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G protein-coupled receptors (GPCRs) respond to diversified extracellular stimuli to modulate cellular function. Despite extensive studies investigating the regulation of single GPCR signaling cascades, the effects of concomitant GPCR activation on downstream signaling and cellular function remain unclear.
We aim to characterize the cellular mechanism by which GPCR cross-talk regulates MAPK activation.
Adrenergic receptors on cardiac fibroblasts were manipulated to examine the role of arrestin in the spatiotemporal regulation of ERK1/2 MAPK signaling. We show a general mechanism in which arrestin activation by one GPCR is capable of regulating signaling originating from another GPCR. Activation of Gq-coupled receptor signaling leads to prolonged ERK1/2 MAPK phosphorylation, nuclear accumulation and cellular proliferation. Interestingly, co-activation of these receptors with the β adrenergic receptors (ARs) induced transient ERK signaling localized within the cytosol, which attenuated cell proliferation. Further studies revealed that recruitment of arrestin3 to the β2AR orchestrates the sequestration of Gq-coupled receptor-induced ERK to the cytosol through direct binding of ERK to arrestin.
This is the first evidence showing that arrestin3 acts as a coordinator to integrate signals from multiple GPCRs. Our studies not only provide a novel mechanism explaining the integration of mitogenic signaling elicited by different GPCRs, but also underscore the critical role of signaling cross-talk among GPCRs in vivo.
G protein-coupled receptors (GPCRs) respond to diversified extracellular stimuli to modulate cellular function. Traditionally, activated receptors couple to G proteins, which transduce downstream signals via second messengers and membrane channels 1. Active GPCRs are phosphorylated by specific G protein-coupled receptor kinases (GRKs) leading to receptor desensitization. Arrestin proteins then bind to the phosphorylated receptor initializing clathrin-mediated internalization 2. Despite extensive studies investigating the regulation of single GPCR signaling cascades, the effect of concomitant GPCR activation by endogenous stimuli on downstream signaling remains poorly understood. There is a great deal of evidence supporting functional cross-talk between different GPCRs both in vitro and in vivo 3–5. The majority of these studies focus on short-term stress responses involved in modulation of common effectors such as G proteins, phospholipases, and adenylyl cyclases 4. However, chronic activation of multiple GPCR signaling pathways during maladaptive tissue and organ remodeling suggests the potential of downstream cross-talk away from the plasma membrane.
One potential nexus for GPCR signaling cross-talk are the multi-functional scaffold proteins known as arrestins. Arrestins not only scaffold proteins for the activation of different MAPK families under single receptor activation, but also mediate transactivation of epidermal growth factor receptor signaling pathways 2, 6 and activation of many other non-GPCR signaling cascades 7. In the case of GPCR-induced ERK MAPK activation, both G proteins and arrestins are capable of mediating ERK activation via independent mechanisms, with each pathway leading to unique spatiotemporal consequences 8. While G protein-dependent ERK translocates to the nucleus for gene transcription, arrestin-dependent ERK remains within the cytoplasm. Since arrestins preferentially bind to some, but not all, GPCRs in a ligand-dependent manner, we envision that arrestins may play a role in GPCR crosstalk by coordinating MAPK activation in distinct subcellular compartments. Such a regulatory mechanism is essential for modulating MAPK signaling in divergent cellular functions such as cell proliferation and growth 9, mobility 10, and apoptosis 11.
We chose cardiac fibroblasts as a model to study GPCR signaling cross-talk. Both α1ARs and βARs are expressed in cardiac tissue and are activated by catecholamines to modulate maladaptive cardiac remodeling, including cardiac fibroblast proliferation, by activation of distinct pathways. These pathways transduce their proliferative signal via members of the MAPK family, including ERK1/2. Stimulation of the α1AR leads to Gq coupling and subsequent phospholipase C (PLC) and protein kinase C (PKC) activation 12. PKC has been shown to directly activate the Raf-MEK1-ERK axis. The activated ERK translocates to the nucleus to activate gene transcription necessary for cellular differentiation, proliferation, and growth 13. Meanwhile, stimulation of βAR signaling leads to ERK activation in a Gi-dependent manner via Gβγ subunits 9. Alternatively, activated βARs associate with arrestins, leading to receptor internalization and arrestin-mediated ERK activation from both the β1AR 14 and the β2AR 8. The later G protein-independent mechanism leads to cytosolic ERK retention for phosphorylation of cytosolic targets.
