Abstract
The angiotensin II type 1 receptor (AT1R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT1R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) β via its association with Gαq/11, leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT1R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT1R, but that RalA expression is required for AT1R-stimulated IP formation but not 5-HT2A receptor-mediated IP formation. AT1R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the β-arrestin-dependent plasma membrane translocation of RalGDS. Gαq/11 small interfering RNA (siRNA) treatment also significantly attenuates both AT1R- and 5-HT2A receptor-stimulated IP formation after 30 min of agonist stimulation. PLC-δ1 has been reported to be activated by RalA, and we show that AT1R-stimulated IP formation is attenuated after PLC-δ1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC-δ1 stimulation.
Footnotes
This work was supported by the Heart and Stroke Foundation of Ontario [Grant T-5933]; an Ontario Graduate Student Science and Technology Studentship (to C.G.); a Tier I Canada Research Chair in Molecular Neurobiology (to S.S.G.F.); and Career Investigator Status with the Heart and Stroke Foundation of Ontario (to S.S.G.F.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.061069
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ABBREVIATIONS:
- AT1R
- angiotensin II type 1 receptor
- GPCR
- G protein-coupled receptor
- AngII
- angiotensin II
- IP3
- inositol-1,4,5-trisphosphate
- PLC
- phospholipase C
- CaM
- calmodulin
- mGluR
- metabotropic glutamate receptor
- IP
- inositol phosphate
- siRNA
- small interfering RNA
- RalGDS
- Ral guanine nucleotide dissociation stimulator
- HEK
- human embryonic kidney
- BRET
- bioluminescence resonance energy transfer
- IP
- inositol phosphate
- GFP
- green fluorescent protein
- YFP
- yellow fluorescent protein
- DMEM
- Dulbecco's modified Eagle's medium
- HBSS
- Hanks' balanced salt solution
- 5-HT
- serotinin
- 5-HT2AR
- serotinin 2A receptor
- DMSO
- dimethyl sulfoxide.
- Received September 16, 2009.
- Accepted December 16, 2009.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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