Published online before print November 16, 2006, doi: 10.1161/01.ATV.0000252667.53790.4e
© 2007 American Heart Association, Inc.
Vascular Biology |
Regulation of Thromboxane Receptor Trafficking Through the Prostacyclin Receptor in Vascular Smooth Muscle Cells
Role of Receptor Heterodimerization
From the Institute of Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia.
Correspondence to Dr Emer M Smyth, Institute of Translational Medicine and Therapeutics, University of Pennsylvania, 808 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104. E-mail emer{at}spirit.gcrc.upenn.edu
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Abstract |
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Background— Prostacyclin (PGI2) and thromboxane (TxA2) effect disparate outcomes for atherogenesis and the response to vascular injury; PGI2, a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA2, a vasoconstrictor and platelet activator. Dimerization of their G protein-coupled receptors, IP and TP, evokes a modified cellular response through which IP/TP counter-balance may be effected. We examined the consequence of IP/TP interaction for the regulatory pathways of both receptors.
Methods and Results— TP
overexpressed in HEK293 cells or expressed endogenously in aortic smooth muscle cells (ASMCs) was internalized after selective activation of either TP or IP. Homologous trafficking of TP was unaltered by coexpression of IP. Heterologous sequestration of TP required the physical presence of activated IP, in transfected and native cells, but was independent of IP signaling to adenylyl cyclase. Reciprocal heterologous regulation of IP, via activated TP, was evident in both HEK293 cells and ASMCs. Homologous TP internalization led to receptor retention and degradation. In contrast, when internalization was IP-induced, TP was recycled to the cell surface in coexpressing HEK293 cells, but not in ASMCs, in accord with the postendocytotic pathway of IP.
Conclusions— IP/TP interaction permits reciprocal regulation of receptor endocytosis via the trafficking pathway determined by the activated dimeric partner.
Interaction of the G protein-coupled receptors for prostacyclin (IP) and thromboxane (TP) permits reciprocal regulation of receptor endocytosis via a trafficking pathway determined by the activated dimeric partner. This represents a further mechanism by which dimerization may effect the IP-TP counterbalance.
Key Words: prostacyclin • thromboxane • G protein-coupled receptor • heterodimer • internalization
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Introduction |
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Prostacyclin (PGI2) and thromboxane (TxA2) the predominant products of COX metabolism of arachidonic acid formed in the macrovascular endothelium and platelets, respectively,1,2 effect opposing actions within the vasculature. PGI2, a potent vasodilator, inhibitor of platelet aggregation and vascular smooth muscle cell proliferation in vitro,3,4 has been established firmly as an important antithrombotic mediator in vivo.5,6 Conversely, TxA2 stimulates platelet aggregation, amplifies the response to other platelet agonists, stimulates mitogenesis and vasoconstriction, and exerts potent effects on platelet function and vascular tone in vivo.5
The interplay between these two mediators is a critical factor in vascular disease; PGI2 limits specifically the proliferative and platelet effects of TxA2 in response to vascular injury.3 Similarly, atherogenesis is oppositely affected by these mediators; deletion of the IP accelerated,6 whereas antagonism7 or deletion6 of the TP retarded, atherogenesis in mice. Indeed, the marked depression of PGI2 biosynthesis, with unrestricted TxA2 generation, that accompanies inhibition of COX-2 is the leading, and most parsimonious, explanation of the cardiovascular hazard associated with specific enzyme inhibitors such as rofecoxib, celebcoxib, and valdecoxib.8
The responses to PGI2 and TxA2, mediated through their G protein-coupled receptors (GPCRs) termed IP and TP, respectively, are regulated tightly via distinct desensitization and internalization pathways. In response to agonist activation, IP is desensitized rapidly, in a PKC-dependent manner,9 before internalization via an arrestin-independent pathway10 that requires interaction of IP with the delta subunit of phosphodiesterase 6.11 In contrast, both the and ß splice variants of human TP are desensitized via specific GPCR kinases and are internalized in an arrestin dependent manner.12,13
Cross regulation between IP and TP has been described; IP activation evokes PKA-dependent phosphorylation of TP, but not TPß, resulting in receptor desensitization.14 We recently reported a novel mechanism by which PGI2 may limit the cellular functions of TxA2 in vascular smooth muscle cells; IP, an adenylyl cyclase-coupled receptor, heterodimerized with the TP, primarily a phospholipase C-coupled receptor, facilitating TP-induced cAMP generation and thus promoting a "PGI2-like" cellular response.15 Reports of functional changes after heterodimerization are increasingly common, for example association of the ß1 and ß2 adrenergic receptors in the heart16 and association of the EP1 receptor for PGE2 with the ß2 adrenergic receptor in airway smooth muscle.17 Indeed, it is apparent that heterodimerization between distinct GPCRs is integral to their function in normal and disease settings.
