Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.

Leukotriene B4 (LTB4) is a potent chemoattractant that is primarily involved in inflammation, immune responses and host defence against infection. LTB4 activates inflammatory cells by binding to its cell-surface receptor (BLTR). LTB4 can also bind and activate the intranudear transcription factor PPAR alpha, resulting in the activation of genes that terminate inflammatory processes. Here we report the cloning of the complementary DNA encoding a cell-surface LTB4 receptor that is highly expressed in human leukocytes. Using a subtraction strategy, we isolated two cDNA clones (HL-1 and HL-5) from retinoic acid-differentiated HL-60 cells. These two clones contain identical open reading frames encoding a protein of 352 amino acids and predicted to contain seven membrane-spanning domains, but different 5'-untranslated regions. Membrane fractions of Cos-7 cells transfected with an expression construct containing the open reading frame of HL-5 showed specific LTB4 binding, with a K(d) (0.154nM) comparable to that observed in retinoic acid-differentiated HL-60 cells. In CHO cells stably expressing this receptor, LTB4 induced increases in intracellular calcium, D-myo-inositol-1,4,5-triphosphate (InsP3) accumulation, and inhibition of adenylyl cyclase. Furthermore, CHO cells expressing exogenous BLTR showed marked chemotactic responses towards low concentrations of LTB4 in a pertussis-toxin-sensitive manner. Our findings, together with previous reports, show that LTB4 is a unique lipid mediator that interacts with both cell-surface and nuclear receptors.

Like neutrophilic leukocytes, differentiated HL-60 cells respond to chemoattractant by adopting a polarized morphology, with F-actin in a protruding pseudopod at the leading edge and contractile actin-myosin complexes at the back and sides. Experiments with pharmacological inhibitors, toxins, and mutant proteins show that this polarity depends on divergent, opposing "frontness" and "backness" signals generated by different receptor-activated trimeric G proteins. Frontness depends upon Gi-mediated production of 3'-phosphoinositol lipids (PI3Ps), the activated form of Rac, a small GTPase, and F-actin. G12 and G13 trigger backness signals, including activation of a second GTPase (Rho), a Rho-dependent kinase, and myosin II. Functional incompatibility causes the two resulting actin assemblies to aggregate into separate domains, making the leading edge more sensitive to attractant than the back. The latter effect explains both the neutrophil's ability to polarize in uniform concentrations of chemoattractant and its response to reversal of an attractant gradient by performing a U-turn.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase-and Rac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 cells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N 17 -Cdc42, but not N 19 -RhoA, suppressed S1P-and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.

Abstract-Ca2ϩ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinasedependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca 2ϩ -dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations.

Class II α-isoform of phosphatidylinositol 3-kinases (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles, however, its functional role is little understood. Global or endothelial cell (EC)-specific targeted disruption of PI3K-C2α resulted in embryonic lethality due to defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in ECs induced decreased phospatidylinositol 3-phosphate-enriched endosomes, impaired endosomal trafficking, and defective delivery of VE-cadherin to EC junctions and its assembly. PI3K-C2α knockdown also impeded cell signaling including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. These together led to defective EC migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deletion suppressed post-ischemic and tumor angiogenesis, and diminished vascular barrier function, with greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α plays a crucial role in vascular formation and barrier integrity, and represents a new therapeutic target for vascular diseases. 3Formation of the vascular network by vasculogenesis and angiogenesis is essential for embryonic development, repair and remodeling of tissues in adults, as well as tumor growth. The angiogenic response to vascular endothelial growth factor (VEGF) and other factors begins with vascular leakage and dissolution of the subendothelial basement membrane, followed by proliferation and migration of vascular EC 1,2 . Then, formation of the intercellular junctions results in initial sprouts from existing vessels. The newly formed endothelial tubes are associated with mural cells, i.e. smooth muscle cells (SMC) and pericytes, thus becoming mature and stabilized 3 . Tightness of the intercellular junctions, particularly adherens junctions composed of VE-cadherin, controls vascular permeability 4,5 . Quiescent, stabilized vasculature with intact barrier integrity dominates in the healthy condition. In contrast, in pathological conditions, such as tumors, the vasculature is generally inmaturate and leaky. In the case of vascular insult such as excessive angiotensin II (Ang II) activity, increased vascular permeability is asssociated with leukocyte infiltration in the vascular wall and vascular disruption 6,7 . Therefore, stabilization of the vasculature and maintenance of vascular integrity is essential for vascular and tissue homeostasis 8,9 .PI3Ks are an enzyme family that phosphorylates membrane inositol lipids at the 3' position of the inositol ring. The lipid products of PI3Ks serve as important intracellular messengers by interacting with effector proteins, which include protein kinases, guanine nucleotide exchangers for G proteins, and actin cytoskeleton-regulating proteins. Through these actions, PI3Ks regulate a diverse array of cellular processes 10-12 .PI3Ks comprise three classes. Class I PI...

