Abstract
In response to 5-hydroxytryptamine (5-HT), the type 1 serotonin receptors (5-HT1Rs) preferentially couple to the inhibitory G protein and elicit many physiological and behavioral processes. However, their regulation by intracellular protein kinases has not been fully investigated. In this study, we identified that glycogen synthase kinase-3 (GSK3) differentially regulates 5-HT1Rs. In receptor-expressing cells and brain slices, activation of both 5-HT1AR and 5-HT1BR reduced forskolin-stimulated cAMP production, but only the effect of 5-HT1BR was abolished by selective GSK3 inhibitors, deletion of GSK3β by RNAi, or overexpression of impaired GSK3β mutants (R96A and K85,86A). A consensus GSK3 phosphorylation sequence was identified between the serine-154 and threonine-158 in the second intracellular loop of 5-HT1BR. Mutation of either serine-154 or threonine-158 to alanine significantly reduced response of 5-HT1BR to 5-HT. Active GSK3β interacted with resting 5-HT1BR to form a protein complex. The interaction was enhanced by receptor activation, abolished by GSK3 inhibitors, and dependent on the phosphorylation state of serine-154. In addition, regulation of 5-HT1BR by GSK3 changed the dynamics of agonist-induced cell surface receptor internalization, in which lack of phosphorylation at Ser154 resulted in sustained reduction of 5-HT1BR at the cell surface. Although the physiological consequences of selective regulation of 5-HT1BR by GSK3 remain to be identified, findings in this study reveal a new function of GSK3 as a protein kinase that is able to selectively regulate G protein-coupled receptors.
Footnotes
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This research was supported by the National Institutes of Health National Institute of Mental Health [Grants MH064555, MH073723].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.056994
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ABBREVIATIONS:
- 5-HT
- 5-hydroxytryptamine, serotonin
- ANOVA
- analysis of variance
- BIO
- 6-bromoindirubin-3′-oxime
- BRET
- bioluminescence resonance energy transfer
- CHO
- Chinese hamster ovary
- FACS
- fluorescence-activated cell sorting
- GFP
- green fluorescent protein
- GPCRs
- G protein-coupled receptors
- GRKs
- G protein-coupled receptor kinases
- GSK3
- glycogen synthase kinase-3
- HEK
- human embryonic kidney
- 5-HT1R
- type-1 serotonin receptor
- 5-HT1AR
- type-1 serotonin receptor subtype A
- 5-HT1BR
- type-1 serotonin receptor subtype B
- K85,86A
- mutation of lysine-85 and -86 to alanine
- 8-OH-DPAT
- 8-hydroxy-2-(dipropylamino)tetralin
- PBS
- phosphate-buffered saline
- Rluc
- Renilla reniformis luciferase
- SB216641
- N-[3-[3-(dimethylamino)ethoxy]-4-methoxyphenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1′-biphenyl]-4-carboxamide
- SB216763
- 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione
- SB224289
- 1′-methyl-5-[[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4′-piperidine] oxalate
- shRNA
- short hairpin RNA
- YFP
- yellow fluorescent protein
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- HA
- hemagglutinin
- Cdk
- cyclin-dependent kinase
- i2
- second intracellular loop
- WT
- wild type.
- Received April 10, 2009.
- Accepted September 8, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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