Transduction of intracellular calcium signals through G protein-mediated activation of phospholipase C by recombinant sphingosine 1-phosphate receptors.
Sphingosine 1-phosphate (S1P) increases intracellular Ca2+ concentration in many cell types, but the signaling mechanism remains uncertain. The recent identification of three closely related seven-transmembrane domain receptors for S1P, termed Edg1, H218, and Edg3, support the extracellular ligand role of S1P and allowed examination of Ca2+ responses mediated specifically by each receptor subtype. To substantiate each subtype in S1P-induced Ca2+ responses and to study the transductional mechanisms, we applied the aequorin luminescence method and the fura-2 fluorescence method in two transfected mammalian cell systems. We showed that H218 and Edg3 were capable of mediating S1P-induced mobilization of intracellular Ca2+ when transiently transfected in human TAg-Jurkat T cells. Ca2+ responses mediated by Edg1 in TAg-Jurkat cells required coexpression of the Gqi5 chimeric G protein that links Gi-coupled receptors to Gq. When H218 and Edg3 were stably expressed in rat HTC4 hepatoma cells, S1P induced Ca2+ responses with nanomolar EC50 values. Edg3, but not H218, elicited a sustained influx of extracellular Ca2+. The coincident formation of inositol phosphates and the complete inhibition of Ca2+ responses by the phospholipase C inhibitor U73122 indicated that H218 and Edg3 mobilized Ca2+ through activation of phospholipase C. Partial inhibition of Ca2+ responses and inositol phosphates formation by pertussis toxin implied that H218 and Edg3 transduce phospholipase C activation and Ca2+ responses only partially through Gi proteins. Although these results did not dismiss that S1P may function as an intracellular second messenger in other settings, they definitively proved that S1P can mobilize Ca2+ as an extracellular ligand for G protein-coupled receptors.
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Department of Medicine, University of California, San Francisco, California 94143-0711, USA. songzhu@itsa.ucsf.edu
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Zheng
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins / Q000378:metabolism; D019169:Jurkat Cells; D000072000:NF-KappaB Inhibitor alpha; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D017874:Immediate-Early Proteins; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011994:Recombinant Proteins; D000072000:NF-KappaB Inhibitor alpha; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium