Heterodimerization of the alpha and beta isoforms of the human thromboxane receptor enhances isoprostane signaling

Isoprostanes are free radical catalyzed products of arachidonic acid that are elevated in pro-oxidant disease states. Two isoprostanes, 8-isoprostaglandin F(2alpha) (iPF(2alpha)III) and 8-isoprostaglandin E2 (iPE2III), act at the receptor for thromboxane A2 (the TP) to mediate pro-atherogenic effects in vivo. We confirmed dimerization of the human TP isoforms, TPalpha and TPbeta, and determined the impact on isoprostane signaling. No overt changes in ligand binding at the TP were observed as a result of TPalpha/TPbeta coexpression. The response to iPF(2alpha)III or iPE2III was enhanced in HEK293 cells stably coexpressing TPalpha and TPbeta, as measured by inositol phosphate generation or intracellular calcium mobilization, relative to cells expressing TPalpha or TPbeta individually. In contrast, the response to traditional thromboxane analogs was unaltered. Augmented isoprostane signaling was similarly observed in HEK 293 cell transiently transfected with TPalpha and TPbeta. These results indicate that TPalpha/TPbeta dimerization enhances isoprostane-mediated signal transduction.     

The alpha, but not the beta, isoform of the human thromboxane A2 receptor is a target for prostacyclin-mediated desensitization

In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A(2) receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TPalpha or TPbeta isoforms. Consistent with previous studies, cicaprost abrogated U46619-mediated platelet aggregation and mobilization of intracellular calcium ([Ca(2+)](i)). In HEK 293 cells, signaling by TPalpha, but not TPbeta, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TPalpha signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of G(q) protein. Signaling by TP(Delta)(328), a truncated variant of TP devoid of the divergent residues of the TPs, or by TPalpha(S329A), a site-directed mutant of TPalpha, were insensitive to IP agonist activation. Whole cell phosphorylations established that TPalpha, but not TPbeta or TPalpha(S329A), is subject to IP-mediated phosphorylation and that TPalpha phosphorylation is inhibited by H-89. Thus, we conclude that TPalpha, but not TPbeta, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser(329) and propose that TPalpha may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.     

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.     

Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor: independent modulation of TPalpha signaling by prostacyclin and nitric oxide

In humans, thromboxane (TX) A(2) signals through the TPalpha and TPbeta isoforms of the TXA(2) receptor that exhibit common and distinct roles. For example, Gq/phospholipase (PL)Cbeta signaling by TPalpha is directly inhibited by the vasodilators prostacyclin and nitric oxide (NO) whereas that signaling by TPbeta is unaffected. Herein, we investigated whether TPalpha and/or TPbeta regulate G(12)/Rho activation and whether that signaling might be differentially regulated by prostacyclin and/or NO. Both TPalpha and TPbeta independently regulated RhoA activation and signaling in clonal cells over-expressing TPalpha or TPbeta and in primary human aortic smooth muscle cells (1 degrees AoSMCs). While RhoA-signaling by TPalpha was directly impaired by prostacyclin and NO through protein kinase (PK)A- and PKG-dependent phosphorylation, respectively, signaling by TPbeta was not directly affected by either agent. Collectively, while TPalpha and TPbeta contribute to RhoA activation, our findings support the hypothesis that TPalpha is involved in the dynamic regulation of haemostasis and vascular tone, such as in response to prostacyclin and NO. Conversely, the role of TPbeta in such processes remains unsolved. Data herein provide essential new insights into the physiologic roles of TPalpha and TPbeta and, through studies in AoSMCs, reveal an additional mode of regulation of VSM contractile responses by TXA(2).     

Palmitoylation of the TPbeta isoform of the human thromboxane A2 receptor. Modulation of G protein: effector coupling and modes of receptor internalization