Here we have identified a novel mechanism regulating Gq-coupled receptor-induced MAPK signaling via cross-talk with β2AR-recruited arrestin3 in mouse cardiac fibroblasts and embryonic fibroblasts (MEFs). This is the first evidence suggesting that arrestin activation by one GPCR is capable of regulating a signaling pathway originating from another GPCR. These studies also provide a novel mechanism explaining the coordination of subcellular mitogenic signaling elicited from different GPCR stimuli.
Animal care and use was in accordance with institutional guidelines. Neonatal cardiac fibroblasts were isolated from new born mice utilizing a collagenase dispersion procedure with a pre-plating step, as previously described. Cardiac fibroblasts or MEF cells were then transfected, if indicated, with either arrestin, GRK, SRC or adrenergic receptor constructs utilizing Lipofectamine2000 according to the manufacturer’s instructions. For Western blotting, cells were pretreated with the indicated drugs and then stimulated with adrenergic agonists or antagonists for the indicated times. Cell lysates were then subjected to Western blotting with antibodies accordingly. The cell proliferation ELISA was carried out utilizing bromodeoxyuridine Labeling and Detection Kit III (Roche) according to the manufacturer’s instructions. Student’s t test was performed using Prism software.
Both α1ARs and βARs are implicated in promoting ERK activation upon stimulation with catecholamines 15. Utilizing cardiac fibroblasts, we investigated the mechanism leading to the modulation of ERK signaling upon potential cross-talk between these GPCRs. We examined agonist-dependent ERK phosphorylation induced by epinephrine (Epi). Since α1ARs are the primary adrenergic receptors responsible for ERK activation in cardiac tissues, we used the α1AR-specific agonist phenylephrine (Phe). Both Epi and Phe activated ERK by increasing the phosphorylation of ERK (phospho-ERK). The maximal phospho-ERK levels peaked at 5 minutes after stimulation, with the peak levels induced by Phe significantly lower than that by Epi. Moreover, the Epi-induced phospho-ERK underwent a rapid decrease to baseline, whereas, the Phe-induced phospho-ERK signal was prolonged and returned to baseline levels slowly (Fig. 1A).
Previous studies have indicated that subcellular ERK distribution may shape the temporal profile of ERK activation 16. We found that Epi-induced phospho-ERK accumulated in the cytosol and returned to baseline levels rapidly (Fig. 1B). Interestingly, Phe-induced phospho-ERK translocated to the nucleus and remained elevated throughout stimulation (Fig. 1B). The subcellular distribution of phospho-ERK was confirmed in fractionation studies. The Epi-induced phospho-ERK was enriched in the cytosolic fraction; however, the Phe-induced phospho-ERK was enhanced in both the cytosolic and nuclear fractions (Fig. 1C). Thus, concomitant activation of theβ and α1ARs by Epi induces a distinct spatiotemporal ERK signal than that elicited by activation of the α1AR alone by Phe.
