Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae- eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 insphingolipid signal transduction

Plasmalemmal caveolae are membrane microdomains that are specifically enric hed in sphingolipids and contain a wide array of signaling proteins, includ ing the endothelial isoform of nitric-oxide synthase (eNOS). EDG-1 is a G p rotein-coupled receptor for sphingosine 1-phosphate (S1P) that is expressed in endothelial cells and has been implicated in diverse vascular signal tr ansduction pathways. We analyzed the subcellular distribution of EDG-1 in C OS-7 cells transiently transfected with cDNA constructs encoding epitope-ta gged EDG-1. Subcellular fractionation of cell lysates resolved by ultracent rifugation in discontinuous sucrose gradients revealed that similar to 55% of the EDG-1 protein was recovered in fractions enriched in caveolin-1, a r esident protein of caveolae, Co-immunoprecipitation experiments showed that EDG-1 could be specifically precipitated by antibodies directed against ca veolin-1 and vice versa. The targeting of EDG-1 to caveolae-enriched fracti ons was markedly increased (from 51 +/- 11% to 93 +/- 14%) by treatment of transfected cells with S1P (5 mu M, 60 min). In co-transfection experiments expressing EDG-1 and eNOS cDNAs in COS-7 cells, we found that S1P treatmen t significantly and specifically increased nitric-oxide synthase activity, with an EC50 of 30 nM S1P. Overexpression of transfected caveolin-1 cDNA to gether with EDG-1 and eNOS markedly diminished S1P-mediated eNOS activation ; caveolin overexpression also attenuated agonist-induced phosphorylation o f 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 S1 P pathway, even as the targeting of EDG-1 to caveolae facilitates the inter actions of this receptor with ligands and effecters that are also targeted to caveolae. The agonist-modulated targeting of EDG-1 to caveolae and its d ynamic inhibitory interactions with caveolin identify new points for regula tion of sphingolipid-dependent signaling in the vascular wall.