Cell Signalling Through Thromboxane A2 Receptors Jin-Sheng Huang, Santosh K

Cellular Signalling 16 (2004) 521–533 www.elsevier.com/locate/cellsig Review article

Cell signalling through thromboxane A2 receptors Jin-Sheng Huang, Santosh K. Ramamurthy, Xin Lin, Guy C. Le Breton*

Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Avenue (Mail Code 868), Chicago, IL 60612, USA Received 19 August 2003; accepted 6 October 2003

Abstract

Thromboxane A2 receptors (TPs) are widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. Following the initial cloning of this receptor class from human placenta, the deduced amino acid sequence predicted seven-transmembrane spanning regions, four extracellular domains and four intracellular domains, making TP a member of the seven-transmembrane G-protein-coupled receptor (GPCR) super family. A single gene on chromosome 19p13.3 leads to the expression of two separate TP isoforms: TPa which is broadly expressed in numerous tissues, and a splice variant termed TPh which may have a more limited tissue distribution. Mutagenesis, photoaffinity labelling, and immunological studies have indicated that the ligand binding domains for this receptor may reside in both the transmembrane (TM) and extracellular regions of the receptor protein. In addition, separate studies have provided evidence that this receptor can couple to at least four separate G protein families. As a consequence, TP signalling has been shown to result in a broad range of cellular responses including phosphoinositide metabolism, calcium redistribution, cytoskeletal arrangement, integrin activation, kinase activation, and the subsequent nuclear signalling events involved in DNA synthesis, cell proliferation, cell survival and cell death. While activation of these different signalling cascades can all derive from TP stimulation, the relative signalling preference for a given cascade appears to be both tissue and cell specific. Finally, separate studies have indicated that TP signalling capacity can be both down-regulated by protein kinase activation and up-regulated by GPCR cross-signalling. Thus, the multitude of signalling events which derive from TP activation can themselves be modulated by endogenous cellular messengers. D 2003 Elsevier Inc. All rights reserved.

Keywords: Thromboxane A2 receptors; Cell signalling; Platelets; Smooth muscle cells; Endothelial cells; Oligodendrocytes; Schwann cells

1. Introduction Historically, TP involvement in blood platelet function has received the greatest attention. However, it is now clear that The initial purification and cloning of the thromboxane TPs exhibit a wide distribution in different cell types and A2 receptor (TP) established this protein as a member of the among different organ systems (Table 1). For example, TPs super family of G-protein-coupled seven-transmembrane have been localized in cardiovascular, reproductive, im- receptors [1]. The originally cloned TP from placenta (343 mune, pulmonary and neurological tissues, among others. amino acids in length) is known as the a isoform, and the Over the years, different biological roles for TP signalling splice variant cloned from endothelium (with 407 amino have been established in both homeostatic and pathological acids) is termed the h isoform. Comparison of the two processes (Table 2). Thus TP activation is thought to be sequences reveals that even though the first 328 amino acids involved in thrombosis/hemostasis, modulation of the im- are the same for both isoforms [2–4], the h isoform exhibits mune response, acute myocardial infarction, inflammatory an extended C-terminal cytoplasmic domain. Furthermore, it lung disease, hypertension, nephrotic disease, etc. Based on is worth noting that expression of each protein is not equal this consideration, attempts have been made to define the within or across different cell types. Thus while platelets distinct signalling pathways by which TPs elicit their express high concentrations of the a isoform (and possess biological and pathological effects. In this regard, it is well residual RNA for the h isoform), expression of the h documented that TPs have the capacity to activate a multi- isoform protein has not been documented in these cells. tude of different signalling cascades which regulate cellular ion flux, cytoskeletal arrangement, cell adhesion, motility, nuclear transcription factors, proliferation, cell survival, and * Corresponding author. Tel.: +1-312-996-4929; fax: +1-312-996- apoptosis. They are known to couple to at least four G 4929. proteins, which in turn activate numerous downstream

Table 1 studies have been performed. Specifically, Funk et al. [56] Thromboxane A2 Receptor Distribution obtained four mutants with point mutations at TM7, i.e., Organ/Tissue [references] between L291 and W299. Three of these mutants complete- Spleen [8,9] Thymus [8–10] ly lost binding activity to both antagonists and agonists. In Uterus [10] Kidney [9,11–13] Placenta [14] Heart [15] addition, Chiang et al. [57] reported that mutations of S201A Aorta [16] Spinal cord [17] and S255A in TM5 and TM6, respectively, caused altered Intestine [10] Brain [10,16] affinity to the agonist, I-BOP, but had no effect on the Liver [10] Lung [8,9] antagonist, SQ29548 binding. Taken together, these latter Eye [18] results indicate that amino acid substitutions in either TM5, Cells/Cell lines [references] TM6 or TM7 regions can diminish ligand binding to TPs. Platelets [19–21] Endothelial cells [2] Other mutagenesis studies [58] employing TP chimeras led Glomerular mesangial cells [22] Trophoblasts [10] the authors to conclude that residues in TM1 constitute an Oligodendrocytes [17,23] Schwann cells [24] important portion of the TP binding site. Finally, reports Cardiac myocytes [25] Astrocytes [26–28] from two different groups suggested that the putative disul- Epithelial cells [13,18] Megakaryocytes [29] Hela cells [30] Hepatoblastoma HepG2 cells [10] fide bond between C105 and C183 in the first and second Smooth muscle cells [10] Immature thymocytes [31] extracellular loops, respectively, plays a critical role in TP- Kupffer cells [32] EL-4 (human T cell line) [9] ligand binding [57,59]. These groups also suggested that Human erythroleukemic C102, which is conserved in most GPCRs, including the TP, megakaryocyte (HEL) [33] also plays an important, yet unspecified role in ligand K562 (Human chronic myelogenous leukemia) cells [34] binding. Consequently, the previous results indicate multiple residues in TPs which may serve as potential ligand coordination sites, i.e., TM regions 1, 3–7, as well as cysteine effectors, including second messenger systems such as residues in the first and second extracellular loops (EL). ˚ inositol triphosphate (IP3)/DAG, cAMP, small G proteins However, since TP ligands are of limited size (c15 A), it is (Ras, Rho), phosphoinositide-3(PI3) kinase, as well as unlikely that all of the suggested regions participate in protein kinase C (PKC) and protein kinase A (PKA). ligand coordination. Rather, the reported loss of binding Furthermore, it has also become apparent that the signalling activity, in at least some of the previous mutagenesis studies, preferences between these different TP-mediated pathways may derive from alterations in gross TP structure. vary in both a cell- and organ-specific manner. Consequent- Evidence has also accumulated that TP ligands can coor- ly, TP activation in one cell type may lead to quite different dinate with extracellular domains. In particular, results signalling events than its activation in a separate cell type. obtained using a biotinylated TP antagonist SQB [60] sug- This review discusses the process of ligand-induced TP gested that the TP ligand coordination sites are not deeply activation, its signal transduction and its downstream effec- embedded in a TM region, but must reside on the external tor activation in selected cells. For additional TP-related aspect of the plasma membrane. Further evidence in support background information, several previously published of ligand interaction at extracellular sites was provided in reviews [5–7] are recommended. subsequent NMR studies which demonstrated that the TP antagonist SQ29548 produced a change in conformation of a constrained peptide containing amino acids representing the 2. TP ligand interaction TP EL2 and EL3 regions [61,62]. Recently, a specific amino acid sequence contained within EL2 has been identified as a Much of the initial work to elucidate the ligand binding domain(s) of TPs centred on the participation of the transmembrane (TM) regions (Fig. 1). This is in large part due to Table 2 earlier findings in bacteriorhodopsin and its GPCR homo- Biological roles/clinical links of TXA2/TP logue, rhodopsin, which revealed a binding pocket in the Hemostasis/thrombosis [35–37] seventh spanning region of the receptor [51]. In addition, Sickle cell disease [38] h Cardiovascular disease [39] separate studies with a2- and 2-adrenergic receptors [52– Lupus nephritis [40] 54] indicated that the highly conserved TM7 region forms Acute myocardial infarction [41] one critical segment of the ligand binding domain. On this Nephrotic syndrome [42] basis, it was originally proposed that the binding domain Hypertension [43] may also reside in the TM7 region. In addition to participa- Immune complex glomerulonephritis [44] Pregnancy-induced hypertension (PIH) [45] tion of TM7, other investigations have suggested [55] Asthma [46] coordination of the ligand head group with TM3 and Inflammatory lung diseases [47] interaction of the ligand alkyl chains with the TM4/TM5 Regulating acquired immunity [48] regions. As a separate approach to define further the ligand Chronic inflammation in atopic diseases [49] coordination sites for TPs, various site-directed mutagenesis Chronic inflammatory bowel diseases [50] J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 523