The functional changes that result from GPCR heterodimerization can result not only from modified signaling but also modified regulation of the individual receptors. For example, heterodimerization of the dopamine D1 and Adenosine A1 receptors results in desensitization of D1-mediated cAMP accumulation on combined exposure to D1 and A1 agonists, but not to either agent alone.18 Similarly, the 
-opioid receptor when coexpressed with the ß2-adrenoceptor was internalized after activation of the latter, whereas, in contrast, neither the ß2-adrenoceptor nor the 
-opioid receptor are sequestered when coexpressed regardless of the activating agent.19 The pathways involved in the regulation of both IP and TP have been described previously; however, the regulatory characteristics of the IP/TP heterodimer have not been elucidated. In the present study we demonstrate that IP/TP heterodimerization facilitates reciprocal regulation between the dimeric partners such that the endocytotic control of TP is rendered sensitive to IP agonists and follows the post-endocytotic fate of IP.
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Methods |
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Cell Culture and Transfection
HEK293 cells (ATTC, Rockville, Md) and human (h) or mouse (m) aortic (A) smooth muscle cells (SMC; Biowhittaker Inc, Md) were maintained as described previously.15 SMCs from passage numbers 3 to 12 were used in experiments.
IP and TP were tagged with the hemagglutinin (HA) or Myc epitope tags as described.9,15 Expression constructs for the receptors, tagged with a triple HA (3xHA; UMR cDNA Resource Center), were used where indicated. For stable transfections, HEK293 cells were transfected with 10 µg DNA by liposome-mediated transfer (DOTAP) and selected with G418 (0.5 to 1.5 mg/mL) and or hygromycin (50 to 75 µg/mL), as described.9 For transient transfections HEK293 were transfected transiently using Fugene 6 (Roche Biochemicals).
In Vivo Model of Endovascular Injury
The left femoral artery was damaged using a curved flexible wire (0.35 mm diameter; Cook Inc), followed by a catheter (0.54 mm diameter; Cole-Parmer), as described.5 The right femoral artery was used as a control. Control and injured femoral arteries were harvested 14 days after surgery, embedded in OCT, frozen at –80°C, and 8-µm cross sections prepared. Sections were fixed with acetone, dried, and rehydrated in PBS before staining with a 1:200 dilution of polyclonal rabbit anti-mTP or anti-mIP (Cayman Chemicals) overnight at 4°C. Receptors were visualized with Cy-3–labeled Donkey anti-rabbit IgG (Jackson Immunoresearch) and nuclei were counterstained with DAPI (Molecular Probes).
