B. Therese Kinsella

Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms.

The human thromboxane A2 receptor (TP), a G protein-coupled receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR)-based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell/tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell/tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha: TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVECs were found to express: (i) low levels of TPbeta and (ii) approximately 6-fold greater levels of TPalpha than TPbeta. These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue/cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H]SQ29,548.

The α, but Not the β, 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 A2 receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TPα or TPβ isoforms. Consistent with previous studies, cicaprost abrogated U46619-mediated platelet aggregation and mobilization of intracellular calcium ([Ca2+] i ). In HEK 293 cells, signaling by TPα, but not TPβ, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TPα signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of Gq protein. Signaling by TPΔ 328, a truncated variant of TP devoid of the divergent residues of the TPs, or by TPαS329A, a site-directed mutant of TPα, were insensitive to IP agonist activation. Whole cell phosphorylations established that TPα, but not TPβ or TPαS329A, is subject to IP-mediated phosphorylation and that TPα phosphorylation is inhibited by H-89. Thus, we conclude that TPα, but not TPβ, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser329 and propose that TPα may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.

Thromboxane A2 receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells

Both thromboxane (TX) A2 and 8-epi prostaglandin (PG) F2α have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA2 and 8-epiPGF2α 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 TXA2 receptor (TP) mediated mitogenic signalling in cultured human vascular SMCs. Both the TP agonist U46619 and 8-epiPGF2α 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-epiPGF2α mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF2α 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, TXA2 signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPα and TPβ independently directed U46619 and 8-epiPGF2α 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-epiPGF2α mediated ERK and JNK activation through both TPα and TPβ in HEK 293 cells providing further evidence that 8-epiPGF2α may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.

The α, but not the β, isoform of the human thromboxane A2 receptor is a target for nitric oxide-mediated desensitization. INDEPENDENT MODULATION OF TPα SIGNALING BY NITRIC OXIDE AND PROSTACYCLIN.

In humans, thromboxane A2 signals through two thromboxane A2 receptor (TP) isoforms termed TPα and TPβ. Signaling by TPα, but not TPβ, is subject to prostacyclin-induced desensitization mediated by direct protein kinase (PK) A phosphorylation where Ser329 represents the phosphotarget (Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412-20423). In the current study, the effect of the vasodilator nitric oxide (NO) on intracellular signaling by the TP isoforms was investigated. The NO donor 3-morpholinosydnonimine, HCl (SIN-1) and 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) functionally desensitized U46619-mediated calcium mobilization and inositol 1,4,5-trisphosphate generation by TPα whereas signaling by TPβ was unaffected by either agent. NO-mediated desensitization of TPα signaling occurred through a PKG-dependent, PKA- and PKC-independent mechanism. TPα, but not TPβ, was efficiently phosphorylated by PKG in vitro and underwent NO/PKG-mediated phosphorylation in whole cells. Deletion/site-directed mutagenesis and metabolic labeling studies identified Ser331 as the target residue of NO-induced PKG phosphorylation of TPα. Although TPαS331A was insensitive to NO/PKG-desensitization, similar to wild type TPα its signaling was fully desensitized by the prostacyclin receptor agonist cicaprost occurring through a PKA-dependent mechanism. Conversely, signaling by TPαS329A was insensitive to cicaprost stimulation whereas it was fully desensitized by NO/PKG signaling. In conclusion, TPα undergoes both NO- and prostacyclin-mediated desensitization that occur through entirely independent mechanisms involving direct PKG phosphorylation of Ser331, in response to NO, and PKA phosphorylation of Ser329, in response to prostacyclin, within the unique carboxyl-terminal tail domain of TPα. On the other hand, signaling by TPβ is unaffected by either NO or prostacyclin.

Investigation of the Mechanisms of G Protein: Effector Coupling by the Human and Mouse Prostacyclin Receptors IDENTIFICATION OF CRITICAL SPECIES-DEPENDENT DIFFERENCES

We recently identified a novel mechanism explaining how the mouse (m) prostacyclin receptor (IP) couples to Gαs, Gαi, and Gαq(Lawler, O. A., Miggin, S. M., and Kinsella, B. T. (2001) J. Biol. Chem. 276, 33596–33607) whereby mIP coupling to Gαi and Gαq is dependent on its initial coupling to Gαs and subsequent phosphorylation by cAMP-dependent protein kinase A (PKA) on Ser357. In the current study, the generality of that mechanism was investigated by examining the G protein coupling specificity of the human (h) IP. The hIP efficiently coupled to Gαs/adenylyl cyclase and to Gαq/phospholipase C activation but failed to couple to Gαi. Coupling of the hIP to Gαq, or indeed to Gαs or Gαi, was unaffected by the PKA or protein kinase C (PKC) inhibitors H-89 and GF 109203X, respectively. Thus, mIP and hIP exhibit essential differences in their coupling to Gαi and in their dependence on PKA in regulating their coupling to Gαq. Analysis of their primary sequences revealed that the critical PKA phosphorylation site within the mIP, at Ser357, is replaced by a PKC site within the hIP, at Ser328. Conversion of the PKC site of the hIP to a PKA site generated hIPQL325,326RP that efficiently coupled to Gαs and to Gαi and Gαq; coupling of hIPQL325,326RP to Gαi but not to Gαs or Gαq was inhibited by H-89. Abolition of the PKC site of the hIP generated hIPS328A that efficiently coupled to Gαs and Gαq but failed to couple to Gαi. Finally, conversion of the PKA site at Ser357 within the mIP to a PKC site generated mIPRP354,355QL that efficiently coupled to Gαs but not to Gαi or Gαq. Collectively, our data highlight critical differences in signaling by the mIP and hIP that are regulated by their differential phosphorylation by PKA and PKC together with contextual sequence differences surrounding those sites.