Fig. 1. Putative structures of the TPa and TPh receptor proteins. critical participant in TP ligand binding [63]. Specifically, respective members. For example, antibody studies have through the use of photoaffinity labelling techniques and site- shown platelet expression of Gq,G16 (Gq family), G12,G13 directed antibodies, it was established that TP residues (G12 family), Gs, as well as Go,Gi and Gz (Gi family) [71– C183–D193 are essential for both ligand interaction and 78]. While it has been shown that two of these families are TP functional response. The importance of this region for TP involved in platelet TP signalling, i.e., Gq and G12, the ligand coordination was subsequently confirmed by studies relative contributions of these G proteins to the individual of So et al. [64]. In these experiments, high resolution NMR platelet functional responses is unclear. Nevertheless, defin- of a TP EL2 peptide segment suggested ligand contact with itive evidence has been provided that TP-mediated activa- V176, L185, T186 and L187. Furthermore, mutation of these tion of selected G protein pathways leads to recruitment of residues (V176 to D, L or R; L185 and L187 to A, D or R; and numerous downstream effector targets. T186 to A, R or S) led to a decrease in SQ29548 binding. The Probably the most well-characterized TP signal transduc- only exception was the V176L mutant which exhibited tion pathway is through Gq which was the first G protein normal binding activity. shown to couple functionally and physically to TPs [73,74]. In summary, while the precise location of ligand inter- Specifically, TP agonists have been shown to cause activation action with TP transmembrane regions remains to be more of the Gq heterotrimer and its subsequent dissociation into a clearly defined, it appears that the C-terminal segment of the and hg subunits. Even though the functional consequences of TP EL2 and possibly EL3 form critical coordination sites activated Gaq are uncertain, dissociated Ghg has been linked for this receptor protein. The existence of such a potential to activation of PLCh [79–83], recruitment of the phosphoi- binding domain in EL2 is consistent with results obtained nositide second messenger system [84,85], and the genera- for both the bradykinin B1/B2 [65] and PGE [66,67] tion of diacylglycerol (DAG) [85,86]. The resulting IP3 then receptor classes. leads to intraplatelet calcium mobilization [84–86] and the liberated DAG causes activation of protein kinase C (PKC) [85,87–89]. What is less clear are the specific contributions 3. TP signalling in platelets of IP3 and DAG to the overall process of platelet activation. For example, the calcium ionophore A23187 is capable of The heterotrimeric G proteins, consisting of a, h and g eliciting the full range of platelet responses, i.e., shape subunits, can be divided into four major families, Gq,G12, change, aggregation and secretion [90–92], suggesting that Gi and Gs [68–70], each of which contains various mem- an increase in intracellular calcium is itself sufficient to cause bers. Previous studies have established that platelets possess activation of the signalling pathways involved in these all of the major G protein families and many of their events. On the other hand, DAG production has been asso- 524 J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 ciated with the process of the platelet secretion [93], and Mention should also be made of the potential ability of inhibition of PKC leads to a reduction in dense granule TPs to signal through other, less well-characterized, G release [85,93–95]. In addition, separate studies have pro- proteins. In this regard, early work using immunoaffinity vided evidence that Ghg activation of PI3 kinase [96–98] is chromatography of platelet membranes revealed the pres- also involved in the platelet secretion response [99]. With ence of a high molecular weight protein (approximately these considerations in mind, it is most likely that each of 85 kDa) which co-purified with TPs in the column eluate these activation pathways contributes to the other, and that [74]. This protein blotted with a Ga common antibody the overall platelet response is a composite of their individual and became GTP labelled upon TP activation with contributing factors. U46619. Based on these findings, it was suggested [74] When examining the complete response profile for TP- that TPs may functionally couple to a previously uniden- mediated platelet activation, one must also take into tified G protein. In subsequent studies, Vezza et al. [116] account contributions from the G12/13 pathway. In this demonstrated TP signalling through a high molecular connection, it was initially demonstrated that activation weight Ga subunit, i.e., Gh [117–119]. It was shown of platelet TPs caused GTP binding to both Ga12 and that Gh (molecular weight 74 kDa) is present in platelets, Ga13, suggesting TP coupling to each of these Ga sub- couples to transfected TPs in COS-7 cells and is func- units. However, these studies also showed that the time tionally linked to inositol phosphate formation [116]. course for photolabelling of Ga12 significantly lagged While the above findings indicate that TPs may also behind that of Ga13 and seemed to be inconsistent with signal through a G protein separate from Gq and G13, the time course for the initial platelet activation response the consequences of such putative signalling are presently [71]. In confirmation of TP signalling through Ga13,a unknown. subsequent report demonstrated the direct physical and As indicated above, the linkage of specific TP–G functional coupling of platelet TPs to the Ga13 subunit protein signalling pathways to specific platelet activation [78,100,101]. On the other hand, these experiments failed events is not well defined. Nevertheless, several reports to show a similar physical association of platelet TPs with have indicated that certain of these events may be Ga12 subunit [78]. Taken together, the above findings associated with selected pathways. For example, in Gq- suggest that platelet TP signalling through the G12/13 deficient mice, the platelet aggregation response to family may disproportionately proceed through G13, and U46619 is inhibited, indicating that TP-mediated aggre- that the TP-mediated activation of G12 occurs as a sec- gation requires signalling through the Gq pathways [120]. ondary rather than a primary event of TP signalling. A On the other hand, in these studies there appeared to be a similar preference for G12 or G13 coupling has been divergence between TP-mediated platelet responses, since described for other seven-transmembrane receptors, includ- U46619-induced platelet shape change was still observed ing the hydroxytryptamine receptor (5-HT4 receptor), the in Gq knockout mice [120]. This observation, coupled lysophosphatidic acid receptor (LPA) and the thrombin with the finding that TP-mediated shape change was receptor (PAR), among others [102–104]. Regarding the blocked by the Rho-kinase inhibitor Y-27632, led to the downstream consequences of TP–G13-mediated signalling, conclusion that TP-induced shape change is a G12/13- evidence has been provided for Ga13 stimulation of the mediated event [105]. This suggestion would seem to Rho/Rho kinase pathway and subsequent myosin light be consistent with earlier observations that TP agonists chain (MLC) phosphorylation in platelets [105,106].Such can induce platelet shape change in the absence of events could lead to alterations in actin formation, platelet measurable calcium mobilization [121] (which is presum- cytoskeletal rearrangement [107–110] and inside-out sig- ably a Gq-mediated event). However, platelet shape nalling [105,111–113]. change can be elicited by extremely low levels of TP Aside from Gq and G12/13, evidence has also been activation, e.g., 2–35 nM [121,122], and the presence of provided that TPs can couple to Gi. In this case, however, undetectable calcium fluxes at these low levels cannot be controversy exists regarding whether such coupling occurs excluded. In addition, it would seem difficult simply to in human platelets. Thus, while Ushikubi et al. [114] assign a single platelet response to a single G protein showed that Gi2 functionally couples to TPs in phospho- pathway, because platelet shape change can also result lipid vesicles, and Gao et al. [115] reported TP–Gi from Gq signalling. Thus, ADP is incapable of inducing signalling in ECV 304 cells (a human endothelial cancer shape change in Gq-deficient mouse platelets [120,123]. cell line), separate studies have been unable to demonstrate Since there is no definitive information demonstrating a such an association in platelets. For example, ligand direct coupling of platelet ADP receptors to G12/13, these affinity chromatography purification did not reveal the findings suggest that the shape change response can be physical coupling of platelet TPs to Gi [74], and other elicited by multiple signalling events. Consequently, under experiments could not detect GTP labelling of Gi follow- physiological signalling conditions, i.e., in the presence of ing TP stimulation [71]. Consequently, at present, there are intact Gq and G12/13 signalling, the TP-mediated shape no definitive results which establish direct TP coupling to change response may actually represent a composite of Gi in platelets. signalling through both of these pathways. J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 525