Western Blotting
Whole cell lysates were resolved on NuPAGE (Invitrogen) 10% gels. HA-tagged or Myc-tagged receptors were visualized as described.15
Measurement of Cell Surface HAhIP or HAhTP Expression
Surface expression of the HA-tagged receptor was measured by ELISA, using anti-HA (1:500 in PBS) and alkaline peroxidase-conjugated anti-mouse IgG (1:10,000 in PBS) as described.10
Binding Studies
Indomethacin (3 µmol/L) pretreated h or m ASMCs, were incubated with a saturating concentration of 3H-SQ 29548 (10 nM), a TP antagonist, or 3H-iloprost (50 nM), an IP agonist, in binding buffer (HBSS containing 2 mg/mL BSA) in the presence or absence of excess (10 µmol/L) unlabeled ligand for 1 hour at 37°C or 18 hour at 4°C, respectively. Cells were washed in ice-cold buffer, treated with 1 mol/L NaOH for 30 minutes at 37°C, and cell-associated radioactivity quantified.
cAMP Measurements
hASMC were treated with IBOP for 2 hours at 37°C and then pretreated with IBMX (0.01 mol/L) 15 minutes before addition of cicaprost for 10 minutes. cAMP was extracted and quantified as described.20
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Results |
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Coincident expression of the IP and TP protein in vascular tissue has been inferred from several studies but has not been demonstrated directly. We first confirmed expression of both receptors in the vessel wall. Positive staining for IP and TP was evident in mouse femoral arteries, whereas marked neointimal expression was apparent after injury (Figure 1). The specific limit imposed on TP-dependent VASMC proliferation in vivo via the IP was established in a similar injury model,5 but the molecular mechanisms that direct this interplay remain ill-defined. We reported previously a novel mechanism that may contribute to the IP-dependent restraint on TP-driven events; dimerization of IP and TP
facilitated TP-cAMP generation, which was ablated in ASMCs deficient in IP.15 In the present study we examined whether agonist-induced TP sequestration, and thus cell surface expression of the functional receptor, also was modified secondary to its interaction with IP.
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Agonist Induced Sequestration of HAhTP
Cell surface TP was radiolabeled with a saturating concentration of the TP antagonist 3H-SQ 29548 in intact hASMCs, which express endogenously both the IP and TP. As expected, cell surface TP was substantially reduced (>75%) by treatment with the TP agonist IBOP (Figure 2A), reflecting internalization of the activated receptor. Interestingly, surface TP was similarly reduced by selective activation of the IP with cicaprost. IP-induced TP sequestration was a dose-dependent (Figure 2B) and rapid process, with maximal internalization occurring within 1 hour (Figure 2C).
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Cell surface TP was examined directly, by ELISA, in HEK 293 cells engineered, as previously described,15 to express stably HA-tagged hTP (HAhTP-HEK). Homologous TP sequestration in this model was time- (Figure 3A) and dose- (data not shown) dependent and was abolished by the TP agonist SQ 29548 (Figure 3B). Coexpression of the unactivated IP (HAhTP/IP-HEK) did not modify homologous TP internalization (Figure 3C, gray bars). Similar to the hASMC experiments, TP was subject to heterologous internalization via the IP (Figure 3C, black bars), albeit at a slower rate. Regardless of the agonist used the absolute level of TP internalization reached a significantly lower maximal level (
30%) in transfected HEK293 cells compared with hASMCs. This difference is likely a result of spare receptors21 in the overexpressing cells.
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IP-dependent TP internalization was not a result of nonspecific cicaprost binding to the TP because surface TP expression was unaltered in ASMCs cultured from IP-deficient mice (Figure 4A, black bar) and in HAhTP-HEK (Figure 4A, white bars). Interestingly, treatment of HAhTP-HEK with increasing concentrations of 8-Br-cAMP (Figure 4B), a regime that mimics the signaling events transduced via the activated IP,15 did not evoke TP sequestration. Thus, it appeared that the physical presence of the activated IP, but not subsequent generation of intracellular cAMP, was necessary for heterologous TP sequestration to occur. These data closely resembled our previously published study15 in which the activated IP modified TP signaling through formation of an IP/TP heterodimer.
