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


CONCLUSIONS
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.
Steiner
Jennifer A
JA
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. jennifer.steiner@med.lu.se
Carneiro
Ana Marin D
AM
Wright
Jane
J
Matthies
Heinrich J G
HJ
Prasad
Harish C
HC
Nicki
Christian K
CK
Dostmann
Wolfgang R
WR
Buchanan
Carrie C
CC
Corbin
Jackie D
JD
Francis
Sharron H
SH
Blakely
Randy D
RD
D000818:Animals; D000918:Antibody Specificity / Q000187:drug effects; D000928:Antidepressive Agents / Q000494:pharmacology*; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D014162:Transfection
D000928:Antidepressive Agents; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D050486:Serotonin Plasma Membrane Transport Proteins; C016276:8-bromocyclic GMP; D012701:Serotonin; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D006152:Cyclic GMP