Palmitoylation is a prevalent feature amongst G protein-coupled receptors. In this study we sought to establish whether the TPalpha and TPbeta isoforms of the human prostanoid thromboxane (TX) A2 receptor (TP) are palmitoylated and to assess the functional consequences thereof. Consistent with the presence of three cysteines within its unique carboxyl-terminal domain, metabolic labelling and site-directed mutagenesis confirmed that TPbeta is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377 whereas TPalpha is not palmitoylated. Impairment of palmitoylation did not affect TPbeta expression or its ligand affinity. Conversely, agonist-induced [Ca2+]i mobilization by TPbetaC347S and the non-palmitoylated TPbetaC347,373,377S, but not by TPbetaC373S or TPbetaC373,377S, was significantly reduced relative to the wild type TPbeta suggesting that palmitoylation at Cys347 is specifically required for efficient Gq/phospholipase Cbeta effector coupling. Furthermore, palmitoylation at Cys373,377 is critical for TPbeta internalization with TPbetaC373S, TPbetaC373,377S and TPbetaC347,373,377S failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPbetaC347S underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPbeta. The deficiency in agonist-induced internalization by TPbetaC347S, but not by TPbetaC373,377 nor TPbeta(C347,373,377S), was overcome by over-expression of either beta-arrestin1 or beta-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPbeta at Cys373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys347 has a role in determining which pathway is followed, be it by the beta-arrestin-dependent agonist-induced pathway or by the beta-arrestin-independent tonic internalization pathway.     

Role of the differentially spliced carboxyl terminus in thromboxane A2 receptor trafficking: identification of a distinct motif for tonic internalization

The thromboxane A(2) receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. We have previously shown that TPbeta, but not TPalpha, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPbeta, but not TPalpha, also undergoes tonic internalization. Tonic internalization of TPbeta was temperature- and dynamin-dependent and was inhibited by sucrose and NH(4)Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX(3)phi motif (where X is any residue and phi is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPbeta was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX(2)phi or YX(3)phi motif in the carboxyl-terminal tail of TPalpha induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPbeta undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPbeta. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPbeta and represent the first report of a YX(3)phi motif involved in tonic internalization of a cell surface receptor.     

Oligomerization of the alpha and beta isoforms of the thromboxane A2 receptor: relevance to receptor signaling and endocytosis

Thromboxane A(2) (TXA(2)) is a potent mediator of inflammation, vasoconstriction and oxidative stress. The TXA(2) receptor (TP) is a G protein-coupled receptor (GPCR) that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. For many years GPCRs were assumed to exist and function as monomeric species, but increasing evidence suggests that a dimer is the minimal functional unit of GPCRs. In the present report, using co-immunoprecipitation of differentially tagged TP expressed in HEK293 cells, we demonstrate that TPalpha and TPbeta form homo- and hetero-oligomers. Immunoblotting of lysates from human platelets with an anti-TP specific antibody revealed the presence of endogenously expressed TP oligomers. We show that TP oligomerization is an agonist-independent process highly affected by the reducing agent dithiothreitol suggesting the involvement of disulfide bonds in TP oligomerization. Over-expression of G protein-coupled receptor kinases and arrestins did not modulate the extent of receptor dimerization/oligomerization. Co-expression of two TP signaling-deficient mutants, R60L and E2402R, resulted in rescuing of receptor signal transduction suggesting that dimers/oligomers constitute the functional units of this receptor. Interestingly, TPalpha which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPbeta, indicating that TPalpha can display intracellular trafficking when complexed through hetero-oligomerization with TPbeta.     

Thromboxane A(2) receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells

Both thromboxane (TX) A(2) and 8-epi prostaglandin (PG) F(2alpha) have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA(2) and 8-epiPGF(2alpha) mediated mitogenic signalling has not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA(2) receptor (TP) mediated mitogenic signalling in cultured human vascular SMCs. Both the TP agonist U46619 and 8-epiPGF(2alpha) elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29548 abolished U46619 mediated signalling, it only partially inhibited 8-epiPGF(2alpha) mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF(2alpha) induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the epidermal growth factor (EGF) receptor. In humans, TXA(2) signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPalpha and TPbeta independently directed U46619 and 8-epiPGF(2alpha) mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29548 abolished 8-epiPGF(2alpha) mediated ERK and JNK activation through both TPalpha and TPbeta in HEK 293 cells providing further evidence that 8-epiPGF(2alpha) may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.     

The role of N-linked glycosylation in determining the surface expression,G protein interaction and effector coupling of the alpha (alpha) isoform of the human thromboxane A(2) receptor

In humans, thromboxane (TX) A(2) signals through two TXA(2) receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within the carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn(4) and Asn(16). While it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn(4) or Asn(16) is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalpha(N4,N16-Q4,Q16) is almost completely retained within the endoplasmic reticulum (ER) and remains functionally inactive, failing to associate with its coupling G protein Galpha(q) and, in turn, failing to mediate phospholipase (PL) Cbeta activation.     

Differential regulation of chemokine gene expression by 15-deoxy-delta 12,14 prostaglandin J2

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-Delta(12,14)PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 x 10(-6) M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARgamma-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.