cell signaling by proton-sensing g protein-coupled receptors department of life sciences, national...
TRANSCRIPT
Cell signaling by proton-sensing G protein-coupled receptors
Department of Life Sciences, National Central University, Chungli, TaiwanChia-Wei Huang, Ying-Ju Chen, Wei-Fen Tsai, and Wei-Hsin Sun*
Proton-sensing G protein-coupled receptors (GPCRs) including OGR1, GPR4, TDAG8 and G2A were previously characterized as receptors for lipid mediators, sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine (ref 1-3). The original publications for OGR1, GPR4, and G2A have now been retracted. Whether OGR1, GPR4, and G2A are SPC or LPC receptors remains unclear. In later studies, these four receptors were found to respond to protons with full activation at pH 6.4-6.8. OGR1 and G2A induce pH-dependent IP3 formation, while activation of GPR4 and TDAG8 lead to cAMP accumulation (Ref 4-7). Whether G2A responds to proton is still arguable. Studies carried out by Radu, et al. (2005) have shown that G2A did not generate a significant response after acid stimulation. There is also evidence to suggest oxidized free fatty acid (9-hydroxyoctadecadienoic acid) as a ligand for G2A. To clarify all these discrepancies, we have cloned four proton-sensing GPCR genes and examined their proton and lipid signaling pathways. Except G2A, all the other three receptors respond proton. Interestingly, in the presence of G2A, OGR1-induced calcium signals increased. An intriguing, but speculated possibility is that G2A forms a heterodimer with OGR1 to enhance pH-dependent calcium response.
Introduction
OGR1, GPR4 and TDAG8 are proton-sensing receptors
Results
To confirm the sensitivities of these four genes to proton stimulation, a single cell calcium recording method for OGR1 and G2A, and cAMP assay for GPR4 and TDAG8 were used. HEK293T cells were transfected with pIRES-OGR1, pIRES-G2A, pIRES-GPR4, pIRES-TDAG8, or pIRES for 18-20 hours. For calcium imaging, transfected cells were incubated with Fura-2 (2.5 mM), at 37 for 40 minutes and then stimulated with pH buffers, followed by single cell calcium recording. For c℃AMP assay, the cells were stimulated by balancing buffers containing 30 mM of RO201724 for 30 minutes at 37 , followed by stimulated with .pH buffers (from 7.4 ℃to 5.0). After stimulation, the cells were lysed by pre-cooled ethanol, intracellular cAMP was collected and quantified using ELISA method. Some studies were done in the presence of U73122 and PTX.
OGR1-transfected cells responded proton stimulation to induced a rise of intracellular calcium. The maximum response is reached at pH6.8. OGR1-induced calcium response was blocked by U73122 and PTX. TDAG8-expressing cells responded proton stimulation and cAMP accumulation reached the maximum at pH6.4. TDAG8-induced cAMP accumulation was not blocked by U73122 or PTX. Cells transfected with GPR4 showed pH-dependent cAMP accumulation and the highest response occurred at pH6.8. No calcium increase or cAMP accumulation was detected in cells transefected with G2A or vector only.
To examine whether G2A enhances OGR1 function, HEK293T cells were transfected with pIRES-OGR1 or pIRES-G2A, or co-transfected with pIRES-OGR1 and pIRES-G2A at different doses. Transfected cells were stimulated with different pH buffers (7.6, 7.2, 6.8) to see changes of the intracellular calcium. As shown, OGR1-tranfected and OGR1/G2A co-transfected cells induced similar levels of calcium after stimulation with pH 7.6 buffer. At pH 7.2, the co-transfected cells had a small increase in calcium response, compared to OGR1-transfected. Proton-induced calcium response was largely increased in co-transfected cells at pH6.8. The results suggest that proton-induced calcium responses are enhanced in the presence of OGR1 and G2A.
Acknowledgements
This work was supported by the fund from National Science Council, Taiwan (grant no. NSC 94-2311-B-008-007). We thank the core facility of the biophysics and soft mater at National Central University to support the calcium imaging system.
References
Phase GFP Fura-2
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mG2A IRES
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Co-expression of OGR1 and G2A enhances pH-dependent calcium responses
Conclusions
0.75 mg G2A-I RES / 0.2 mg OGR1-DsredpH 6.8
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0.2 mg OGR1-Dsred / 0.75 mg G2A-I RESpH 7.2
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0.2 mg OGR1-DsredpH 6.8
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1 mg mOGR1-DsredpH 6.8
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*
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cAMPCa2+
Ca2+Ca2+
++ ++++ -
G2A
TDAG8OGR1OGR1 GPR4
H+H+H+
H+
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