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Postendocytotic Processing of Sequestered TP
The fate of the sequestered GPCR varies from receptor to receptor and by cell type, but culminates in two principal outcomes: lysosomal degradation, leading to downregulation of receptor levels, or recycling to the plasma membrane,22 and the return of cellular responsiveness. Reports generally are consistent with downregulation of agonist-sequestered TP23,24 although the postendocytotic fate of TP has not been examined directly. We determined whether agonist withdrawal prompted recycling of internalized TP to the cell membrane. Homologous sequestration of TP was induced in hASMCs or HAhTP-HEK (1 µmol/L IBOP, 2 hours), before their return to agonist-free conditions. TP surface expression was reduced, as expected, in each cell model, and remained depressed after a further 60 minutes in the absence of agonist (Figure 5A, left panel and 5B, left panel). Indeed, in HEK293 cells, HAhTP underwent significant degradation after prolonged agonist treatment (Figure 5B, right panel). Thus, analogous to platelets treated with TP agonist,23 homologous activation induced downregulation of TP expression leading to a sustained loss of the receptor from the plasma membrane.
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Homologously sequestered IP (1 µmol/L cicaprost; 4 hours) was similarly not recycled to the plasma membrane in hASMCs (Figure 5A, right panel). This pattern of postendocytotic IP processing contrasts starkly with that observed in transfected HEK293 cells where sequestered IP did not undergo degradation (data not shown) and was recycled to the plasma membrane after agonist withdrawal restoring IP responsiveness.10 We exploited this difference in IP trafficking between the native and transfected cell models to explore further IP-dependent TP internalization. Strikingly, when TP sequestration was heterologously induced via the IP, in HAhTP/IP-HEK, surface TP expression was fully restored on agonist withdrawal (Figure 5C). These data are consistent with cotrafficking of the IP and TP as a single heterodimeric unit via the pathway taken by the agonist-occupied partner.
Reciprocal Regulation of IP via the TP
We examined further cotrafficking of TP with IP as a single heterodimeric unit by reversing the focus to TP-dependent trafficking of the IP. Surface IP was radiolabeled in intact hASMCs with the IP agonist 3H-iloprost at 4°C (to prevent trafficking in response to iloprost). IBOP reduced surface IP to 77.9±2.9% of control (P<0.01, n=3; Figure 6B), an extent similar to that observed with cicaprost treatment (Figure 5A, right panel, black bar). As expected, this was offset when cells were pretreated with the TP antagonist SQ 29548 (Figure 6B). The antagonist itself did not modify significantly surface IP expression demonstrating that TP activation, and not merely its occupancy, was required for IP internalization. TP-induced sequestration of IP was translated into a functionally significant change in IP signaling: cicaprost-induced cAMP generation was depressed to a similar degree (25%) in hASMCs pretreated with IBOP to induce IP sequestration (Figure 6C). TP-dependent sequestration of IP was examined directly in HEK293 cells transfected with HA-tagged hIP alone or in combination with the untagged TP. IBOP was without effect in cells expressing the IP alone, but induced a small but significant loss of the IP for the cell surface over time (87.3±0.8% of control; P<0.05, n=3; Figure 6A). This was sustained when cells were returned to IBOP-free conditions for 1 hour (data not shown). Taken together with the recycling data in Figure 5C, reciprocal sequestration of IP and TP, in response to each other’s agonists, strongly supports their cotrafficking as a single heterodimeric unit.
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Discussion |
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The interplay between PGI2 and TxA2, an important contributor to cardiovascular physiology and pathophysiology, is directed, at least in part, through complex interactions between their receptors.14,15 The coincident expression of IP and TP in the neointima, after vascular injury (Figure 1), together with augmentation of the TP-dependent proliferative response in IP-deficient mice exposed to a similar injurious insult,5 underscores the relevance of the IP-TP interaction to cardiovascular function in vivo. We and others have examined the molecular mechanisms that contribute to IP-TP interactions; these include heterologous phosphorylation of the TP, secondary to IP-induced PKA activation,14 and modified TP signaling after its physical association with the IP as heterodimer.15 Reports indicate that formation of GPCR heterodimers can modify receptor internalization,25,26 a critical step in the shut-off mechanisms that limit GPCR activation.22 Therefore, we examined whether expression of the TP at the cell surface, and its intracellular trafficking, was also modified via its interaction with the IP. We report internalization of the TP through the activated IP, in a manner that is consistent with cotrafficking of the two receptors as a single heterodimeric unit.
