Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction.
Plasmalemmal caveolae are membrane microdomains that are specifically enriched in sphingolipids and contain a wide array of signaling proteins, including the endothelial isoform of nitric-oxide synthase (eNOS). EDG-1 is a G protein-coupled receptor for sphingosine 1-phosphate (S1P) that is expressed in endothelial cells and has been implicated in diverse vascular signal transduction pathways. We analyzed the subcellular distribution of EDG-1 in COS-7 cells transiently transfected with cDNA constructs encoding epitope-tagged EDG-1. Subcellular fractionation of cell lysates resolved by ultracentrifugation in discontinuous sucrose gradients revealed that approximately 55% of the EDG-1 protein was recovered in fractions enriched in caveolin-1, a resident protein of caveolae. Co-immunoprecipitation experiments showed that EDG-1 could be specifically precipitated by antibodies directed against caveolin-1 and vice versa. The targeting of EDG-1 to caveolae-enriched fractions was markedly increased (from 51 +/- 11% to 93 +/- 14%) by treatment of transfected cells with S1P (5 microm, 60 min). In co-transfection experiments expressing EDG-1 and eNOS cDNAs in COS-7 cells, we found that S1P treatment significantly and specifically increased nitric-oxide synthase activity, with an EC(50) of 30 nm S1P. Overexpression of transfected caveolin-1 cDNA together with EDG-1 and eNOS markedly diminished S1P-mediated eNOS activation; caveolin overexpression also attenuated agonist-induced phosphorylation of EDG-1 receptor by >90%. These results suggest that the interaction of the EDG-1 receptor with caveolin may serve to inhibit signaling through the S1P pathway, even as the targeting of EDG-1 to caveolae facilitates the interactions of this receptor with ligands and effectors that are also targeted to caveolae. The agonist-modulated targeting of EDG-1 to caveolae and its dynamic inhibitory interactions with caveolin identify new points for regulation of sphingolipid-dependent signaling in the vascular wall.
Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
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J
Igarashi
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T
Michel
D000818:Animals; D019556:COS Cells; D021941:Caveolae / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008246:Lysophospholipids; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014162:Transfection
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D017874:Immediate-Early Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011993:Recombinant Fusion Proteins; C060506:sphingosine 1-phosphate; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D013110:Sphingosine