Recombinant Human G Protein-Coupled Lysophosphatidic Acid Receptors Mediate Intracellular Calcium Mobilization
- Departments of Medicine and Microbiology-Immunology, University of California, San Francisco, California 94143-0711
Abstract
Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca2+ bound to aequorin.N-Palmitoyl-l-serine-phosphoric acid andN-palmitoyl-l-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2+ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca2+ concentration was followed by a sustained influx of extracellular Ca2+. The coincident production of inositol phosphates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca2+ mobilization largely through pertussis toxin-sensitive Gi proteins, whereas Edg4 requires both Gi and Gq.
Footnotes
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Send reprint requests to: Dr. Songzhu An, Division of Immunology and Allergy, Department of Medicine, Box 0711, Room UB-8, University of California, San Francisco, CA 94143-0711. E-mail:songzhu{at}itsa.ucsf.edu
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This work was supported in part by research grants from the American Heart Association (Grant-in-Aid 96007190 to S.A.) and the National Institutes of Health (Grant HL31809 to E.J.G.).
- Abbreviations:
- LPA
- lysophosphatidic acid
- [Ca2+]i
- intracellular calcium concentration
- Vzg
- ventricular zone gene
- Edg
- endothelial differentiation gene
- GPCR
- G protein-coupled receptor
- NPSPA
- N-palmitoyl-l-serine-phosphoric acid
- NPTyrPA
- N-palmitoyl-l-tyrosine-phosphoric acid
- PLC
- phospholipase C
- IP3
- inositol-1,4,5-trisphosphate
- PTX
- pertussis toxin
- PA
- dioleoylphosphatidic acid
- LPC
- lysophosphatidylcholine
- LPS
- lysophosphatidylserine
- LPG
- lysophosphatidylglycerol
- LPE
- lysophosphatidylethanolamine
- S1P
- sphingosine-1-phosphate
- cLPA
- oleoyl-cyclic-2,3-phosphate-lysophosphatidic acid
- IP
- inositol phosphate
- alkenyl-GP
- alkenyl-glycerol phosphate
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- [cAMP]i
- intracellular cAMP concentration
- PBS
- phosphate-buffered saline
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- Received April 29, 1998.
- Accepted July 20, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