To investigate the mechanism underlying the modulation of ERK signaling upon adrenergic receptor cross-talk, we first examined which adrenergic receptor was responsible for ERK phosphorylation. Stimulation with Epi and Phe induced potent ERK activation (Figs. 2A). Stimulation with the βAR agonist isoproterenol (Iso) resulted in minimal ERK activation, whereas, stimulation of the α2ARs with clonidine (Clo) did not activate ERK (Fig. 2A). In addition, pretreatment with the α1AR antagonist prazosin (Prz), but not the βAR antagonist timolol (Tim) or the α2AR antagonist yohimbine (Yoh), significantly blocked Epi-induced ERK activation (Fig. 2B and data not shown). As a control, Prz and Yoh, as well as a panel of β-blockers including Tim, propanolol, alprenolol, and cavedilol did not alter basal ERK levels (Supplementary Figure I). Previous studies show that activation of βARs can induce cell-specific ERK activation 17. Similar to neonatal cardiac fibroblasts, Iso induced minimal ERK phosphorylation in MEFs, and in both neonatal and adult cardiac myocytes. In contrast, stimulation of βARs with Iso induced robust ERK phosphorylation in HEK293 cells (Supplementary Figure II). Together, these results confirmed that ERK activation resulted primarily from α1AR stimulation in cardiac fibroblasts, which was supported by the expression of both α1AAR and α1BAR genes (Supplementary Figure III). Accordingly, stimulation of Gq with Pasteurella multocida toxin and stimulation of PKC with phorbol myristate acetate (PMA) was sufficient to induce potent ERK activation (Fig. 2 and data not shown). In contrast, direct inhibition of Gi with Pertussis toxin (PTX) had no effect on agonist-induced ERK activation (Supplementary Figure IV). Pretreatment with the PKA inhibitor H89, which blocks Gs signaling, also had no significant effect on agonist-induced ERK activation (Supplementary Figure IV). Together, these data confirm that the α1ARs are the primary adrenergic subtypes responsible for ERK phosphorylation via Gq activation in cardiac fibroblasts.
Classic α1AR/Gq coupling activates phospholipase C to produce diacyl glycerol, leading to PKC activation. Pretreatment with the PLC inhibitor U73122 significantly blocked both Epi- and Phe-induced ERK phosphorylation (Fig. 2C and 2D). Similarly, pretreatment with a myristolated PKC inhibitory peptide (PKCi) significantly prevented ERK activation upon either Epi or Phe stimulation (Fig. 2C and 2D). Moreover, inhibition of MEK with U1026 also prevented both Epi and Phe-induced ERK activation (Fig. 2C and 2D). These data confirm that α1AR-induced ERK signaling is dependent on activation of PLC and PKC leading to the Raf-MEK pathway 18.
One possible explanation for the differences in the spatiotemporal ERK activation profile between Epi and Phe is that βAR activation by Epi may lead to cytosolic retention of α1AR-induced phospho-ERK. To test this hypothesis, we blocked the βARs with Tim before Epi stimulation. Inhibition of the βARs with Tim redistributed the Epi-induced phospho-ERK to both the cytosol and the nucleus, resulting in a similar spatial profile to that induced by Phe (Figs. 3A and 3B). As a result, βAR blockade significantly prolonged the phospho-ERK signal (Fig. 3C). Moreover, simultaneous stimulation of βARs with Iso prevented nuclear translocation of Phe-induced phospho-ERK and promoted phospho-ERK signal attenuation (Supplemental Figure V). Together, our data suggest that activation ofβARs can prevent nuclear translocation of phospho-ERK induced by the α1AR/Gq signaling pathway.
To understand the physiologic consequence of cytosolic ERK sequestration upon α1AR and βAR cross-talk in cardiac fibroblasts, we examined cell proliferation utilizing bromodeoxyuridine incorporation upon different ligand stimulation. Activation of the α1ARs with Phe, but not theβARs with Iso, induced a significant increase in bromodeoxyuridine incorporation, which was blocked by the MEK inhibitor U1026. Further, activation of both the α1AR and βARs with Epi did not significantly enhance bromodeoxyuridine incorporation. However, inhibition of βARs with Tim enabled Epi to stimulate bromodeoxyuridine incorporation (Fig. 3D), presumably due to the redistribution of phospho-ERK to the nucleus for gene transcription (Fig. 3A). This increase in bromodeoxyuridine incorporation was again blocked by U1026.