Separate studies have linked TP–G12/13 signalling to the 4. TP signalling in other cells and tissues platelet aggregation response. Specifically, it was found that the Rho kinase inhibitor Y-27632 abolished TP-dependent 4.1. Smooth muscle cells shape change in Gq-deficient platelets, but had no significant influence on the aggregation response [113].This Since its initial identification as the ‘‘rabbit aorta con- finding led the authors to suggest that G12/13 can contribute tracting substance (RCS)’’, TXA2-mediated contraction has to integrin activation by regulating a Rho kinase-indepen- been examined in many smooth muscle types including dent mechanism. Furthermore, it was also shown that vascular, respiratory, uterine and intestinal [134–140]. U46619-induced aggregation could occur in Gq-deficient While not completely understood, certain results have mice if Gi signalling was simultaneously activated. Based provided evidence that the signalling pathway of TXA2- on these findings, it was concluded that co-stimulation of induced contraction appears to involve activation of the G12/13 and Gi is sufficient to induce aIIb h3 activation. small GTPase Rho, Rho kinase, myosin light chain kinase, Similar conclusions were reached by a subsequent study and to a lesser extent PKC [141,142]. On the other hand, from another group [124]. In these experiments, attempts another study using canine pulmonary vascular smooth were made to eliminate the participation of Gq signalling in muscle found that an inhibitors of tyrosine kinases (genis- the TP response by using a low concentration of U46619 tein) and Rho kinase (Y-27632) abolished U46619-induced which is presumably selective for the TP–G12/13 pathway. contraction, whereas PKC (calphostin C), p38 MAPK (SB- Under these conditions, simultaneous activation of Gi sig- 203580) and MAPK kinase (PD-98059) inhibitors were nalling resulted in platelet aggregation at a concentration of ineffective [143]. Most recently, U46619-induced contrac- U46619 which itself could not induce the aggregation tion was studied in isolated rat mesenteric resistance arteries response. [144]. In this case, however, a tyrosine kinase inhibitor Finally, TP-mediated G protein signalling is known to (tyrphostin A25) was ineffective in blocking contraction, result in the activation of numerous of downstream kinases but both PKC inhibitors (GF109203X) and p38 MAPK as part of the platelet response. Thus, TP stimulation has inhibitors (SB-203580) were found to block the contraction been associated with activation of Ras [125],Rho response. Based on these findings, it therefore appears that [105,126],Rac[126] and related effectors such as TXA2-induced contraction involves activation of different p160ROCK, as well as the Ca/calmodulin system [127]. signalling mechanisms depending on both the species and Other studies have demonstrated TP-mediated activation of the type of smooth muscle being investigated. pp72(syk), pp60(c-src) tyrosine kinase and mitogen-activat- Aside from its role in stimulating smooth muscle ed protein kinase (MAPK)(p38MAPK, p42MAPK) contraction, TP activation has been shown to have mito- [105,128–133]. Since, however, the process of platelet genic effects. For example, using rat vascular smooth activation leads to multiple feedback signalling mecha- muscle, it was found that TP agonists increased thymidine nisms, it has been difficult to determine whether the incorporation and proliferation by stimulating cell cycle stimulation of a specific kinase serves as an initiating event, progression from the S to G2/M phase [145,146]. Simi- or an event which results as a consequence of the overall larly, work by Morinelli et al. [147] showed that TP platelet response. Nevertheless, recent work by Li et al. [99] stimulation in guinea pig coronary artery smooth muscle suggests that PI3 kinase is linked to the process of TP- produced increased DNA synthesis, activation of the mediated platelet secretion. In these studies it was shown ERK1/2 MAPKs, and the S6 kinase p85RSK. Further that U46619 causes PI3 kinase-dependent phosphorylation work in rat aortic smooth muscle cells [148] provided of Akt, and that platelets deficient in PI3 kinase exhibit an evidence that stable TP agonists induced the expression of impaired U46619-induced second wave of secretion. How- c-fos and early growth response gene-1 (egr-1) mRNA. ever, in this case as well, it is not clear whether the PI3 These studies also revealed that TP activation potentiated kinase-dependent secretion derives from downstream events the mitogenic effects of platelet derived growth factor associated with the TP-mediated signalling process or by (PDGF). In contrast, separate results have indicated that direct activation via Ghg. Clearly, additional studies will be even though TP stimulation of rat aortic smooth muscle required to define more fully the precise TP signalling increases proto-oncogene expression and accelerates pro- events linked to the activation of different platelet kinases tein synthesis, it does so in the absence of DNA synthesis and the functional consequences of such activation. or cell proliferation [149,150]. Specifically, in these In summary, platelet TPs have the capacity to couple experiments the results indicated that TXA2 stimulates functionally to different G protein families and elicit a smooth muscle hypertrophy by increasing the synthesis multitude of downstream signalling events. While it appears and release of endogenous basic fibroblast growth factor that activation of certain of these pathways can be associated (bFGF). Other work has also demonstrated that rat aortic with selected platelet responses, it seems likely that in the smooth muscle proliferation induced by angiotensin II or fully functional platelet, each of these pathways contributes tumour necrosis factor alpha (TNFa) is mediated by a to the overall shape change, aggregation and secretion MAPK-dependent induction of cyclooxgenase 2 (COX-2) response. and increased TXA2 production [151]. 526 J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533

Additional experiments have investigated the TP signal- growth factor (VEGF) [157]. However, these findings appear ling pathways mediating the mitogenic response in human to be in contrast to those reported by a separate group [158] uterine vascular smooth muscle cells. In this connection, who found that TP stimulation inhibited human endothelial Miggin and Kinsella [152] showed that TP stimulation not cell migration and in vitro capillary formation. Additional only caused the activation of ERKs but also caused the work by this group provided evidence that TP signalling activation of c-Jun N-terminal kinases (JNKs). This re- through both TP variants induces apoptosis of endothelial sponse was found to be dependent on PKC, PKA, PI3- cells and inhibits the phosphorylation of Akt kinase which is kinase, and appeared to be linked to transactivation of the involved in cell survival [159].Furthermore,therealso epidermal growth factor (EGF) receptor. More recently, appeared to be a divergence in the signalling pathways by Gallet et al. [153] showed that TP-mediated ERK activation which these events occurred, since activation of adenylyl involves both pertussis toxin-sensitive and -insensitive G cyclase and PKA caused inhibition of apoptosis induced by proteins, src kinase, as well as transactivation of matrix the TPh isoform, but did not block apoptosis induced by TPa metalloproteinases. In short, previous studies have provided activation. Most recently, this group examined the interaction evidence that TP activation not only causes smooth muscle between TXA2 and TNFa signalling as it may occur under cell contraction, but also initiates a host of nuclear signalling inflammatory or ischemic conditions [160]. It was found that events. These studies also reveal apparent differences in the TP agonists stimulated the expression of leukocyte adhesion nuclear signalling profile for TP-mediated responses, and molecule (LAM) on endothelial cells via TPh. In contrast, TP while the reasons for these disparities are unknown, they signalling had the opposite effect in the presence of TNFa,in may at least in part relate to species differences and/or that TP activation was found to reduce TNF-mediated LAM experimental culture conditions. expression and enhance endothelial cell apoptosis. This effect was ascribed to a reduction in the prosurvival NFnB activa- 4.2. Endothelial cells tion pathway induced by TNFa. Consequently, TP activation appears to recruit a number of endothelial cell responses Following the cloning of the placental TP receptor, a ranging from the expression of surface adhesive molecules second splice variant with a divergent cytoplasmic tail was involved in cell migration, angiogenesis and tumour metas- cloned from a human endothelial cell library [2]. However, tasis to the induction of apoptosis in inflammatory conditions. even though it was determined that endothelial cells express this splice variant termed TPh as well as the platelet/ 4.3. CNS and PNS cell types placenta variant TPa, the specific roles of these variants in endothelial cell function remain unclear. Nevertheless, var- More recently, evidence has been provided that TPs ious studies have made significant contributions to defining foster both physiological and pathological responses in the endothelial cell TP signalling. Ishizuka et al. [154] deter- CNS and PNS. For example, CNS TPs contribute to the mined that activated human vascular endothelial cells ex- angiotensin II-mediated dipsogenic response [161],the press intracellular adhesion molecule-1 (ICAM-1) following stimulation of foetal adrenocorticotropic hormone (ACTH) the production and release of endogenous TXA2, and that secretion [162], the central adrenomedullary outflow, and this TP-dependent expression of ICAM-1 is PKC-depen- adrenaline release [163]. In addition, TXA2 has been shown dent. In addition, the surface expression of other adhesion to elevate blood pressure by multiple mechanisms including proteins, i.e., vascular cell adhesion molecule-1 (VCAM-1), activation of specific CNS and PNS TPs as well as interac- and endothelial leukocyte adhesion molecule (ELAM-1), tion with vascular smooth muscle TPs leading to vasocon- were also induced by TP stimulation in a PKC-dependent striction in the systemic circulation [162,164,165]. Aside manner [155]. On the other hand, there appear to be differ- from hormonal or cardiovascular effects which may impact ences in the downstream signalling events associated with homeostasis, TPs also appear to contribute to pathological the TP-mediated expression of these adhesive molecules. conditions such as cerebral ischemia–reperfusion injury. Thus, ICAM-1 or ELAM-1 expression was found to be This effect may be due to several considerations including dependent on activation of the NFnB and AP-1 transcription an increase in the generation of oxygen radicals [166], local factors, whereas VCAM-1 was found only to involve NFnB vasoconstriction and/or platelet deposition [167–171].A activity. similar involvement of TPs has been linked to secondary Separate evidence has been provided that endothelial cell damage following spinal cord injury [172,173], as shown by TPs are linked to the processes of cell migration, survival, the finding that TP antagonists provide protective effects on angiogenesis and tumour metastasis. Specifically, it was spinal cord perfusion following experimental cord injury found that TX2 produced via COX-2 is a mediator of human [174]. microvascular endothelial cell migration (HMVEC) and While the cellular function of TP signalling in neurons angiogenesis [156] Similarly, other studies reported that has not been extensively studied, several potentially impor- TXA2 synthesis and release is associated with the migration tant features of this pathway have been identified. For of human umbilical vein endothelial cells (HUVECs) or example, hippocampal TPs seem to play a functional role HMVECs in response to bFGF or vascular endothelial in both neuronal excitability and synaptic transmission. J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 527

