were immunoprecipitated with either SERT 48 immune or preimmune sera, and the resulting blot was probed for PP2Ac. B, Dose–response of OA for the disruption of SERT/PP2Ac association in whole-cell preparations. 293-hSERT cells were treated with increasing concentrations of OA for 30 min at 37°C. Detergent extraction, immunoprecipitation with SERT antibody 48, and autoradiography were performed as described in Materials and Methods. Bottom panel shows a representative Western blot of total PP2Ac protein from 293-hSERT cell extracts treated with 0, 0.5, or 1.0 μm OA. C, OA pretreatment diminishes PP2Ac in SERT immunoprecipitates from mouse midbrain synaptosomes. Synaptosomes were incubated with 1 μm OA for 30 min and immunoprecipitated with either preimmune (PI) or SERT immune sera (48), and the resulting blot was probed for PP2Ac as described in Materials and Methods.

Fig. 4.

PKC activation disrupts SERT/PP2Ac associations.A, Treatment of cells with phorbol esters decreases SERT/PP2Ac associations. 293-hSERT cells were incubated for 30 min in the presence or absence of 1 μm β-PMA. Cells were solubilized and immunoprecipitated with SERT antibody (48), and the resulting blot was probed for PP2Ac as described in Materials and Methods. The PKC inhibitor staurosporine (1 μm) was added 30 min before the addition of β-PMA. Averaged data from three separate experiments are presented ± SEM. *p< 0.05, as determined by Student's t test. Representative blots are shown of SERT/PP2Ac coimmunoprecipitation using preimmune sera (PI) or SERT antibody (48) from cells treated with β-PMA (1 μm) or β-PMA plus staurosporine (1 μm) and total cell PP2Ac under various conditions. B, 5-HT attenuates the β-PMA-induced decrease in associated PP2Ac. SERT/PP2Ac complexes were immunoprecipitated from 293-hSERT cells treated with 250 nm5-HT or buffer for 20 min at 37°C. The resulting blot was probed for PP2Ac as described. The bar plot displays averaged data from three separate experiments and are presented ± SEM. *p < 0.05, as determined by a Student'st test.

Fig. 5.

PP2Ac associates with cocaine-sensitive norepinephrine and dopamine transporters. A, Norepinephrine (NE) uptake in rat vas deferens is sensitive to PKC activation and PP2A inhibition. Pretreatment of rat vas deferens slices with 1 μm β-PMA or 1 μm OA for 30 min significantly reduces specific NE uptake. Specific NE uptake was determined in rat vas deferens as described in Materials and Methods. Data are presented as mean ± SEM of three separate experiments performed in triplicate.B, OA (1 μm) treatment reduces DA clearance in rat vas deferens. Representative effect of 1 μm OA (30 min) on DA oxidation signals is presented. Application and electrochemical recordings were performed as described in Materials and Methods. Darker arrow indicates time of DA application. The amplitude (micrometers),T₈₀ (seconds), time course (seconds), and clearance rate (micrometers per second) are 5.4 ± 0.62, 52 ± 4.0, 63 ± 7, and 0.22 ± 0.01, respectively, for controls and 5.3 ± 0.31, 68 ± 3*, 76 ± 5*, and 0.14 ± 0.01* in the presence of OA. Desipramine (DMI; 1 μm), but not 0.3 μm GBR 12909, alters DA signal (amplitude,T₈₀, time course, and clearance rate are 5.3 ± 0.2, 42 ± 4, 59 ± 3, and 0.18 ± 0.12, respectively, for controls and 5.9 ± 0.2, 58 ± 5*, 76 ± 5*, and 0.11 ± 0.11* in the presence of DMI). * indicates significant difference as compared with controls, p< 0.05, Student's unpaired t test. C, β-PMA- and OA-sensitive coimmunoprecipitation of PP2Ac with NETs in rat vas deferens. Triton X-100-soluble membrane fractions were incubated overnight with NET antisera (43411), and the resulting immunoblot was probed for PP2Ac as described in Materials and Methods. Pretreatment of rat vas deferens slices with 1 μm β-PMA or 1 μm OA markedly reduces association of PP2Ac with NETs, with no alteration in total PP2Ac levels. D, Pretreatment of rat vas deferens slices with 1 μm β-PMA or 1 μm OA produces a significant reduction in the association of PP2Ac with NETs. Averaged band density ± SEM of three separate experiments as percentage of vehicle controls is presented. Asterisks represent a significant difference (p < 0.05, one-tailed Student'st test) as compared with vehicle (Veh) controls. E, Specificity of NET/PP2Ac association. Triton X-100-soluble membrane fractions of rat vas deferens were incubated overnight with affinity-purified 43411 NET antibody, and the resulting immunoblot was probed for PP2Ac as described in Materials and Methods. Preadsorption of antibody with immunogenic peptide abolishes the coimmunoprecipitation of PP2Ac. F, Immunoprecipitation of PP2Ac with DAT antisera in mouse striatal synaptosomes. Triton X-100-soluble synaptosomal membrane extracts were incubated with either nonimmune (NI) or DAT-immune (I) sera, and the resulting blot was probed for PP2Ac monoclonal antibody as described in Materials and Methods.