Regulation of cell migration is critical in such diverse biological processes as organogenesis, neuronal axon pathfinding, wound healing, inflammatory responses, vascular remodeling, and tumor cell dissemination (21). Extracellular cues called attractants and repellants, which are either soluble or membrane bound, instruct cells to advance and to retreat, respectively (36,40). A number of chemokines, growth factors, cytokines, and other inflammatory mediators have been shown to stimulate directed cell migration, whereas a much more limited number of biological mediators have been shown to inhibit cell motility in a manner dependent on their concentration gradients. The latter include metastin (28), Slit, semaphorins, ephrins (44), and a lipid mediator, sphingosine 1-phosphate (S1P) (42). S1P is a bioactive lysophospholipid that exerts a wide variety of biological activities, most of which are mediated via Edg family G protein-coupled receptors (GPCRs), including S1P 1 /Edg1, S1P 2 /Edg5/AGR16/H218, and S1P 3 /Edg3 (7,16,39,43). S1P has been demonstrated to be quite unique as an extracellular regulator of motility in that it exerts either stimulatory or inhibitory actions on cell motility (42). These bimodal actions are apparently cell type specific; thus, S1P stimulates chemotaxis in vascular endothelial cells (22) and embryonic fibroblasts (24), whereas it inhibits cell migration in vascular smooth muscle cells (3, 33) and melanoma cells (34). We recently showed that this bimodal regulation by S1P is based upon a diversity of S1P receptor isotypes, which mediate either stimulatory or inhibitory regulation for cell migration (31, 42). Thus, we found that S1P 2 acts as a repellant receptor to mediate inhibition of chemotaxis toward attractants, whereas S1P 1 and S1P 3 act as attractant receptors to mediate migration directed toward S1P. Elimination of the S1P receptor gene in mice (24) and development of a drug to target S1P receptors (4, 25) have revealed that S1P is involved in regulation of cell migration in vivo, thus contributing to morphogenesis and regulation of lymphocyte homing.Small GTPases of the Rho family, primarily Rac, Cdc42, and Rho, are well-known regulators of actin organization and myosin motor function and thereby of cell motility (10,14,47). These Rho GTPases show distinct activities on actin cytoskeletons: Rho mediates stress fiber formation and focal adhesion, while Rac and Cdc42 direct peripheral actin assembly that results in formation of lamellipodia and filopodia, respectively. Despite limitation of our understanding of intracellular signaling from the membrane to the cytoskeleton, a model has emerged from the observations in a variety of cell types that attractive extracellular cues activate Rac or Cdc42, while repulsive cues inhibit Rac or Cdc42 and stimulate Rho (9,38,42,48). In fact, the repellant receptor S1P 2 negatively regulates cellular Rac activity through mechanisms involving stimulation of a GTPase-activating protein (GAP) for Rac (31). In contrast, the attractant receptors ...

Recent studies (1-3) provide increasing evidence of roles for lysosphingolipids as mediators to elicit a variety of physiological and pathophysiological responses. Thus, the lysosphingolipids SP 1 and SPC have been shown to evoke diverse cellular responses in various cell types, including mitogenesis (1, 2), inhibition of migration (4, 5), cell shape change (6), and microfilament reorganization (6, 7). Stimulation of cells with the lysosphingolipids triggers the activation of multiple intracellular signaling molecules, including phospholipase C (2, 5, 8, 9), phospholipase D (8), PKC (10), MAPK (5, 11), and K ϩ channel (muscarinic K ϩ current) (12). Many of the lysosphingolipidinduced responses are demonstrated to be inhibited by PTX pretreatment (5, 8 -13). In addition, either an increase or a decrease in cellular cAMP content in response to SP has been reported, depending on cell types used (5, 13). These observations suggest the existence of multiple G protein-coupled cell surface receptors for SP and SPC.Recently, the orphan G protein-coupled receptor EDG2 was identified as a functional receptor for LPA (14). Moreover, EDG4 was very recently identified to be the second LPA receptor (15). EDG2 and EDG4 are members of the EDG family of receptors comprising EDG1 (16), EDG3 (17), and AGR16 (18)/ H218 (19), which have 36 -58% homology in amino acid sequences with each other. SP is related in its structure to LPA, and in some cell types, LPA and SP have been suggested to share a cell surface receptor (20, 21). These observations prompted us to examine the possibility that members of the EDG family receptors could function as a receptor for the lysosphingolipids. Many of cell lines usually used for expression of exogenous genes, including COS, NIH3T3 and HEK293 cells, respond to SP (13), which hampered expression cloning of SP receptor gene and functional analysis of cloned SP receptor gene. In the present study, by using carefully selected mammalian cell expression systems, we found that EDG1 is a functional receptor with a high specificity and affinity for SP. We demonstrate that EDG1 is coupled via a G i/o protein to multiple effector pathways, including phospholipase C, adenylate cyclase, and Ras/MAPK. MATERIALS AND METHODS Cells-CHO-K1(CHO) and HEL cells, obtained from RIKEN CellBank and the Japanese Cancer Research Resources Bank (Tokyo, Japan), respectively, were grown in Ham's F-12 (CHO) and RPMI (HEL) media supplemented with 10% fetal calf serum (Equitech-Bio, Ingram, TX), 100 units/ml penicillin, and 100 g/ml streptomycin (Wako Pure Chemicals, Osaka, Japan). Before each experiment, cells were switched to the respective medium supplemented with 1% fetal calf serum.