cGMP-dependent protein kinase Ialpha associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

Abstract

Background: The Na(+)/Cl(-)-dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.

Results: In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIalpha specifically associates with hSERT.

Conclusion: Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIalpha that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIalpha supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.

Figure 1

Colocalization of SERT and PKGI in RN46A cells. A) Total RNA from RN46A cells or rat midbrain was isolated and subjected to RT-PCR as described in Methods. Figure shows agarose gel analysis of cDNA products, revealing expression of PKGI but very little PKGII. B-E) Double immunocytochemical labeling of SERT, PKGI, and PKGII. RN46A cells were fixed, stained, and imaged by confocal microscopy as described in Methods. SERT staining was achieved with an ectodomain antibody (Advanced Targeting AB-N09). NP, nonpermeabilized. P, permeabilized by 0.2% NP40. NP/B, nonpermeabilized with antibody preabsorbtion by peptide (Advanced Targeting PR-03). DIC image is inset in B, middle, to demonstrate presence of cells. Inset image in D, right, shows staining in process. Closed arrows indicate perinuclear staining while open arrows indicate process staining. Scale bars represent 10 μm. Data are representative of at least three independent experiments.

Figure 2

Quantitative analysis of SERT/PKGI colocalization. Cells were fixed and stained as shown representatively in Figure 1, and the Intensity Correlation Quotient was calculated for both cell bodies and processes under either nonpermeabilized (NP) or permeabilized (P) conditions, as described in Methods. Data represent mean values ± SEM from multiple cells in 4–6 fields, replicated in at least three experiments. Statistical analysis was conducted by one-sample t test. *, p < 0.001.

Figure 3

DT-2 sensitive, PKGI activity in RN46A cells. A) DEAE chromatography and phosphorylation assays of RN46A cell extracts were performed as described in Methods. Chromatogram displays cGMP-dependent (background-subtracted) kinase activity and quantitative protein measurement. cGMP-independent kinase activity was measured as 27.3% of total kinase activity. Representative immunoblot of fractions with detectable kinase activity is shown below chromatogram. B) Evaluation of DT-2 sensitivity in fractions containing detectable (13–17) or nondetectable (18–22) PKGI protein by immunoblot. Both fraction bins 13–17 and 18–22 displayed DT-2-sensitive kinase activity (***, p < 0.001; **, p < 0.01), with fractions overlaying PKGI protein being significantly more sensitive to DT-2 (***, p < 0.001). Dashed line represents cGMP-independent kinase activity equivalent to 27.3%. Data shown are from a single experiment, are representative of three independent experiments, and are presented as means ± SEM. Statistical significance was calculated via two-way ANOVA, followed by Bonferroni's post-test.

Figure 4

Treatment of transfected HeLa cells with PKGI siRNA prevents 8-Br-cGMP-induced SERT stimulation. Cells were transfected with hSERT and siRNA directed against human PKGI and assayed for 8-Br-cGMP stimulation of SERT activity as described in Methods. Upon 8-Br-cGMP stimulation, cells expressing hSERT (closed bar) demonstrate elevated 5-HT transport activity (147 ± 3.56%) absent from cells treated with PKGI siRNA (dotted bar, 96.4 ± 6.96%). Cells treated with siRNA in the absence of 8-Br-cGMP (checkered bar) show no change in 5-HT uptake (94.5 ± 1.88%) from control (open bar, 100 ± 0.010%). Each assay was repeated three times. Values are means ± SEM. ***, p < 0.001 versus control, calculated via one-way ANOVA followed by Tukey's post-test.

Figure 5

hSERT forms a stable association with PKGIα. hSERT and HA-PKGIα or HA-PKGIβ were coexpressed in HEK-293 T cells and coimmunoprecipitations were performed as described in Methods. Total cell extracts were blotted for A) HA to reveal PKG expression or B) SERT. C) Co-immunoprecipations of SERT and PKG using anti-HA for immunoprecipitations and anti-SERT for blotting. Experiment shown was replicated in four independent experiments with similar results.