Abstract
G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Gα protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.
Footnotes
This work was supported by the G-COE program and grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); The Support Program for Improving Graduate School Education from MEXT (A.O.), and The Takeda Science Foundation and The Uehara Memorial Foundation (H.O.).
The entire sequences of G2A-a and G2A-b mRNAs were deposited in the DNA Data Bank of Japan, accession number AB465599 for G2A-a and AB465600 for G2A-b.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.158758
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ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- ATRA
- all-trans-retinoic acid
- SRE
- serum response element
- CRE
- cAMP response element
- OGR1
- ovarian cancer G protein-coupled receptor 1
- GPR4
- G protein-coupled receptor 4
- TDAG8
- T-cell death-associated gene 8
- LPC
- lysophosphatidylcholine
- IP
- inositol phosphate
- 9-HODE
- 9-hydroxyoctadecadienoic acid
- 11-HETE
- 11-hydroxyeicosatetraenoic acid
- RACE
- rapid amplification of cDNA ends
- RT
- room temperature
- BSA
- bovine serum albumin
- IP1
- inositol 1-phosphate
- MES
- 2-(N-morpholino)ethanesulfonic acid
- EPPS
- 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid
- NCBI
- National Center for Biotechnology Information
- PCR
- polymerase chain reaction.
- Received July 10, 2009.
- Accepted October 23, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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