Abstract
Activation of A3 adenosine receptors (A3ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A3AR agonist stimulation of SERT activity is lost in A3AR knockout mice. A3AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A3ARs target SERTs among other potential effectors is unknown. Here we present evidence that A3ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A3AR/hSERT cotransfected cells. Treatment of A3AR/SERT-cotransfected Chinese hamster ovary cells with the A3AR agonist N6-(3-iodobenzyl)-N-methyl-5′-carbamoyladenosine (1 μM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A3AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A3AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A3AR activation. Because PKGI and nitric-oxide synthetase are required for A3AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A3AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by the National Institutes of Health National Center for Research Resources [Grant UL1-RR024975] (to C.-B.Z.); the National Institutes of Health National Institute on Drug Abuse [Grant R01-DA07390] (to R.D.B.); the National Institutes of Health National Institute of Mental Health [Grants R01-MH078028, R01-MH94527] (to R.D.B.); the National Science Foundation [Grant NS049261] (to J.A.S.); and the OCD/TS Program at Vanderbilt University (to W.A.H.). The Vanderbilt University Medical Center Cell Imaging Shared Resource is supported by the National Institutes of Health National Cancer Institute [Grant CA68485]; the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grants DK2093,DK58404, DK59637]; the National Institutes of Health National Institute of Child Health and Human Development [Grant HD15052]; and the National Institutes of Health National Eye Institute [Grant EY08126].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.071399.
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ABBREVIATIONS:
- 5-HT
- 5-hydroxytryptamine (serotonin)
- SERT
- serotonin transporter
- OCD
- obsessive-compulsive disorder
- GPCR
- G-protein coupled receptor
- PKGI
- protein kinase G, type I
- PKGII: protein kinase G
- type II
- MAPK
- mitogen-activated protein kinase
- A3AR
- A3 subtype adenosine receptor
- CNS
- central nervous system
- IB-MECA
- N6-(3-iodobenzyl)-N-methyl-5′-carbamoyladenosine
- HA
- hemagglutinin
- PBS
- phosphate-buffered saline
- CHO
- Chinese hamster ovary
- coIP
- coimmunoprecipitation
- PBS/CM
- PBS containing Ca2+ and Mg2+
- GAD
- glutamic acid decarboxylase
- ASD
- autism spectrum disorder
- NOS
- nitric-oxide synthetase
- PCR
- polymerase chain reaction
- ANOVA
- analysis of variance
- DT-2
- YGRKKRRQRRRPP-LRK5H
- MRS1191
- 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate.
- Received February 1, 2011.
- Accepted June 24, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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