We then sought to identify the βAR subtype responsible for cytosolic sequestration of α1AR-induced ERK. Cardiac fibroblasts lacking the β2AR, but not those lacking the β1AR, displayed nuclear phospho-ERK accumulation upon Epi stimulation (Fig. 4A and 4B), which also prolonged the phospho-ERK signal (Fig. 4C). Stimulation of DKO cells, which lack both βAR subtypes, with Epi induced phospho-ERK in both the cytosol and nucleus (Fig. 4D and 4E). We then reintroduced either β1AR or β2AR into DKO cells with similar expression levels (data not shown). Expression of β2AR, but not β1AR, recovered the cytosolic retention of phospho-ERK induced by Epi (Fig. 4D and 4E). Thus, the effect is β2AR-specific, and is not due to the higher endogenous expression levels of the β2AR than the β1AR in cardiac fibroblasts 19. Together, these data suggest that activation of the β2AR modulates both the spatial and temporal profile of α1AR-induced ERK activation in cardiac fibroblasts.
We further examined the molecular mechanism explaining this signaling cross-talk. Previous studies reported that α1DARs form a heterodimer with β2ARs in HEK293 cells, thus altering α1D signaling20, but this α1AR subtype is not expressed in cardiac fibroblasts (Supplementary Figure III). Moreover, neither α1AAR nor α1BAR dimerized with β2AR (Supplementary Figure VI). Alternatively, upon phosphorylation of the β2ARs via GRKs, arrestins are recruited leading to receptor internalization 21; and the internalized β2AR/arrestin complexes propagate numerous signaling pathways, including ERK pathways 22. Due to their role in arrestin recruitment, we investigated the role of GRKs in Epi-induced ERK activation. MEF cells were used for selective knockdown individual GRKs. In wild-type MEF cells, Epi stimulated cytosolic phospho-ERK, similar to cardiac fibroblasts. Selective knockdown of GRK2, but not other GRKs, in MEF cells significantly promoted nuclear accumulation of phospho-ERK induced by Epi (Figure 5A and Supplementary Figure VII). Expression of βARKct, a GRK2 inhibitor that prevents β arrestin recruitment and thus βAR internalization 23, significantly promoted nuclear accumulation of phospho-ERK (Fig. 5B) and prolonged ERK signaling (Fig. 5C). These data indicate that the GRK2-mediated phosphorylation of the β2AR modulates ERK activation, presumably through β arrestin-mediated scaffolding of ERK.
In addition, Src is necessary for arrestin-dependent βAR internalization upon agonist binding 24. Inhibition of Src with either over-expression of dominant negative Src (DN-Src) or treatment with Src inhibitor PP2 prolonged ERK activation induced by Epi, but had no effect on Phe-induced ERK (Supplementary Figure VIII and data not shown). Fractionation studies revealed that DN-Src expression enhanced nuclear phospho-ERK translocation upon Epi stimulation (Supplementary Figure VIII). These data suggest that Src-dependent and arrestin-mediated β2AR internalization is necessary for cytosolic sequestration of the α1AR-induced ERK signal under Epi stimulation.
To identify the arrestin(s) responsible for this cross-talk, MEF cells lacking either arrestin2 (arr2-KO) or arrestin3 (arr3-KO) were used. In comparison to wild-type MEF cells, Epi promoted phospho-ERK translocation to the nucleus in arr3-KO, but not arr2-KO MEF cells (Figs. 6A and 6B). Thus, arrestin3 is primarily responsible for the cross-talk between the β2 and α1ARs. We then examined the association between ERK and the β2AR/arrestin3 complex upon agonist stimulation. Epi induced a significant increase in the association between ERK and the β2AR/arrestin3 complex, which was attenuated by Tim (Fig. 6C and Supplementary Figure IX). In contrast, neither Phe nor Iso enhanced the association between ERK and the β2AR/arrestin3 complex; but co-stimulation with Iso and Phe promoted formation of the complex (Fig. 6C). Further, a dominant-negative arrestin3 (GFP-V54Darr3 25) also formed a complex with the β2AR, but was not sufficient to promote the binding of ERK to the β2AR/arrestin3 complex (Fig. 6C). We then used GFP-V54Darr3 to further perturb the cross-talk between the β2 and α1ARs. Expression of GFP-V54Darr3, but not GFP-arr3, promoted nuclear translocation of the phospho-ERK induced by Epi, which did not colocalize with GFP-V54Darr3 in the cytosol (Figs. 6D and 6E). In contrast, expression of neither GFP-arr3 nor GFP-V54Darr3 affected the Phe-induced nuclear accumulation of phospho-ERK (Fig. 6D and 6E). In cells expressing GFP-V54Darr3, the cytosolic and the nuclear accumulation of Epi-induced phospho-ERK was further confirmed by fractionation studies (Fig. 6F). Not surprisingly, nuclear translocation also prolonged the Epi-induced phospho-ERK signal (Fig. 6G). Together, these data show that internalization of the β2AR via arrestin3 induces the cross-talk between the α1 and β2ARs, leading to ERK sequestration within the cytosol.
