Truncation of the N-terminal ectodomain has implications in the N-glycosylation and transport to the cell surface of Edg-1/S1P1 receptor

The endothelial cell-expressed sphingosine 1-phosphate receptors Edg-1/S1P1 and Edg-3/S1P3 have been implicated in various physiological events such as the regulation of angiogenesis. Since there is an excess of a ligand constitutively in blood, these receptors may have some mechanism(s) avoiding overstimulation. In this study, we found that the N-terminal ectodomains of Edg-1/S1P1 and Edg-3/S1P3 were truncated in overexpressing cells. The truncated form of Edg-1/S1P1 expressed on the cell surface had undergone complex-type oligosaccharide modification at the Golgi. A deletion mutant lacking the N-terminal processing domain of Edg-1/S1P1 accumulated in the endoplasmic reticulum, and was not expressed on the cell surface. When a basic amino acid residue was introduced at the cleavage site of Edg-1/S1P1, the molecular weight of the glycosylated protein was greater in the mutant compared to the wild type, due to the bound oligosaccharide. These results demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. Ectodomain shedding of many membrane proteins has been implicated in various diseases. Therefore, N-terminal processing of Edg-1/S1P1 and Edg-3/S1P3 might play roles in endothelial cell functions.     

Enhancement of sphingosine 1-phosphate-induced migration of vascular endothelial cells and smooth muscle cells by an EDG-5 antagonist

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.     

Sphingosine 1-phosphate inhibits migration of RBL-2H3 cells via S1P2: cross-talk between platelets and mast cells

To analyze the involvement in allergic reactions of platelets and sphingosine 1-phosphate (Sph-1-P), a lysophospholipid mediator released from activated platelets, the effects of Sph-1-P and a supernatant prepared from activated platelets on mast cell line RBL-2H3 were examined. Sph-1-P strongly inhibited the migration of both non-stimulated and fibronectin-stimulated RBL-2H3 cells, which was reversed by JTE-013, a specific antagonist of G protein-coupled Sph-1-P receptor S1P(2); S1P(2) was confirmed to be expressed in these cells. A similar anti-motility effect of Sph-1-P was observed in a phagokinetic assay. Consistent with these results, treatment of RBL-2H3 cells with Sph-1-P resulted in a rounded cell morphology, which was blocked by JTE-013. Under the present conditions, Sph-1-P failed to induce intracellular Ca(2+) mobilization or histamine degranulation, responses postulated to be elicited by intracellular Sph-1-P. Importantly, the Sph-1-P effect, i.e., the regulation of RBL-2H3 cell motility, was mimicked by the supernatant (both with and without boiling) prepared from activated platelets, and this effect of the supernatant was also blocked by JTE-013. Our results suggest that the motility of mast cells can be regulated by Sph-1-P and also platelets (which release Sph-1-P), via cell surface receptor S1P(2) (not through intracellular Sph-1-P actions, postulated previously in the same cells).     

Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins.

Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [(35)S]GTPgammaS binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G(i) family, and not G(s), G(q), G(12), or G(13). We additionally established that Edg-1 activates G(i) in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G(13) as well as G(i). Because several of the biological actions of S1P are assumed to proceed through the G(12/13) family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G(i) but also with G(q) and G(13). These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.     

Fluid shear stress enhances the sphingosine 1-phosphate responses in cell-cell interactions between platelets and endothelial cells

Fluid shear stress modulates the functional responses of platelets and vascular cells, and plays an important role in the pathogenesis of vascular disorders, including atherosclerosis and restenosis. Since shear stress induces activation of platelets, which abundantly store sphingosine 1-phosphate (Sph-1-P), and upregulates the mRNA expression of S1P(1), the most important Sph-1-P receptor expressed on the endothelial cells, we examined the effects of shear stress on the Sph-1-P-related responses involving these cells. Shear stress was found to induce Sph-1-P release from the platelets in a shear intensity- and time-dependent manner. Inhibitors of protein kinase C suppressed this mechanical force-induced Sph-1-P release, suggesting involvement of this kinase. On the other hand, in vascular endothelial cells, shear stress increased S1P(1) protein expression, as revealed by flow-cytometric analysis, and the responsiveness to Sph-1-P, which was assessed by monitoring the intracellular Ca(2+) concentration. These results indicate that shear stress enhances the Sph-1-P responses in cell-cell interactions between platelets and endothelial cells.     

Plasma sphingosine 1-phosphate metabolism and analysis

The importance of sphingosine 1-phosphate (Sph-1-P) as an intercellular sphingolipid mediator has been established in various systems, and this is especially true in the areas of vascular biology and immunology. Blood platelets store Sph-1-P abundantly and release this bioactive lysophospholipid extracellularly upon stimulation, while vascular endothelial cells and smooth muscle cells respond dramatically to this platelet-derived bioactive lipid. Most of the responses elicited by extracellular Sph-1-P are believed to be mediated by G protein-coupled cell surface receptors, i.e., S1Ps. It is likely that regulation of Sph-1-P biological activity could be important for therapeutics, including but not limited to control of vascular disorders. Furthermore, elucidation of the mechanisms by which the levels of Sph-1-P in the blood are regulated seems important. Accordingly, the application of Sph-1-P analysis to laboratory medicine may be an important task in clinical medicine. In this review, Sph-1-P-related metabolism in the plasma will be summarized. Briefly, the levels and bioactivities of plasma Sph-1-P in vivo may be regulated by various factors, including Sph-1-P release from platelets (and red blood cells, based upon the recent reports), Sph-1-P distribution between albumin and lipoproteins, and S1P expression and lipid phosphate phosphatase activity on the cell surface. Then, application of Sph-1-P analysis to laboratory medicine will be discussed.     