Thus, it was found in rat CA1 neurons that TP activation activation of the MAPK cascade and associated nuclear dose-dependently suppressed whole-cell Ca2+ currents signalling events. In particular, activation with low nano- [175]. Additionally, other studies reported that activation molar concentrations of U46619 causes ERK1/2 and CREB of presynaptic TPs increased the release of glutamate, while phosphorylation, up-regulation of c-fos and p-65, as well as activation of postsynaptic TPs was associated with inhibi- stimulation of AP1 and NFnB [182]. tion of synaptic transmission [176]. Both of these effects In addition to their presence on OLGs, TPs have been were found to be mediated by a PKC-dependent pathway identified in other myelinating cells as well. Thus, initial which was both pertussis- and cholera toxin-insensitive. studies revealed dense immunostaining of white matter in The first indication of the presence of TP signalling in the rat spinal cord [17], and subsequent experiments dem- glial tissue was the observation that a TXA2 analogue, STA2 onstrated immunocytochemical and immunoblot labelling of (9,11-epithio-11,12-methano-thromboxane A2), stimulates TPs in rat Schwann cells (rSC) [24]. Interestingly, these rSC phospholipase C and inositol phosphate accumulation in TPs were not only localized on the plasma membrane but 1321N1 human astrocytoma cells via a pertussis toxin- were also associated with the cytoplasm and the nucleus. insensitive G protein [177]. Using radioligand binding, it Functional analysis of these TPs revealed that unlike OLGs, was subsequently demonstrated that TPs exist on primary U46619 stimulation of rSC cells did not cause measurable cultured rabbit astrocytes, and that activation of TPs also Ca2+ release, but did result in a marked increase in both results in phosphoinositide hydrolysis [26]. Furthermore, cAMP levels and CREB phosphorylation [24]. using immunoaffinity chromatography purification, this Taken together, the above studies provide evidence for group later showed that 1321N1 cells expressed two TPs the presence of functional TPs in both the CNS and PNS, (MW 58 and 55 kDa), which copurified with both Gq/11 and and even though the precise TP signalling mechanisms in G12 proteins [178]. Additional studies provided evidence these organ systems remain to be established, it appears that that differentiation of 1321N1 cells by dibutyryl-cAMP was TP activation can modulate both neuronal and glial cell accompanied by a reduction in TPR density and phosphoi- responses through cytoplasmic and/or nuclear signalling nositide mobilization in response to agonist stimulation events. [179]. On the other hand, the differentiated cells revealed a marked STA2-mediated activation of the MAPK cascade 4.4. Other cell types via stimulation of a phosphatidylcholine-specific phospholipase C and PKC [180]. Collectively, these results therefore Aside from the organ systems described above, TPs are suggest a possible shift in TP signalling pathways upon cell also known to be functionally involved in the immune differentiation. system. In this regard, Namba et al. [8] cloned the mouse In separate experiments with antibodies against specific TP and examined its expression in various tissues by TP sequences, it was noted that TPs are concentrated in Northern blot analysis. Of the mouse tissues examined, discrete rat brain regions. Thus, unlike the diffuse labelling the thymus appeared to possess the highest expression of pattern which would be expected from astrocytes, the TPs, followed by the spleen and lung. In later studies, it was labelling was distinctly associated with myelin-enriched determined that the receptor density in immature thymo- white matter structures, e.g., the striatum, the internal cytes is comparable to that found in platelets, and that TP capsule and the optic tract [16]. These observations there- stimulation mediates apoptosis and DNA fragmentation of fore provided the first indication that TPs are highly CD4+/CD8+ cells [31]. Recently, this group has generated concentrated in myelinated regions of the CNS, and sug- TP-deficient mice to study the consequences of TXA2 gested the presence of TPs on oligodendrocytes. This notion signalling in the immune system [50]. It was found that was further supported by RT-PCR analysis which revealed TXA2 produced by dendritic cells (DC) induces the chemo- the presence of TP mRNA in brain glial cells including kinesis of naive T cells, impairs DC–T cell adhesion, and oligodendrocytes [28,181]. Subsequent studies established inhibits DC-dependent proliferation of T cells. These results that cultured OLGs (or their tumour-related cell line, human therefore suggest that TP signalling modulates acquired oligodendroglioma [HOG] cells) express TPs on their plas- immunity by interfering with DC–T cell interaction. ma membranes, and that this protein has an apparent Finally, other studies have provided evidence that kidney molecular weight and ligand affinity comparable to that TPs may play a role in both normal and pathological seen in platelet membranes [17,23] or 1321N1 human conditions [22,40,42,44,183,184]. Specifically, cloning of astrocytoma cells. These studies also revealed that activation the rat kidney TP revealed the presence of TP mRNA in of OLG TPs by U46619 elicited intracellular Ca2+ mobili- various regions of the kidney [11]. The widespread distribu- zation, which is consistent with earlier observations linking tion of kidney TPs was later confirmed by immunological Gq/11 signalling to TPs in astrocytes [27]. Finally, recent studies which demonstrated TP receptor protein immunore- data has linked TP signalling in OLG cell lines to functional activity along the lumen of glomerular capillary loops, as well effects, i.e., enhanced proliferation and survival [182]. as in mesangial cells, podocytes, and epithelial cells [13]. While the signalling pathways associated with these effects Additional experiments have shown that TP stimulation of are unknown, it appears that TP stimulation leads to mesangial cells increases cell contraction, promotes cell 528 J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 proliferation, causes changes in cellular ion fluxes, mobilizes platelet cAMP levels with PGI2 caused a substantial phos- phosphoinositides, activates PKC, and increases fibronectin, phorylation of TP-associated Ga13 [100]. This phosphory- laminin, collagen, tissue plasminogen activator (tPA), plas- lation was shown to be PKA-dependent [100] and to occur minogen activator inhibitor-1 (PAI-1) mRNA levels and at a single site within the functionally important Switch I 203 transforming growth factor synthesis. [22,185–187]. More Region of the Ga13 subunit, i.e., at Thr [101]. These recently, separate studies have [188] determined that TP findings were consistent with an earlier report demonstrat- stimulation has a cytoprotective role in renal epithelial cells ing that cAMP blocks U46619-induced Rho/Rac activation via the activation of the NFnB and AP-1 transcription factors. [126] in platelets, and suggest that signalling through the TP In summary, the above results suggest that TXA2 may have pathway may itself be modulated by activation of second unique roles in kidney function, and that these roles are messenger kinases. dependent upon its particular site of action.

6. Modulation of TP signalling by other GPCRs 5. Modulation of TP signalling by protein kinases: PKC, PKA, PKG Even though different GPCRs are known to function through distinct signal transduction pathways, it is also Early investigations into modulation of TP signalling by apparent that there are certain points along the activation cyclic nucleotides revealed that elevation of platelet cAMP cascade by which these pathways communicate. Thus, levels caused a suppression of U46619-induced intraplatelet agonist–GPCR interaction can influence the cellular re- calcium mobilization. This effect was interpreted as evidence sponse to a separate agonist interacting with a different for one potential mechanism by which cAMP may inhibit TP- GPCR. One example of such ‘‘cross-talk’’ between path- mediated blood platelet activation [90,189,190], i.e., en- ways is the phenomenon of synergism. Although synergism hanced calcium sequestration through PKA-mediated phos- is widely observed in pharmacological therapeutics, the phorylation of a 22-kDa protein [191–196].Oncethe molecular mechanisms leading to such disproportionate primary structure of TPs was resolved, it became apparent responses have not been extensively studied. However, that TPs possess one PKA/PKG phosphorylation site and four there is increasing evidence to suggest that communication PKC phosphorylation sites. Based on this consideration, between GPCRs can occur through different mechanisms experiments were next undertaken to determine whether including heterodimerization [200–206], cross-reactions TPs can serve as substrates for PKC, PKA and/or PKG. among GPCR downstream effectors [207] and G protein The initial studies in this area revealed that PKC and PKA redistribution [208–212]. caused phosphorylation of a fusion protein spanning the latter While the processes of heterodimerization or downstream third extracellular loop of TPs [197], leading the authors to effector cross-signalling have not been defined for TP- suggest that PMA or cAMP (PKC and PKA, respectively) mediated responses, recent evidence suggests that signalling may desensitize TP-induced platelet activation through phos- between TPs and other GPCRs can occur through agonist- phorylation of TPs themselves. While a subsequent report induced redistribution of G proteins. In this regard, initial using transfected HEK293 cells questioned physiological studies focused on the classical synergism which occurs significance of this effect [198], a later study [4] concluded between platelet PAR1 receptors and TPs. It was found that that PKC-mediated receptor phosphorylation may in fact low-dose activation of PAR1 receptors led to a substantial serve as one mechanism for TP desensitization in platelets. increase in ligand binding to TPs [208]. Furthermore, this Regarding the modulatory effects of cGMP, a separate group increased ligand binding was associated with a shift in the used immunoaffinity chromatography to demonstrate that Hill coefficient from a single- to a two-site model, and the PKG has the capacity to phosphorylate TPs [199],ina appearance of a high affinity TP pool [209]. Additional putative G protein coupling domain. On this basis, it was experiments also demonstrated that platelet PAR1 receptor postulated that such phosphorylation may interfere with TP– activation causes a measurable increase in TP-associated G protein signal transduction. Consistent with this suggestion Gaq. Based on these findings, a model (Fig. 2) was devel- was the finding that cGMP-mediated phosphorylation was oped to describe a dynamic equilibrium that may exist associated with decreased U46619-stimulated GTPase activ- between TPs and other GPCRs which couple to the same ity. Even though a net decrease in GTPase activity cannot Ga subunits. In this model, both TPs and PAR1 receptors differentiate between effects on the receptor and/or its asso- exist in at least two different ligand affinity states: uncoupled ciated G protein, these results nevertheless provided a link low-affinity and G-protein-coupled high affinity. Upon ago- between PKG phosphorylation and inhibition of the TP–G nist binding to the high ligand affinity PAR1 receptors, the protein signalling transduction mechanism. Gahg heterotrimer dissociates and interacts with down- As a separate approach to investigating cyclic nucleotide stream effectors. During reassociation, these Ga subunits modulation of TP signalling, other studies have examined may or may not recouple to their original GPCR receptor the possible phosphorylation of TP-coupled G proteins. In classes. Rather, the mass and affinity TP and PAR1 for the particular, these experiments demonstrated that elevation of ‘‘free’’ Ga will determine the final equilibrium of the J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 529

above, recent studies [113,124] demonstrated that co-stimulation of TP-mediated G12/13 and P2Y12 (or a2A-adrenergic)-mediated Gi signalling led to platelet integrin activation. These results are consistent with previous studies which demonstrated synergistic interactions between U46619 and ADP [215–217] or between U46619 and epinephrine [217,218]. While the mechanism of this cross- signalling between TP- and Gi-coupled receptors is unknown, it is tempting to speculate that it may proceed through similar mechanism of hg redistribution as the bradykinin (B2) and P2Y receptors described above. Alter- natively, this synergism may derive from hg activation of downstream effector sites. Clearly, these concepts are at a relatively early stage of development, and additional studies will be required to define further the various mechanisms by which TPs have the capacity to cross-signal and the possible physiological consequences of these events.