To test whether this arrestin-mediated GPCR cross-talk is a general MAPK regulatory mechanism upon concomitant activation of multiple GPCRs, we used MEF cells lacking both arrestin2 and arrestin3 (arr2/3-KO) to investigate phospho-ERK distribution induced by different Gq-coupled receptors in the absence and presence ofβ2 AR activation. Stimulating both wild type MEFs and cardiac fibroblasts with either Phe or PMA induced nuclear accumulation of phospho-ERK. Interestingly, stimulation of wild type MEF and cardiac fibroblast cells with two other Gq-coupled receptor agonists ((Val5) angiotensin II or thrombin) also induced nuclear phospho-ERK accumulation (Figs. 7A and 7B), likely through Gq-dependent pathways 26. However, upon Iso co-stimulation, Phe-, (Val5) angiotensin II-, thrombin-, as well as PMA-induced phospho-ERK was sequestered within the cytoplasm in both wild type MEF and cardiac fibroblast cells (Figs. 7A and 7B). As expected, in MEF cells lacking β arrrestins, or in cardiac fibroblasts expressing the GFP-V54Darr3 mutant, ERK signaling induced by the different stimuli was able to translocate into the nucleus. However, both arrestin deficiency and GFP-V54Darr3 expression blocked the effect of β2AR activation under Iso stimulation (Figs. 7A and 7B). Together, these data suggest that arrestin activation by the β2AR can sequester Gq-coupled receptor-induced phospho-ERK within the cytosol in both cardiac fibroblasts and MEF cells.
In this study, we have identified a novel mechanism regulating Gq-coupled receptor-induced ERK MAPK signaling via cross-talk with β2AR-recruited arrestin3 in cardiac fibroblasts and MEFs (Fig. 8). This is the first evidence suggesting that arrestin activation by one receptor is capable of regulating signaling originating from another GPCR. G protein-independent regulation of GPCR signaling via arrestins is an emerging mechanism explaining the regulation of a growing list of GPCR-mediated signaling including α2AR 16, angiotensin receptors 22, β1AR 14, β2AR 8, opioid receptors 27 and the vasopressin receptors 28. Recruitment of arrestins to a phosphorylated GPCR regulates not only receptor internalization, but also intracellular signaling such as transactivation of receptor tyrosine kinases 2, 6. Arrestin, in addition, scaffolds ERKs leading to activation, cytosolic retention 22, 29, and decreased transcription in the nucleus 30. In reconstituted systems this provides a linear mechanism, however, this model fails to reflect the convoluted signaling networks in vivo. Endogenous ligands, including neurotransmitters and hormonal peptides, bind to multiple receptors present in a cell, activating numerous signaling cascades. Indeed, accumulative evidence supports signal cross-talk among GPCRs, such as between the β1AR and the angiotensin 1 receptor 5, the α2AR and the opioid receptors 31, as well as the c5a receptor and the UDP receptor 32. Here, our results suggest that arrestin functions as a master regulator, coordinating subcellular ERK activation under multiple extracellular stimuli to inhibit nuclear translocation and facilitate signal attenuation. Considering the ability of arrestin to scaffold different cytosolic signaling components besides ERK, such as Src, Jun N-terminal Kinase, and p38 26, and the ability of arrestin to selectively bind to some, but not all GPCRs in an agonist-dependent fashion 33, our data suggest a general mechanism of arrestin-mediated cross-talk among GPCRs with broad implications in physiological responses under neurohormonal regulation in vivo.