In the "canonical" pathway of GPCR regulation, the receptor, phosphorylated by kinases activated secondary to receptor activation, interacts with an adapter protein, arrestin, leading to its uncoupling from the G protein (desensitization) and its sequestration, generally into clatharin-coated pits.27 Thereafter, GPCRs which complex stably with arrestins typically are retained intracellularly, and ultimately degraded, whereas rapid receptor-arrestin dissociation facilitates plasma membrane receptor recycling, an important factor in the return of cellular responsiveness.27 In keeping with this general paradigm, we (Figures 2 and 3
) and others13,24 report sequestration of the TP after its homologous activation in both native and transfected cells. In contrast to a previous study,13 coexpression of arrestin-3 was not required for sequestration of the TP in the HEK293 cell model, a discrepancy that may relate to sensitivity issues in the sequestration assay. After homologous sequestration of TP, reportedly an arrestin-dependent event, the receptor was retained intracellularly leading to its degradation, at least in transfected HEK293 cells (Figure 5B), and downregulation of cell surface receptor sites.13,23,24
A substantial number of GPCRs, including the IP10,28,29 are sequestered via phosphorylation- and/or arrestin-indpendent pathways, underscoring the complexity of these regulatory events. Rapid recycling, and hence restoration of responsiveness, is readily observed for GPCRs that traffic in an arrestin-independent manner.10,13,30 In the case of the IP, its postendocytotic fate appears dependent on cell type: rapid recycling has been reported in platelets,29 fibroblasts,28 and transfected HEK293 cells,10 whereas a slower process, dependent on de novo protein synthesis, contributes to restoration of membrane IP expression in some cells, including vascular SMCs.29,31
Neither homologous internalization of TP (Figure 3C), nor its postendocytotic processing (Figure 5C), were modified overtly when IP and TP were coexpressed. In contrast, TP internalization was induced heterologously after activation of IP in both natively coexpressing and transfected cells, but not IP deficient mASMC or in HEK293 cells expressing TP alone.
One explanation for heterologous internalization of TP via activated IP is that IP-induced PKA-dependent phosphorylation of TP can direct both its desensitization14 and sequestration. However, IP-induced sequestration of TP was not replicated in TP-HEK treated with 8-Br-cAMP (Figure 4B), a cell permeable cAMP mimetic, under conditions that mimicked IP activation. Thus, although dependent on the physical presence of activated IP, IP-dependent sequestration of TP was independent of its second messenger. These findings closely resemble IP-induced TP-signaling changes, which occur secondary to the formation of an IP/TP heterodimer.15 We considered, therefore, whether similar to other GPCR heterodimers, the physical association of IP with TP facilitated trafficking of the former under the control of the latter. If that were true we expected firstly that the TP would accompany the IP along its agonist-evoked endocytotic pathway, and secondly that reciprocal cotrafficking of the IP/TP heterodimer would be initiated through TP activation. We took advantage of the easily discernible difference in post-endocytotic processing of the individually sequestered receptors in HEK293 cells, namely that TP is retained intracellularly and IP recycled after agonist withdrawal. As we predicted, when TP sequestration in HEK293 cells was initiated via the IP, the former was rapidly recycled to the plasma membrane (Figure 5C). The second question was examined in both native hASMCs and cotransfected HEK293 cells—in both models significant IP sequestration was initiated secondary to TP activation and recycling was absent in HEK293 cells. Given that IP trafficking is phosphorylation- and arrestin-independent in transfected10 and native28 cells, it is unlikely that TP-induced signaling was responsible for TP-dependent trafficking of IP supporting further the conclusion that the physical association of these receptors facilitates their cotrafficking as a single entity.