Palmitoylation of the human prostacyclin receptor. Functional implications of palmitoylation and isoprenylation.

We have previously established that isoprenylation of the prostacyclin receptor (IP) is required for its efficient G protein coupling and effector signaling (Hayes, J. S., Lawler, O. A., Walsh, M. T., and Kinsella, B. T. (1999)J. Biol. Chem. 274, 23707–23718). In the present study, we sought to investigate whether the IP may actually be subject to palmitoylation in addition to isoprenylation and to establish the functional significance thereof. The human (h) IP was efficiently palmitoylated at Cys308 and Cys311, proximal to transmembrane domain 7 within its carboxyl-terminal (C)-tail domain, whereas Cys309 was not palmitoylated. The isoprenylation-defective hIPSSLC underwent palmitoylation but did not efficiently couple to Gs or Gq, confirming that isoprenylation is required for G protein coupling. Deletion of C-tail sequences distal to Val307 generated hIPΔ307 that was neither palmitoylated nor isoprenylated and did not efficiently couple to Gs or to Gq, whereas hIPΔ312 was palmitoylated and ably coupled to both effector systems. Conversion of Cys308, Cys309, Cys311, Cys308,309, or Cys309,311 to corresponding Ser residues, while leaving the isoprenylation CAAX motif intact, did not affect hIP coupling to Gs signaling, whereas mutation of Cys308,311 and Cys308,309,311 abolished signaling, indicating that palmitoylation of either Cys308or Cys311 is sufficient to maintain functional Gs coupling. Although mutation of Cys309 and Cys311 did not affect hIP-mediated Gq coupling, mutation of Cys308 abolished signaling, indicating a specific requirement for palmitoylation of Cys308 for Gq coupling. Consistent with this, neither hIPC308S,C309S, hIPC308S,C311S, nor hIPC308S,C309S,C311S coupled to Gq. Taken together, these data confirm that the hIP is isoprenylated and palmitoylated, and collectively these modifications modulate its G protein coupling and effector signaling. We propose that through lipid modification followed by membrane insertion, the C-tail domain of the IP may contain a double loop structure anchored by the dynamically regulated palmitoyl groups proximal to transmembrane domain 7 and by a distal farnesyl isoprenoid permanently attached to its carboxyl terminus.

Investigation of the role of the carboxyl-terminal tails of the α and β isoforms of the human thromboxane A2 receptor (TP) in mediating receptor:effector coupling

We have investigated the functional coupling of α and β isoforms of the human thromboxane A2 receptor (TP) to Gα16 and Gα12 members of the Gq and G12 families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.α10 or HEK.β3, stably over-expressing TPα and TPβ, respectively. Moreover, using HEK.TPΔ328 cells which over-express a variant of TP truncated at the point of divergence of TPα and TPβ, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPα and TPβ couple similarly to Gα16 to affect increases in inositol 1,4,5-trisphosphate (IP3) and mobilisation of intracellular calcium ([Ca2+]i) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca2+]i mobilisation in cells co-transfected with Gα12, neither receptor generated corresponding increases in IP3, indicating that the Gα12-mediated increases in [Ca2+]i do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca2+ channels, reduced [Ca2+]i mobilisation in TPα and TPβ cells co-transfected with Gα12 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 Ca2+ release, had no effect on [Ca2+]i mobilisation by either receptor isoform co-transfected with Gα12. Despite the lack of differential coupling specificity by TPα and TPβ, TPΔ328 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 Gα11, Gα12 or Gα16 subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPα but not TPβ, TPΔ328 coupled to Gαs, leading to increased adenosine 3′,5′-cyclic monophosphate (cAMP), rather than to Gαi. Whereas TPΔ328 signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPα, co-transfection of Gαs did not augment cAMP generation by TPΔ328. Hence, from these studies involving the wild type TPα, TPβ and TPΔ328, we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Gα11 and Gα16 members of the Gq family or to Gα12; it may play a role in determining Gs versus Gi coupling and may act as a determinant of coupling efficiency.