Fig. 2. Dynamic equilibrium between TPs and other GPCRs. 7. Summary reassociation process. In the case of PAR1 receptor activation, both the PAR1 mass and its Ga affinity would decrease Over the years, it has become increasingly apparent that because of PAR1 internalization and because of PAR1– TPs are involved in a multitude of physiological and ligand interaction. Acting together, these effects would foster pathological processes. They are present on a variety of increased Ga coupling to TPs and their consequent shift to a cell types and have been localized to both plasma membrane higher ligand affinity state. Thus, it appears that there can be and cytosolic compartments. As a member of the seven- a cycling of G proteins not only within a specific receptor transmembrane receptor class, TPs are known to couple to class but also between receptors that share common Ga (or and signal through several different G protein families. As a possibly hg) subunits. This process of mass/affinity-directed consequence, these receptors have been shown to participate TP–G protein coupling also suggests that a competition can in the activation of a number of different signalling cas- exist between TPs and other GPCRs, and that this compe- cades. Even though many of the details regarding these tition may define the predominant signalling pathways signalling events are presently unknown, certain character- through which TPs signal under different experimental istics are apparent. For example, TP signalling preference conditions and in different cell types. For example, even among these different pathways is not uniform across all cell though TPs signal through the Gq,G12/13 pathway or Gi types, or in certain circumstances, even within a given cell pathways in most cells, TP-mediated Gs signalling has been type. Thus, blood platelet TP activation does not result in shown to be a significant pathway in transfected COS-7 cells elevated cAMP levels, whereas such activation does pro- [213,214] and rat Schwann cells [24]. duce measurable cAMP increases in PNS Schwann cells. On the other hand, there is no direct evidence for TP Furthermore, TP signalling produces apoptosis in certain cross-signalling through a redistribution of hg G protein cell types and prolonged survival in others. This difference subunits. Nevertheless, previous results have indicated that in signalling preference also seems to extend to different this process can occur for other GPCRs [210]. In these stages of the cell cycle where its activation of various experiments, two receptor groups were studied: the adeno- nuclear transcription events changes during proliferation sine A(1)/a2C adrenergic receptors which couple to Gai and and differentiation. Consequently, it would seem that cau- the bradykinin B(2) or P2Y (UTP-responding) receptors tion should be exercised when attempting to define the which couple to Gaq. It was found that activation of Gai- signalling pathways of this eicosanoid receptor. The accu- coupled receptors increased the potency and the efficacy of mulation of data thus far compiled across different cell lines inositol phosphate production induced by bradykinin or establishes the signalling capacity of TPs and not necessar- UTP activation. Furthermore, the overexpression of ily that which occurs in a given cell at a particular stage of Gh1g2 also resulted in increased potency and efficacy of its development. bradykinin or UTP, and that almost all possible combina- tions of Gh(1–3) with Gg(2–7)produced similar effects. On h this basis, the authors proposed that G g subunits could References redistribute from an activated Gai-coupled receptor to an activated Gaq-coupled receptor and enhance the receptor- [1] M. Hirata, Y. Hayashi, F. Ushikubi, Y. Yokota, R. Kageyama, S. stimulated GDP/GTP exchange of Gq [210]. As mentioned Nakanishi, et al., Nature 349 (1991) 617–620. 530 J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533