The phenomenon of GPCR cotrafficking has been described previously for several receptors including the V1a/V2 vasopressin receptor heterodimer.25 Individually, both receptors undergo arrestin-dependent internalization; however, whereas arrestin dissociates rapidly from the V1aR, thereby allowing recycling to occur, formation of a stable complex with the V2R leads to its intracellular retention. Sequestration of that heterodimer is evoked by either V1aR or V2R selective agonists, but the postendocytotic pathway taken faithfully reflects the preference of the activated dimeric partner.25 In the same way, distinct endocytotic pathways contribute to homologous regulation of IP and TP, whether they are expressed alone or in combination, but regulation of the heterodimer appears dependant on which of the dimeric partner is stimulated and, by inference, the associated regulatory proteins recruited.
The IP and TP direct contrasting outcomes in animal models of atherosclerosis6 and neointima formation.5 To our knowledge this is the first report that the molecular interplay between these two receptors extends to control of their activation-dependent trafficking. Thus, through its physical association with TP, IP may limit the cellular effect of TP in several ways: (1) by evoking a PGI2-like cellular response,15 (2) by augmenting sequestration of TP from the cell surface limiting TP-PLC signaling, and (3) by restoring, in cells where IP recycling occurs, responsiveness of the IP/TP heterodimer, and its attendant PGI2-like signaling. Interestingly, the TP was substantially more responsive than the IP to cicaprost-induced sequestration in hASMCs (Figure 5A), suggesting that a substantial proportion of the TP is associated with the IP in normal cells, and supporting the likely functional impact of this cross-regulatory event. We have not examined directly how TP signaling is modified secondary to IP-dependent sequestration because of the confounding rapid IP-evoked phosphorylation and desensitization of TP.14 However, TP-induced loss of 25% to 30% surface IP in hASMC (Figure 6A and 6B) was accompanied by comparable reduction in IP signaling efficiency (Figure 6C), supporting the relevance of reciprocal cotrafficking of these receptors for cellular responsiveness to their agonists.
The biological outcome of this mutual regulation of the IP/TP heterodimer in a given cell or tissue likely is a combination of many factors, including the cellular complement of accessory proteins, the expression levels of the individual receptors, and their propensity to heterodimerize under physiological or pathophysiological conditions, as well as the milieu of activating ligands. Little is known with regard to the regulation of IP and TP expression in the setting of cardiovascular disease. However, IP is downregulated in the thoracic aorta of spontaneously hypertensive rats,32 whereas TP expression may be upregulated in human cardiovascular disease.33 Interestingly, the interplay between at least one heterodimeric pair of GPCRs, the AT1 and AT2 angiotensin receptors, may be relevant to the gender-specific response to vascular injury. Thus, upregulation of the AT2, which antagonizes signaling of the AT1 through heterodimerization,34 after vascular injury, was greater in female mice compared with males.35 This divergence was offset after AT2 deletion. The interplay of IP and TP, which is critical in atherogenesis and the response to vascular injury, also may contribute to gender-based cardiovascular events. Indeed PGI2, acting through IP accounts, at least in part, for the atheroprotective effect afforded by estrogen,36 whereas TP, but not TxA2, is responsible for the increased blood pressure observed in male spontaneously hypertensive rats compared with females.37 Further investigation will determine the extent to which dysregulation of IP/TP heterodimerization contributes to the development of cardiovascular disease.
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Acknowledgments |
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Sources of Funding
This work was supported by National Institutes of health grant HL066233.
Disclosures
None.
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Footnotes |
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Original received November 30, 2005; final version accepted October 31, 2006.
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References |
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