Involvement of sphingosine 1-phosphate, a platelet-derived bioactive lipid, in contraction of mesangium cells

Platelet-derived mediators may play an important role in the development of renal diseases through interaction with glomerular mesangial cells (MCs), and we, in this study, examined the effect of sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, on the contraction of MCs. Sph-1-P was found to induce MC contraction through mediation of Rho kinase both in cell shape change and collagen gel contraction assays. The specific antagonist of the Sph-1-P receptor S1P(2) inhibited the response. Similar results were obtained when the supernatant from activated platelet suspensions were used instead of Sph-1-P. Our findings suggest that platelet-derived Sph-1-P may be involved in MC contraction via S1P(2) and that regulation of this receptor might be useful therapeutically.     

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.     

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca(2+) mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.     

Sphingosine 1-phosphate breakdown in platelets

We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.     

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.     

Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells.

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [(3)H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca(2+)](i) increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca(2+) responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca(2+) system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.     

Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Edg-6 as a putative sphingosine 1-phosphate receptor coupling to Ca(2+) signaling pathway

The endothelial differentiation gene-6 (Edg-6) was recently identified as an orphan G-protein-coupled receptor. Its predicted amino acid sequence is very close to Edg family of receptor proteins whose ligand is supposed to be lysophosphatidic acid (LPA) or lysosphingolipid such as sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). Transfection of the Edg-6 into Chinese hamster ovary (CHO) cells and K562 cells resulted in the appearance of high-affinity [(3)H]S1P binding activity. Among lipids employed, S1P and, even though less potent, SPC, displaced the [(3)H]S1P binding, but LPA was inactive. In Edg-6-transfected CHO cells, an increase in cytosolic Ca(2+) concentration in response to S1P or SPC was clearly enhanced without change in the LPA-induced action as compared with the vector-transfected cells. The enhancement of the Ca(2+) response was associated with a significant accumulation of inositol phosphate, reflecting activation of phospholipase C. Similar enhancement of Ca(2+) response to S1P or SPC was also observed in Edg-6-expressing K562 cells. These lipid-induced actions in CHO cells and K562 cells expressing Edg-6 were markedly suppressed by pertussis toxin treatment. We conclude that Edg-6 is one of S1P or lysosphingolipid receptors that couple to phospholipase C-Ca(2+) system through pertussis toxin-sensitive G-proteins.     

Dual Actions of Sphingosine-1-Phosphate: Extracellular through the Gi-coupled Receptor Edg-1 and Intracellular to Regulate Proliferation and Survival

Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein–coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552–1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin–sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125^FAK, independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers.     

Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P₂ and S1P₃ receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P₃ receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P₃R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P₂R and S1P₃R stimulation using Smad-independent pathways.     

G(i)-mediated Cas tyrosine phosphorylation in vascular endothelial cells stimulated with sphingosine 1-phosphate: possible involvement in cell motility enhancement in cooperation with Rho-mediated pathways

Since blood platelets release sphingosine 1-phosphate (Sph-1-P) upon activation, it is important to examine the effects of this bioactive lipid on vascular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions. In the present study, we examined Sph-1-P-stimulated signaling pathways related to human umbilical vein endothelial cell (HUVEC) motility, with a special emphasis on the cytoskeletal docking protein Crk-associated substrate (Cas). Sph-1-P stimulated tyrosine phosphorylation of Cas, which was inhibited by the G(i) inactivator pertussis toxin but not by the Rho inactivator C3 exoenzyme or the Rho kinase inhibitor Y-27632. Fyn constitutively associated with and phosphorylated Cas, suggesting that Cas tyrosine phosphorylation may be catalyzed by Fyn. Furthermore, upon HUVEC stimulation with Sph-1-P, Crk, through its SH2 domain, interacted with tyrosine-phosphorylated Cas, and the Cas-Crk complex translocated to the cell periphery (membrane ruffles), through mediation of G(i) (Fyn) but not Rho. In contrast, tyrosine phosphorylation of focal adhesion kinase, and formation of stress fibers and focal adhesion were mediated by Rho but not G(i) (Fyn). Finally, Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay using gold sol-coated plates and a Boyden's chamber assay, was markedly inhibited not only by pertussis toxin (or the Fyn kinase inhibitor PP2) but also by C3 exoenzyme (or Y-27632). In HUVECs stimulated with Sph-1-P, these data suggest the following: (i) cytoskeletal signalings may be separable into G(i)-mediated signaling pathways (involving Cas) and Rho-mediated ones (involving FAK), and (ii) coordinated signalings from both pathways are required for Sph-1-P-enhanced HUVEC motility. Since HUVECs reportedly express the Sph-1-P receptors EDG-1 (coupled with G(i)) and EDG-3 (coupled with G(13) and G(q)) and the EDG-3 antagonist suramin was found to block specifically Rho-mediated responses, it is likely that Cas-related responses following G(i) activation originate from EDG-1, whereas Rho-related responses originate from EDG-3.