[2] M.K. Raychowdhury, M. Yukawa, L.J. Collins, S.H. McGrail, K.C. [36] M. Randomski, Acta Physiol. Pol. 36 (1985) 153–164. Kent, J.A. Ware, J. Biol. Chem. 269 (1994) 19256–19261. [37] M.L. Ogletree, Fed. Proc. 46 (1987) 133–138. [3] T. Hirata, F. Ushikubi, A. Kakizuka, M. Okuma, S. Narumiya, J. [38] I. Foulon, D. Bachir, F. Galacteros, J. Maclouf, Arterioscler. Clin. Invest. 97 (1996) 949–956. Thromb. 13 (1993) 421–426. [4] A. Habib, G.A. FitzGerald, J. Maclouf, J. Biol. Chem. 274 (1999) [39] S.D. Katugampola, A.P. Davenport, Br. J. Pharmacol. 134 (2001) 2645–2651. 1385–1392. [5] R.F. Shen, H.H. Tai, J. Biomed. Sci. 5 (1998) 153–172. [40] R.F. Spurney, P.Y. Fan, P. Ruiz, F. Sanfilippo, D.S. Pisetsky, T.M. [6] P.V. Halushka, Prostaglandins Other Lipid Mediat. 60 (2000) Coffman, Kidney Int. 41 (1992) 973–982. 175–189. [41] G.W. Dorn, N. Liel, J.L. Trask, D.E. Mais, M.E. Assey, P.V. Ha- [7] R.A. Armstrong, N.H. Wilson, Gen. Pharmacol. 26 (1995) lushka, Circulation 81 (1990) 212–218. 463–472. [42] T. Kobayashi, J. Suzuki, M. Watanabe, S. Suzuki, K. Yoshida, K. [8] T. Namba, Y. Sugimoto, M. Hirata, Y. Hayashi, A. Honda, A. Kume, et al., Nephron. 77 (1997) 309–314. Watabe, et al., Biochem. Biophys. Res. Commun. 184 (1992) [43] J. Geoffroy, D. Benzoni, J. Sassard, J. Hypertens. Suppl. 7 (1989) 1197–1203. S272–S273. [9] B. Batshake, C. Nilsson, J. Sundelin, Biochem. Biophys. Res. [44] H. Saito, T. Ideura, J. Takeuchi, Nephron. 36 (1984) 38–45. Commun. 256 (1999) 391–397. [45] N. Liel, I. Nathan, T. Yemiyahu, Z. Zolotov, J.R. Lieberman, P.V. [10] S.M. Miggin, B.T. Kinsella, Biochim. Biophys. Acta 1425 (1998) Halushka, Thromb. Res. 70 (1993) 205–210. 543–559. [46] S. Kohno, Nippon Rinsho 54 (1996) 551–558. [11] T. Abe, K. Takeuchi, N. Takahashi, E. Tsutsumi, Y. Taniyama, K. [47] C. Martin, S. Uhlig, V. Ullrich, Am. J. Respir. Cell. Mol. Biol. 24 Abe, J. Clin. Invest. 96 (1995) 657–664. (2001) 139–145. [12] G.P. Brown, R.C. Venuto, Prostaglandins Other Lipid Mediat. 57 [48] K. Kabashima, T. Murata, H. Tanaka, T. Matsuoka, D. Sakata, N. (1999) 179–188. Yoshida, et al., Nat. Immunol. 4 (2003) 694–701. [13] B.A. Bresnahan, G.C. Le Breton, E.A. Lianos, Kidney Int. 49 (1996) [49] K. Tanaka, M.H. Roberts, N. Yamamoto, H. Sugiura, M. Uehara, 1207–1213. X.Q. Mao, et al., Biochem. Biophys. Res. Commun. 292 (2002) [14] A. Hedberg, P.F. Mento, E.C. Liu, B.M. Wilkes, Am. J. Physiol. 256 776–780. (1989) E256–E263. [50] G. Muller, C. Taube, U. Senger, L. Wagner, Dtsch Z Verdau Stoff- [15] G.L. Stahl, H. Darius, A.M. Lefer, Life Sci. 38 (1986) 2037–2041. wechselkr. 46 (1986) 243–246. [16] C. Borg, C.T. Lim, D.C. Yeomans, J.P. Dieter, D. Komiotis, E.G. [51] J.B.C. Findlay, D.J.C. Pappin, Biochem. J. 238 (1986) 625–642. Anderson, et al., J. Biol. Chem. 269 (1994) 6109–6116. [52] B.K. Kobilka, T.S. Kobilka, K. Daniel, J.W. Regan, M.J. Caron, R.J. [17] S.C. Blackman, C. Borg, D.C. Yeomans, G.C. Le Breton, Methods Lefkowitz, Science 4857 (1988) 1310–1316. Mol. Biol. 120 (1999) 145–171. [53] T.M. Savarese, C.M. Fraser, Biochem. J. 283 (1992) 1–19. [18] Z. Chen, S. Prasad, M. Cynader, Br. J. Ophthalmol. 78 (1994) [54] S.K.-F. Wong, C. Slaughter, A. Ruoho, E. Ross, J. Biol. Chem. 263 921–926. (1988) 7925–7928. [19] G.C. Le Breton, D.L. Venton, S.E. Enke, P.V. Halushka, Proc. Natl. [55] S. Narumiya, N. Hirata, T. Namba, Y. Hayashi, F. Ushikubi, Y. Acad. Sci. U. S. A. 76 (1979) 4097–4101. Sugimoto, et al., J. Lipid. Mediators 6 (1993) 155–161. [20] S.C. Hung, N.I. Ghali, D.L. Venton, G.C. Le Breton, Biochim. [56] C.D. Funk, L. Furci, N. Moran, G.A. FitzGerald, Mol. Pharmacol. Biophys. Acta 728 (1983) 171–178. 44 (1993) 934–939. [21] R.A. Armstrong, R.L. Jones, N.H. Wilson, Br. J. Pharmacol. 79 [57] N. Chiang, W.M. Kan, H.H. Tai, Arch. Biochem. Biophys. 334 (1983) 953–964. (1996) 9–17. [22] R.F. Spurney, J.J. Onorato, F.J. Albers, T.M. Coffman, Am. J. [58] G.W. Dorn, M.G. Davis, D.D. D’Angelo, J. Biol. Chem. 272 (1997) Physiol. 264 (1993) F292–F299. 12399–12405. [23] S.C. Blackman, G. Dawson, K. Antonakis, G.C. Le Breton, J. Biol. [59] D.D. D’Angelo, J.J. Eubank, M.G. Davis, G.W. Dorn, J. Biol. Chem. Chem. 273 (1998) 475–483. 271 (1996) 6233–6240. [24] N. Muja, S.C. Blackman, G.C. Le Breton, G.H. DeVries, J. Neuro- [60] D. Komiotis, J.D. Wencel-Drake, J.P. Dieter, C.T. Lim, G.C. Le chem. 78 (2001) 446–456. Breton, Biochem. Pharmacol. 52 (1996) 763–770. [25] Y. Li, J.X. Kang, A. Leaf, Prostaglandins 54 (1997) 511–530. [61] K.-H. Ruan, S.-P. So, J. Wu, D. Li, A. Huang, J. Kung, Biochemistry [26] N. Nakahata, H. Ishimoto, M. Kurita, K. Ohmori, A. Takahashi, H. 40 (2001) 275–280. Nakanishi, Brain. Res. 583 (1992) 100–104. [62] J. Wu, S.P. So, K.H. Ruan, Arch. Biochem. Biophys. 414 (2003) [27] N. Nakahata, A. Miyamoto, S. Ohkubo, H. Ishimoto, K. Sakai, H. 287–293. Nakanishi, et al., Res. Commun. Mol. Pathol. Pharmacol. 87 (1995) [63] J.W. Turek, T. Halmos, N.L. Sullivan, K. Antonakis, G.C. Le Bre- 243–251. ton, J. Biol. Chem. 277 (2002) 16791–16797. [28] J. Kitanaka, H. Hashimoto, Y. Sugimoto, M. Sawada, M. Negi- [64] S.P. So, J. Wu, G. Huang, A. Huang, D. Li, K.H. Ruan, J. Biol. shi, A. Suzumura, et al., Biochim. Biophys. Acta 1265 (1995) Chem. 278 (2003) 10922–10927. 220–223. [65] S.A. Alla, U. Quitterer, S. Grigoriev, A. Maidhof, M. Haasemann, [29] G.W. Dorn 2nd, Am. J. Physiol. 262 (1992) C991–C999. K. Jarnagin, et al., J. Biol. Chem. 271 (1996) 1748–1755. [30] S. Nigam, S. Eskafi, A. Roscher, H. Weitzel, FEBS Lett. 316 (1993) [66] L. Audoly, R.M. Breyer, J. Biol. Chem. 272 (1997) 13475–13478. 99–102. [67] B.A. Stillman, L. Audoly, R.M. Breyer, Eur. J. Pharmacol. 357 [31] F. Ushikubi, Y. Aiba, K. Nakamura, T. Namba, M. Hirata, O. Mazda, (1998) 73–82. et al., J. Exp. Med. 178 (1993) 1825–1830. [68] A.G. Gilman, Ann. Rev. Biochem. 56 (1987) 615–649. [32] A. Fennekohl, H.L. Schieferdecker, K. Jungermann, G.P. Puschel, [69] J.R. Hepler, A.G. Gilman, Trends Biochem. Sci. 17 (1992) J. Hepatol. 30 (1999) 38–47. 383–387. [33] P.R. Mayeux, D.E. Mais, C. Carr, P.V. Halushka, J. Pharmacol. Exp. [70] Y. Kaziro, H. Itoh, T. Kozasa, M. Nakafuku, T. Satoh, Annu. Rev. Ther. 250 (1989) 923–927. Biochem. 60 (1991) 349–400. [34] D.D. D’Angelo, M.G. Davis, S. Ali, G.W. Dorn, J. Pharmacol. Exp. [71] S. Offermanns, K.L. Laugwitz, K. Spicher, G. Schultz, Proc. Natl. Ther. 271 (1994) 1034–1041. Acad. Sci. U. S. A. 91 (1994) 504–508. [35] D.W. Thomas, R.B. Mannon, P.J. Mannon, A. Latour, J.A. Oliver, [72] S. Offermanns, Y.H. Hu, M.I. Simon, J. Biol. Chem. 271 (1996) M. Hoffman, et al., J. Clin. Invest. 102 (1998) 1994–2001. 26044–26048. J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 531

