Abstract
BackgroundThe 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.ResultsIn 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, PKGIα specifically associates with hSERT.ConclusionOur findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα 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.
Highlights
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 5HT after release
Expression and colocalization of PKGI with SERT in RN46A cells To initiate studies of PKG gene products engaged in neuronal SERT regulation, we explored the expression of PKG isoforms in RN46A cells, a serotonergic cell line that we have previously found to display 8-Br-cGMP-stimulated SERT activity
In keeping with a lack of detection of PKGII in the RN46A cells, low signal for PKGII was evident by immunofluorescence (Fig. 1E, middle panel) and was detected as a nuclear pattern surrounded by diffuse cytoplasmic labeling
Summary
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 5HT after release. Further specification and localization of the key molecules involved in SERT regulation, clarification of whether they form stable or transient associations with SERT, and evaluation of how loss of regulation contributes to SERT dysfunction may offer important insights into 5-HT-linked brain disorders such as depression, autism, and obsessive-compulsive disorder (OCD) [12,13,14,15] In this regard, we and others have shown that multiple human SERT coding variants display compromised regulation through protein kinase C (PKC)-, cGMP-dependent protein kinase (PKG)-, and p38 mitogen-activated protein kinase (p38 MAPK)-linked pathways [16,17,18,19]. Among these coding variants are mutants found to associate with autism and OCD [20,21]
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