Abstract
Serotonin (5-HT)2C receptor is a Gq-coupled receptor exhibiting a high degree of constitutive activity toward phospholipase C effector pathway, a process regulated by receptor mRNA editing. In addition to G protein-dependent signaling, 5-HT2C receptors also activate the extracellular signal-regulated kinase (ERK) 1/2 pathway independently of receptor coupling to G proteins. Constitutive activity at ERK signaling has not yet been explored. Transient expression of unedited 5-HT2C-INI receptors in human embryonic kidney (HEK) 293 cells resulted in a marked increase in ERK1/2 phosphorylation compared with nontransfected cells. No increase in ERK1/2 phosphorylation was measured in cells expressing fully edited (5-HT2C-VGV) receptors. Basal ERK1/2 phosphorylation in 5-HT2C-INI receptor-expressing cells was abolished by 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553), a 5-HT2C inverse agonist toward phospholipase C. This effect was prevented by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which alone did not alter basal activity. Similar observations were made in vivo in mouse choroid plexus, a structure rich in constitutively active 5-HT2C receptors. Reminiscent of agonist-induced ERK1/2 phosphorylation, basal activity in HEK 293 cells was unaffected by cellular depletion of Gαq/11 and Gα13 proteins but strongly reduced in cells expressing a dominant-negative β-arrestin or depleted of β-arrestin by RNA interference and in cells expressing a dominant-negative calmodulin or a 5-HT2C-INI receptor mutant not capable of interacting with calmodulin. The tetracyclic antidepressants mirtazapine and mianserin likewise reduced basal ERK activation. On the other hand, the m-chlorophenylpiperazine derivative trazodone and the selective serotonin reuptake inhibitor fluoxetine were inactive alone but blocked 5-HT-induced ERK1/2 phosphorylation. Together, these data provide the first evidence of constitutive activity of a G protein-coupled receptor toward G-independent, β-arrestin-dependent, receptor signaling.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Servier Pharmaceuticals; la Fondation pour la Recherche Médicale [Grants Equipe FRM 2005; Equipe FRM 2009]; and fellowships from the French Minister of Research and la Fondation pour la Recherche Médicale (to M.L.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.066035.
-
ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- CaM
- calmodulin
- ERK
- extracellular signal-regulated kinase
- HEK
- human embryonic kidney
- 5-HT
- 5-hydroxytryptamine
- IP
- inositol phosphate
- PL
- phospholipase
- SSRI
- selective serotonin reuptake inhibitor
- DMSO
- dimethyl sulfoxide
- YFP
- yellow fluorescent protein
- GFP
- green fluorescent protein
- ANOVA
- analysis of variance
- PBS
- phosphate-buffered saline
- DN
- dominant negative
- SL327
- a-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile
- BIM-46,187
- 7-[2-amino-1-oxo-3-thio-propyl]-8-cyclohexylmethyl-2-phenyl-5,6,7,8-tetrahydro-imidazo-[1,2a]-pyrazine dimer, hydrochloride
- SB206,553
- 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole
- SB242,084
- antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline.
- Received May 3, 2010.
- Accepted August 10, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|