[73] A. Shenker, P. Goldsmith, C.G. Unson, A.M. Spiegel, J. Biol. Chem. [109] A. McNicol, T.S. Shibou, C. Pampolina, S.J. Israels, Thromb. Res. 266 (1991) 9309–9313. 103 (2001) 25–34. [74] I. Knezevic, C. Borg, G.C. Le Breton, J. Biol. Chem. 268 (1993) [110] J.E. Fox, S.J. Shattil, R.L. Kinlough-Rathbone, M. Richardson, 26011–26017. M.A. Packham, D.A. Sanan, J. Biol. Chem. 271 (1996) 7004–7011. [75] L.F. Brass, J.A. Hoxie, T. Kieber-Emmons, D.R. Manning, M. [111] T.M. Leisner, J.D. Wencel-Drake, W. Wang, S.C. Lam, J. Biol. Poncz, M. Woolkalis, Adv. Exp. Med. Biol. 344 (1993) 17–36. Chem. 274 (1999) 12945–12949. [76] A.N. Giesberts, M. van Ginneken, G. Gorter, E.G. Lapetina, J.W. [112] S.J. Shattil, H. Kashiwagi, N. Pampori, Blood 91 (1998) Akkerman, G. Van Willigen, Biochem. Biophys. Res. Commun. 234 2645–2657. (1997) 439–444. [113] B. Nieswandt, V. Schulte, A. Zywietz, M.P. Gratacap, S. Offer- [77] N. Nakahata, A. Miyamoto, S. Ohkubo, H. Ishimoto, K. Sakai, H. manns, J. Biol. Chem. 277 (2002) 39493–39498. Nakanishi, et al., Res. Commun. Mol. Pathol. Pharmacol. 87 (1995) [114] F. Ushikubi, K. Nakamura, S. Narumiya, Mol. Pharmacol. 46 (1994) 243–251. 808–816. [78] Y. Djellas, J.M. Manganello, K. Antonakis, G.C. Le Breton, J. Biol. [115] Y. Gao, S. Tang, S. Zhou, J.A. Ware, J. Pharmacol. Exp. Ther. 296 Chem. 274 (1999) 14325–14330. (2001) 426–433. [79] M. Camps, A. Carozzi, P. Schnabel, A. Scheer, P.J. Parker, P. Giers- [116] R. Vezza, A. Habib, G.A. FitzGerald, J. Biol. Chem. 274 (1999) chik, Nature 360 (1992) 684–686. 12774–12779. [80] A. Katz, D. Wu, M.I. Simon, Nature 360 (1992) 686–689. [117] M.J. Im, R.M. Graham, J. Biol. Chem. 265 (1990) 18944–18951. [81] D.Y. Jhon, H.H. Lee, D. Park, C.W. Lee, K.H. Lee, O.J. Yoo, et al., [118] M.J. Im, R.P. Riek, R.M. Graham, J. Biol. Chem. 265 (1990) J. Biol. Chem. 268 (1993) 6654–6661. 18952–18960. [82] D. Park, D.Y. Jhon, C.W. Lee, K.H. Lee, S.G. Rhee, J. Biol. Chem. [119] H. Nakaoka, D.M. Perez, K.J. Baek, T. Das, A. Husain, K. Misono, 268 (1993) 4573–4576. et al., Science 264 (1994) 1593–1596. [83] Y. Banno, T. Asano, Y. Nozawa, Thromb. Haemost. 79 (1998) [120] S. Offermanns, C.F. Toombs, Y.H. Hu, M.I. Simon, Nature 389 1008–1013. (1997) 183–186. [84] L.F. Brass, C.C. Shaller, E.J. Belmonte, J. Clin. Invest. 79 (1987) [121] A.W. Simpson, T.J. Hallam, T.J. Rink, FEBS Lett. 201 (1986) 1269–1275. 301–305. [85] M.J. Berridge, Biochem. J. 220 (1984) 345–360. [122] T.A. Morinelli, S. Niewiarowski, J.L. Daniel, J.B. Smith, Am. J. [86] T.J. Rink, Experientia 44 (1988) 97–100. Physiol. 253 (1987) H1035–H1043. [87] K. Kaibuchi, K. Sano, M. Hoshijima, Y. Takai, Y. Nishizuka, Cell [123] P. Ohlmann, A. Eckly, M. Freund, J.P. Cazenave, S. Offermanns, C. Calcium 3 (1982) 323–335. Gachet, Blood 96 (2000) 2134–2139. [88] K. Sano, Y. Takai, J. Yamanishi, Y. Nishizuka, J. Biol. Chem. 258 [124] R.T. Dorsam, S. Kim, J. Jin, S.P. Kunapuli, J. Biol. Chem. 277 (1983) 2010–2013. (2002) 47588–47595. [89] D.E. MacIntyre, M. Bushfield, A.M. Shaw, FEBS Letters 188 (1985) [125] D.D. Shock, K. He, J.D. Wencel-Drake, L.V. Parise, Biochem. J. 321 383–388. (Pt 2) (1997) 525–530. [90] N.E. Owen, G.C. Le Breton, Am. J. Physiol. 239 (1981) [126] M.P. Gratacap, B. Payrastre, B. Nieswandt, S. Offermanns, J. Biol. H613–H619. Chem. 276 (2001) 47906–47913. [91] P. Massini, E.F. Luscher, Biochim. Biophys. Acta 372 (1974) [127] B.Z. Paul, J.L. Daniel, S.P. Kunapuli, J. Biol. Chem. 274 (1999) 109–121. 28293–28300. [92] M.B. Feinstein, C. Fraser, J. Gen. Physiol. 66 (1975) 561–581. [128] C. Gallet, J.P. Rosa, A. Habib, M. Lebret, S. Levy-Toledano, J. [93] T.J. Rink, A. Sanchez, T.J. Hallam, Nature 305 (1983) 317–319. Maclouf, J. Biol. Chem. 274 (1999) 23610–23616. [94] Y. Patel, V.V. Kakkar, K.S. Authi, Biochim. Biophys. Acta 1224 [129] A. Oda, B.J. Druker, M. Smith, E.W. Salzman, J. Biol. Chem. 267 (1994) 480–488. (1992) 20075–20081. [95] S.H. Chung, J. Polgar, G.L. Reed, J. Biol. Chem. 275 (2000) [130] J. Saklatvala, L. Rawlinson, R.J. Waller, S. Sarsfield, J.C. Lee, L.F. 25286–25291. Morton, et al., J. Biol. Chem. 271 (1996) 6586–6589. [96] B. Stoyanov, S. Volinia, T. Hanck, I. Rubio, M. Loubtchenkov, D. [131] C. Dangelmaier, J. Jin, J.L. Daniel, J.B. Smith, S.P. Kunapuli, Eur. J. Malek, et al., Science 269 (1995) 690–693. Biochem. 267 (2000) 2283–2289. [97] J. Zhang, J. Zhang, J.L. Benovic, M. Sugai, R. Wetzker, I. Gout, et [132] P. Minuz, S. Gaino, V. Zuliani, R.M. Tommasoli, D. Benati, R. al., J. Biol. Chem. 270 (1995) 6589–6594. Ortolani, et al., Thromb. Haemost. 87 (2002) 888–898. [98] L.R. Stephens, A. Eguinoa, H. Erdjument-Bromage, M. Lui, F. [133] S. Ohkubo, N. Nakahata, Y. Ohizumi, Prostaglandins 52 (1996) Cooke, J. Coadwell, et al., Cell 89 (1997) 105–114. 403–413. [99] Z. Li, G. Zhang, G.C. Le Breton, X. Gao, A.B. Malik, X. Du, J. Biol. [134] T. Tuvemo, K. Strandberg, M. Hamberg, B. Samuelsson, Adv. Pros- Chem. 278 (2003) 30725–30731. taglandin Thromboxane Res. 1 (1976) 425–428. [100] J.M. Manganello, Y. Djellas, C. Borg, K. Antonakis, G.C. Le Breton, [135] S. Bunting, S. Moncada, J.R. Vane, Br. J. Pharmacol. 57 (1976) J. Biol. Chem. 274 (1999) 28003–28010. 462P–463P. [101] J.M. Manganello, J.S. Huang, T. Kozasa, T.A. Voyno-Yasenetskaya, [136] J. Svenssen, K. Strandberg, T. Tuvemo, M. Hamberg, Prostaglandins G.C. Le Breton, J. Biol. Chem. 278 (2003) 124–130. 14 (1977) 425–436. [102] E.G. Ponimaskin, J. Profirovic, R. Vaiskunaite, D.W. Richter, T.A. [137] L.E. LeDuc, P. Needleman, J. Pharmacol. Exp. Ther. 211 (1979) Voyno-Yasenetskaya, J. Biol. Chem. 277 (2002) 20812–20819. 181–188. [103] A. Gohla, R. Harhammer, G. Schultz, J. Biol. Chem. 273 (1998) [138] R.A. Coleman, P.P. Humphrey, I. Kennedy, G.P. Levy, P. Lumley, Br. 4653–4659. J. Pharmacol. 73 (1981) 773–778. [104] Y. Yamaguchi, H. Katoh, M. Negishi, J. Biol. Chem. 278 (2003) [139] L. Wilhelmsson, M. Wikland, N. Wiqvist, Prostaglandins 21 (1981) 14936–14939. 277–286. [105] B. Klages, U. Brandt, M.I. Simon, G. Schultz, S. Offermanns, J. Cell [140] P.T. Horn, J.D. Kohli, G.C. Le Breton, D.L. Venton, J. Cardiovasc. Biol. 144 (1999) 745–754. Pharmacol. 6 (1984) 609–613. [106] M. Bauer, M. Retzer, J.I. Wilde, P. Maschberger, M. Essler, M. [141] X. Fu, M.C. Gong, T. Jia, A.V. Somlyo, A.P. Somlyo, FEBS Lett. Aepfelbacher, et al., Blood 94 (1999) 1665–1672. 440 (1998) 183–187. [107] M.B. Zucker, V.T. Nachmias, Arteriosclerosis 5 (1985) 2–18. [142] S. Sakurada, H. Okamoto, N. Takuwa, N. Sugimoto, Y. Takuwa, [108] J.E. Fox, Thromb. Haemost. 70 (1993) 884–893. Am. J. Physiol. Cell. Physiol. 281 (2001) C571–C578. 532 J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533