In cardiac fibroblasts and MEFs, our data indicate that the α1ARs make the primary contribution to ERK activation, supporting the dominant roles of these receptor subtypes in cardiac remodeling 34. Consistent with previous studies 35, stimulation of the α1ARs with Phe induces the classic Gq-dependent activation of PLC and PKC, leading to ERK activation through Raf-MEK1 kinase cascade. Under this signaling cascade, activated ERK translocates to the nucleus 35. Interestingly, this scenario is completely reshaped when β2ARs are co-activated with the α1AR upon Epi stimulation. Co-activation of the α1 and β2ARs leads to sequestration of phospho-ERK within the cytosol. Under Epi stimulation, it was possible that two pools of ERK existed; a transient pool activated by the β2AR and a prolonged pool activated by the α1AR. The first pool becomes dominant simply because β2ARs are more prominent in cardiac tissues than α1ARs. This explanation is unlikely for several reasons. First, α1AR antagonist prazosin blocked Epi-induced ERK phosphorylation. Second, stimulation with isoproterenol (a βAR agonist) alone induces minimal ERK activation. Third, blockade of the β2AR prolonged ERK activation and only slightly decreased maximal ERK levels (Fig. 3C). Taken together, these data support that ERK activation by Epi and Phe originates from α1AR activation. However, under Epi stimulation, β2AR activation changes the spatiotemporal profile of ERK signaling by policing ERK to the β2AR/arrestin3 complex. Cytosolic retention may allow targeting to non-nuclear ERK substrates involved in different cellular stress processes 36 and receptor desensitization 37. This unique observation highlights the potential diversified physiological consequences under a given extracellular environment when multiple receptors are activated simultaneously. As expected, this arrestin3-mediated cross-talk is dependent on the receptor phosphorylation by GRK. Inhibiting GRK2, but not other GRKs, is sufficient to prevent the β2AR-dependent modulation of α1AR-induced ERK signaling. In addition to sequestering ERK within proximity of cytosolic ERK targets, our data indicate that interaction with arrestin3 may facilitate the attenuation of ERK signaling in the cytosol 38. However, inhibition of phosphatase 2A or dual-specificity phosphatases failed to prevent ERK dephosphorylation (data not shown), thus the phosphatases involved remain to be identified.
The distinct spatiotemporal profile of ERK activation induced by Gq-coupled receptors in the presence or absence of β2AR (and potentially other GPCRs) activation has broad implications in the maladaptative remodeling of different tissues in vivo. Indeed, in cardiac myocytes, the β1AR, but not the β2AR appears to cross-talk with Gq-coupled receptors in modulating ERK activation (unpublished data). Gq-coupled receptor signaling and ERK activation play essential roles in long-term pathophysiological cardiac remodeling 9, 34. Traditionally, activated ERK MAPK translocates to the nucleus to activate transcription factors including Elk-1 39 and GATA-4 40 leading to fibroblast proliferation and myocyte hypertrophy. Here, this nuclear translocation of ERK is abrogated by arrestin binding to activated β2ARs in cardiac fibroblasts; interestingly it can be restored with the clinically-relevant β blocker timolol. Our data thus provide insights into understanding the tissue remodeling observed in patients under chronic treatment with β-blockers in various clinical and physiological conditions.
In summary, our findings provide the first evidence for the role of β2AR-recruited arrestin in regulating signaling from another GPCR. These findings provide a novel mechanism to significantly advance our understanding of the growing profiles of GPCR regulatory pathways. These data further underscore the critical role of signaling cross-talk in the complex regulation of receptor signaling via subcellular localization of signaling components, which will have significant implications in numerous clinical and physiological conditions.
We thank Ryan Mitacek, Ruijie Liu, and Dr. Vania De Arcangelis for technical assistance, and Drs. Qin Wang, Gang Pei, Randy Hall, and Robert Lefkowitz for reagents.
Sources of Funding
This study was supported by NIH HL082646 and AHA 0635331N to YX.