Regulation of the human prostanoid TPα and TPβ receptor isoforms mediated through activation of the EP1 and IP receptors

The intermolecular cross‐regulation mediated by the prostanoid IP‐receptor (IP)/EP1 receptor (EP1) agonists PGI2 and 17 phenyl trinor PGE2 on TP receptor (TP) signalling within platelets was compared to that which occurs to the individual TPα and TPβ receptors over‐expressed in human embryonic kidney (HEK) 293 cells. Ligand mediated TP receptor activation was monitored by analysing mobilization of intracellular calcium ([Ca2+]i) following stimulation with the selective thromboxane (TX) A2 mimetic U46619. Consistent with previous studies, in platelets, PGI2 acting through endogenous IP receptors completely inhibited U46619‐mediated TP receptor signalling in a protein kinase (PK) A‐dependent, PKC‐independent manner. In HEK 293 cells, PGI2, acting through endogenous AH6809 sensitive EP1 rather than IP receptors, and the selective EP1 receptor agonist 17 phenyl trinor PGE2 antagonized U46619‐mediated signalling by both TPα and TPβ receptors in a PKC‐dependent, PKA‐independent manner. The maximum response induced by either ligand was significantly (P<0.005) greater for the TPα receptor than the TPβ receptor, pointing to possible physiologic differences between the TP isoforms, although the potency of each ligand was similar for both TP receptors. TPΔ328, a truncated variant of TP receptor lacking the C‐tail sequences unique to TPα or TPβ receptors, was not sensitive to EP1 receptor‐mediated regulation by PGI2 or 17 phenyl trinor PGE2 In conclusion, these data confirm that TPα and TPβ receptors are subject to cross regulation by EP1 receptor signalling in HEK 293 cells mediated by PKC at sites unique to the individual TP receptors and that TPα receptor responses are significantly more reduced by EP1 receptor regulation than those of the TPβ receptor.

The Lipoxin A4 Receptor Is Coupled to SHP-2 Activation IMPLICATIONS FOR REGULATION OF RECEPTOR TYROSINE KINASES

Mesangial cell proliferation is pivotal to the pathology of glomerular injury in inflammation. We have previously reported that lipoxins, endogenously produced eicosanoids with anti-inflammatory and pro-resolution bioactions, can inhibit mesangial cell proliferation in response to several agents. This process is associated with elaborate receptor cross-talk involving modification receptor tyrosine kinase phosphorylation (McMahon, B., Mitchell, D., Shattock, R., Martin, F., Brady, H. R., and Godson, C. (2002) FASEB J. 16, 1817–1819). Here we demonstrate that the lipoxin A4 (LXA4) receptor is coupled to activation and recruitment of the SHP-2 (SH2 domain-containing tyrosine phosphatase-2) within a lipid raft microdomain. Using site-directed mutagenesis of the cytosolic domain of the platelet-derived growth factor receptor β (PDGFRβ), we report that mutation of the sites for phosphatidylinositol 3-kinase (Tyr740 and Tyr751) and SHP-2 (Tyr763 and Tyr1009) recruitment specifically inhibit the effect of LXA4 on the PDGFRβ signaling; furthermore inhibition of SHP-2 expression with short interfering RNA constructs blocked the effect of LXA4 on PDGFRβ phosphorylation. We demonstrate that association of the PDGFRβ with lipid raft microdomains renders it susceptible to LXA4-mediated dephosphorylation by possible reactivation of oxidatively inactivated SHP-2. These data further elaborate on the potential mechanisms underlying the anti-inflammatory, proresolution, and anti-fibrotic bioactions of lipoxins.

Characterization of promoter 3 of the human thromboxane A2 receptor gene

The TPα and TPβ isoforms of the human thromboxane A2 receptor (TP) arise by differential splicing but are under the transcriptional control of two distinct promoters, termed Prm1 and Prm3, respectively (Coyle et al. 2002 Eur J Biochem269, 4058–4073). The aim of the current study was to determine the key factors regulating TPβ expression by functionally characterizing Prm3, identifying the core promoter and the cis‐acting elements regulating basal Prm3 activity. Hence, the ability of Prm3 and a series of Prm3 deleted/mutated subfragments to direct reporter gene expression in human erythroleukemia 92.1.7 and human embryonic kidney 293 cells was investigated. It was established that nucleotides −118 to +1 are critical for core Prm3 activity in both cell types. Furthermore, three distinct regulatory regions comprising of an upstream repressor sequence, located between −404 to −320, and two positive regulatory regions required for efficient basal gene expression, located between −154 to −106 and −50 to +1, were identified within the core Prm3. Deletion and site‐directed mutagenesis of consensus Oct‐1/2 and AP‐1 elements within the latter −154 to −106 and −50 to +1 regions, respectively, substantially reduced Prm3 activity while mutation of both elements abolished Prm3 activity. Electromobility shift and supershift assays confirmed the specificity of nuclear factor binding to the latter Oct‐1/2 and AP‐1 elements. Moreover, herein it was established that the core AP‐1 element mediates phorbol myristic acid‐induction of Prm3 activity hence providing a mechanistic explanation of phorbol ester up‐regulation of TPβ mRNA expression.