[143] L.J. Janssen, H. Lu-Chao, S. Netherton, Am. J. Physiol. Lung Cell [178] S. Honma, N. Nakahata, Y. Ohizumi, Prostaglandins 55 (1998) Mol. Physiol. 280 (2001) L666–L674. 159–168. [144] M. Bolla, K. Matrougui, L. Loufrani, J. Maclouf, B. Levy, S. Levy- [179] S. Honma, N. Nakahata, H. Kobayashi, S. Ikeda, N. Takeda, Y. Toledano, et al., J. Vasc. Res. 39 (2002) 353–360. Ohizumi, Prostaglandins Other Lipid Mediat. 58 (1999) 51–62. [145] Y. Uehara, T. Ishimitsu, K. Kimura, M. Ishii, T. Ikeda, T. Sugimoto, [180] H. Kobayashi, S. Honma, N. Nakahata, Y. Ohizumi, J. Neurochem. Prostaglandins 36 (1988) 847–857. 74 (2000) 2167–2173. [146] T. Nagata, Y. Uehara, A. Numabe, T. Ishimitsu, N. Hirawa, T. Ikeda, [181] J. Kitanaka, H. Hashimoto, M. Gotoh, K. Kondo, K. Sakata, Y. et al., Am. J. Physiol. 263 (1992) H1331–H1338. Hirasawa, et al., Brain Res. 707 (1996) 282–287. [147] T.A. Morinelli, L.M. Zhang, W.H. Newman, K.E. Meier, J. Biol. [182] Lin X, Ramamurthy S, Le Breton GC. Society for Neuroscience: Chem. 269 (1994) 5693–5698. Washington, DC, 2002; Program No. 232.2. [148] A. Sachinidis, M. Flesch, Y. Ko, K. Schror, M. Bohm, R. Dusing, et [183] W.J. Welch, C.S. Wilcox, J. Clin. Invest. 89 (1992) 1857–1865. al., Hypertension 26 (1995) 771–780. [184] V.E. Kelley, S. Sneve, S. Musinski, J. Clin. Invest. 77 (1986) [149] G.W. Dorn, M.W. Becker, M.G. Davis, J. Biol. Chem. 267 (1992) 252–259. 24897–24905. [185] P. Mene, G.R. Dubyak, A. Scarpa, M.J. Dunn, Am. J. Physiol. 260 [150] S. Ali, M.G. Davis, M.W. Becker, G.W. Dorn, J. Biol. Chem. 268 (1991) C159–C166. (1993) 17397–17403. [186] L.A. Bruggeman, E.A. Horigan, S. Horikoshi, P.E. Ray, P.E. Klot- [151] W. Young, K. Mahboubi, A. Haider, I. Li, N.R. Ferreri, Circ. Res. 86 man, Am. J. Physiol. 261 (1991) F488–F494. (2000) 906–914. [187] R.K. Studer, H. Negrete, P.A. Craven, F.R. DeRubertis, Kidney Int. [152] S.M. Miggin, B.T. Kinsella, Biochim. Biophys. Acta 1539 (2001) 48 (1995) 422–430. 147–162. [188] T.J. Weber, T.J. Monks, S.S. Lau, Am. J. Physiol. Renal. Physiol. [153] C. Gallet, S. Blaie, S. Levy-Toledano, A. Habib, Biochem. J. 371 278 (2000) F270–F278. (2003) 733–742. [189] M.B. Feinstein, J.J. Egan, R.I. Sha’afi, J. White, Biochem. Biophys. [154] T. Ishizuka, K. Suzuki, M. Kawakami, T. Hidaka, Y. Matsuki, H. Res. Commun. 113 (1983) 598–604. Nakamura, Eur. J. Pharmacol. 312 (1996) 367–377. [190] L.D. Brace, D.L. Venton, G.C. Le Brenton, Am. J. Physiol. 249 [155] T. Ishizuka, M. Kawakami, T. Hidaka, Y. Matsuki, M. Takamizawa, (1985) H8–H13. K. Suzuki, et al., Clin. Exp. Immunol. 112 (1998) 464–470. [191] R. Kaser-Glanzmann, M. Jakabova, J.N. George, E.F. Luscher, Bio- [156] T.O. Daniel, H. Liu, J.D. Morrow, B.C. Crews, L.J. Marnett, Cancer chim. Biophys. Acta 466 (1977) 429–440. Res. 59 (1999) 4574–4577. [192] J.E. Fox, A.K. Say, R.J. Haslam, Biochem. J. 184 (1979) 651–661. [157] D. Nie, M. Lamberti, A. Zacharek, L. Li, K. Szekeres, K. Tang, et [193] R. Kaser-Glanzmann, E. Gerber, E.F. Luscher, Biochim. Biophys. al., Biochem. Biophys. Res. Commun. 267 (2000) 245–251. Acta 558 (1979) 344–347. [158] A.W. Ashton, R. Yokota, G. John, S. Zhao, S.O. Suadicani, D.C. [194] J. Enouf, R. Bredoux, C. Boucheix, M. Mirshahi, C. Soria, S. Levy- Spray, et al., J. Biol. Chem. 274 (1999) 35562–35570. Toledano, FEBS Letters 183 (1985) 398–402. [159] Y. Gao, R. Yokota, S. Tang, A.W. Ashton, J.A. Ware, Circ. Res. 87 [195] J.M. Hettasch, G.C. Le Breton, Biochim. Biophys. Acta 931 (1987) (2000) 739–745. 49–58. [160] A.W. Ashton, G.M. Ware, D.K. Kaul, J.A. Ware, J. Biol. Chem. 278 [196] T.H. Fischer, G.C. White, Biochem. Biophys. Res. Commun. 149 (2003) 11858–11866. (1987) 700–706. [161] C. Kitiyakara, W.J. Welch, J.G. Verbalis, C.S. Wilcox, Am. J. Phys- [197] B.T. Kinsella, D.J. O’Mahony, G.A. FitzGerald, J. Biol. Chem. 269 iol. Regul. Integr. Comp. Physiol. 282 (2002) R865–R869. (1994) 29914–29919. [162] C.E. Wood, T.A. Cudd, C. Kane, K. Engelke, Am. J. Physiol. 265 [198] A. Habib, R. Vezza, C. Creminon, J. Maclouf, G.A. FitzGerald, (1993) R858–R862. J. Biol. Chem. 272 (1997) 7191–7200. [163] Y. Murakami, K. Yokotani, Y. Okuma, Y. Osumi, Neuroscience 87 [199] G.R. Wang, Y. Zhu, P.V. Halushka, T.M. Lincoln, M.E. Mendelsohn, (1998) 197–205. Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 4888–4893. [164] H. Gao, B. Peng, W.J. Welch, C.S. Wilcox, Am. J. Physiol. 272 [200] I. Gomes, B.A. Jordan, A. Gupta, N. Trapaidze, V. Nagy, L.A. Devi, (1997) R1493–R1500. J. Neurosci. 20 (2000) 1–5. [165] T.A. Cudd, Am. J. Physiol. 274 (1998) R1353–R1360. [201] I. Gomes, B.A. Jordan, A. Gupta, C. Rios, N. Trapaidze, L.A. Devi, [166] Y. Matsuo, T. Kihara, M. Ikeda, M. Ninomiya, H. Onodera, K. J. Mol. Med. 79 (2001) 226–242. Kogure, Brain Res. 709 (1996) 296–302. [202] I. Gomes, J. Filipovska, B.A. Jordan, L.A. Devi, Methods 27 (2002) [167] S. Suzuki, E. Sobata, T. Iwabuchi, Neurosurgery 9 (1981) 358–365. 679–685. [203] B.A. Jordan, N. Trapaidze, I. Gomes, R. Nivarthi, L.A. Devi, Proc. [168] S.T. Chen, C.Y. Hsu, E.L. Hogan, P.V. Halushka, O.I. Linet, F.M. Natl. Acad. Sci. U. S. A. 98 (2001) 343–348. Yatsu, Neurology 36 (1986) 466–470. [204] B.A. Jordan, L.A. Devi, Nature 399 (1999) 697–700. [169]R.J.Dempsey,M.W.Roy,K.Meyer,D.E.Cowen,H.H.Tai,J. [205] B.A. Jordan, S. Cvejic, L.A. Devi, Neuropsychopharmacology 23 Neurosurg. 64 (1986) 118–124. (2000) S5–S18. [170] J.J. Jafar, R. Menoni, H. Feinberg, G. LeBreton, R.M. Crowell, [206] C.D. Rios, B.A. Jordan, I. Gomes, L.A. Devi, Pharmacol. Ther. 92 Stroke 20 (1989) 664–667. (2001) 71–87. [171] K. Ichikawa, S. Tazawa, S. Hamano, M. Kojima, S. Hiraku, Phar- [207] Y. Cordeaux, S.J. Hill, Neurosignals 11 (2002) 45–57. macology 59 (1999) 257–265. [208] Y. Djellas, K. Antonakis, G.C. Le Breton, Proc. Natl. Acad. Sci. [172] C.Y. Hsu, P.V. Halushka, E.L. Hogan, N.L. Banik, W.A. Lee, P.L. U. S. A. 95 (1998) 10944–10948. Perot Jr., Neurology 35 (1985) 1003–1009. [209] Y. Djellas, K. Antonakis, G.C. Le Breton, Biochem. Pharmacol. 59 [173] T. Mitsuhashi, T. Ikata, K. Morimoto, T. Tonai, S. Katoh, Paraplegia (2000) 1521–1529. 32 (1994) 524–530. [210] U. Quitterer, M.J. Lohse, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) [174] G.E. Tempel, H.F. Martin 3rd, J. Neurol. Sci. 109 (1992) 162–167. 10626–10631. [175] K.S. Hsu, C.C. Huang, W.M. Kan, P.W. Gean, Am. J. Physiol. 271 [211] M. Shapira, Z. Vogel, Y. Sarne, Cell. Mol. Neurobiol. 20 (2000) (1996) C1269–C1277. 291–304. [176] K.S. Hsu, W.M. Kan, Br. J. Pharmacol. 118 (1996) 2220–2227. [212] C. Vasquez, D.L. Lewis, J. Neurosci. 19 (1999) 9271–9280. [177] N. Nakahata, I. Matsuoka, T. Ono, H. Nakanishi, Eur. J. Pharmacol. [213] S. Muck, A.A. Weber, J. Meyer-Kirchrath, K. Schror, Naunyn 162 (1989) 407–417. Schmiedebergs Arch. Pharmacol. 357 (1998) 10–16. J.-S. Huang et al. / Cellular Signalling 16 (2004) 521–533 533

[214] K.P. Becker, M. Ullian, P.V. Halushka, Biochim. Biophys. Acta 1403 [217] F.M. Pulcinelli, J.L. Daniel, S. Riondino, P.P. Gazzaniga, L. Salga- (1998) 109–114. nicoff, Thromb. Haemost. 73 (1995) 304–308. [215] G. Soslau, L. Arabe, J. Parker, A. Pelleg, Thromb. Res. 72 (1993) [218] H.S. Banga, E.R. Simons, L.F. Brass, S.E. Rittenhouse, Proc. Natl. 127–137. Acad. Sci. U. S. A. 83 (1986) 9197–9201. [216] F.M. Pulcinelli, M. Pesciotti, P. Pignatelli, S. Riondino, P.P. Gazza- niga, FEBS Lett. 435 (1998) 115–118.