Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

Upon cell activation, membrane phospholipids are metabolized into potent lysophospholipid (LP) mediators, such as sphingosine 1-phosphate and lysophosphatidic acid. LPs fulfill signaling roles in organisms as diverse as yeast and humans. The recent discovery of G protein-coupled receptors for LPs in higher eukaryotes, and their involvement in regulating diverse processes such as angiogenesis, cardiac development, neuronal survival, and immunity, has stimulated growing interest in these lipid mediators. LP receptor biology has generated insights into fundamental cellular mechanisms and may provide therapeutic targets for drug development.

The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a 1/2 of ϳ15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a 1/2 of ϳ30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process. INTRODUCTIONThe bioactive sphingolipid mediator sphingosine-1-phosphate (SPP) is synthesized by sphingosine kinasecatalyzed phosphorylation of sphingosine (Buehrer and Bell, 1993). It is produced by many cells, including platelets (Yatomi et al., 1995(Yatomi et al., , 1997, fibroblasts (Olivera and Spiegel, 1993), and neuron-like pheochromocytoma-12 cells (Edsall et al., 1997). SPP elicits a variety of biological responses, such as fibroblast proliferation (Zhang et al., 1991), neurite retraction (Postma et al., 1996), calcium signaling (Ghosh et al., 1990), regulation of apoptosis (Cuvillier et al., 1996;Hung and Chuang, 1996), morphogenetic differentiation (Lee et al., 1998b), inhibition of cell motility (Sadahira et al., 1992;Bornfeldt et al., 1995), induction of activator protein-1 transcription factor activity (Su et al., 1994), regulation of G-protein-dependent cAMP levels (Zhang et al., 1991;Bornfeldt et al., 1995), and mitogen-activated protein (MAP) kinase activity (Wu et al., 1995). Many of the effects of SPP are mediated by plasma membrane receptors that are coupled to G-proteins (reviewed in Hla et al., 1999). For example, SPP induces the calcium transients, which are inhibited by pertussis toxin (Van Koppen et al., 1996). However, some effects of SPP may occur via the intracellular action of this mediator on novel, although unknown, intracellular targets (reviewed in Spiegel and Merrill, 1996) gosine, which inhibits endogenous synthesis of SPP, blocked apoptosis in monocytic cell lines (Cuvillier et al., 1996) and platelet-derived growth factor-induced mitogenesis in Swiss 3T3 fibroblasts (Olivera and Spiegel, 1993). ...

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