β-Arrestin Mediates β1-Adrenergic Receptor-Epidermal Growth Factor Receptor Interaction and Downstream Signaling*

β1-Adrenergic receptor (β1AR) stimulation confers cardioprotection via β-arrestin-de pend ent transactivation of epidermal growth factor receptors (EGFRs), however, the precise mechanism for this salutary process is unknown. We tested the hypothesis that the β1AR and EGFR form a complex that differentially directs intracellular signaling pathways. β1AR stimulation and EGF ligand can each induce equivalent EGFR phos pho ryl a tion, internalization, and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas β1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. β1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. Although catecholamine stimulation mediates the retention of β1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of β1AR with EGFR following activation is dependent upon C-terminal tail GRK phos pho ryl a tion sites of the β1AR and recruitment of β-arrestin. These data reveal a new signaling paradigm in which β-arrestin is required for the maintenance of a β1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.

␤1-Adrenergic receptor (␤1AR) stimulation confers cardioprotection via ␤-arrestin-dependent transactivation of epidermal growth factor receptors (EGFRs), however, the precise mechanism for this salutary process is unknown. We tested the hypothesis that the ␤1AR and EGFR form a complex that differentially directs intracellular signaling pathways. ␤1AR stimulation and EGF ligand can each induce equivalent EGFR phosphorylation, internalization, and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas ␤1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. ␤1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. Although catecholamine stimulation mediates the retention of ␤1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of ␤1AR with EGFR following activation is dependent upon C-terminal tail GRK phosphorylation sites of the ␤1AR and recruitment of ␤-arrestin. These data reveal a new signaling paradigm in which ␤-arrestin is required for the maintenance of a ␤1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.
␤1-Adrenergic receptor (␤1AR) 3 stimulation regulates a number of signaling pathways and has been recently shown to transactivate epidermal growth factor receptor (EGFR) and increase ERK activation in both in vitro cell culture systems and in vivo mouse models (1). The key signaling components required for EGFR transactivation following ␤1AR stimulation include: 1) C-terminal phosphorylation of activated ␤1ARs by GRK5/6, and 2) recruitment of both ␤-arrestins 1 and 2 to phosphorylated ␤1ARs. Recruitment of ␤-arrestins to activated ␤1ARs allow subsequent activation of c-Src and matrix metalloproteinases, cleavage of HB-EGF and activation of EGFR, processes that contribute to transactivation pathways defined for other 7TMRs (2)(3)(4)(5)(6)(7). Trafficking of EGFR has been shown to be critical in defining downstream signaling pathways regulated by ligand-induced activation (8). Although direct EGFR stimulation via its ligand EGF leads to internalization of the receptor as well as ERK1/2 translocation to the nucleus and activation of Elk-1-mediated transcription (9,10), the precise mechanism by which ERK signaling pathways are regulated following ␤1AR-mediated EGFR stimulation is poorly understood.
Recruitment of ␤-arrestin1/2 to activated 7TMRs allows these multifunctional scaffold proteins to target other signaling proteins that are involved in receptor internalization, desensitization, and intracellular signaling complexes. Members of the mitogen-activated protein kinase family such as ERK are among the proteins recruited to receptors by ␤-arrestins (11). Several recent studies have shown that ␤-arrestin1/2 recruitment to activated 7TMRs may direct both cytosolic ERK1/2 signaling, via formation of internalized ␤-arrestin signalosomes containing the receptor and activated ERK (12,13), and nuclear targeting of ERK (14,15), depending upon the 7TMR, cell-type, and culture conditions tested. Although ␤-arrestin recruitment is essential for ␤1AR-mediated transactivation of EGFR, it remains unclear if ␤-arrestins play a role in regulating and targeting the downstream ERK response.
In this study we sought to determine whether the mode of EGFR activation, via ␤1AR-mediated transactivation versus direct ligand activation, induces differential effects on ERK1/2 signaling and if so, to elucidate the mechanism by which this process occurs. Here, we show that ␤1AR and EGFR form a receptor complex at the plasma membrane that is dynamically regulated by ligand stimulation, leading to differential ERK1/2 targeting and intracellular effects. Moreover, the recruitment of ␤-arrestin to the agonist-occupied ␤1AR is required to maintain prolonged ␤1AR-EGFR interaction during simultaneous receptor internalization and to retain ERK1/2 activation in the cytosol. These findings illustrate the importance of ␤-arrestin in mediating receptor-receptor interaction and the targeting of downstream signaling pathways.
Luciferase Assay-WT-␤1AR cells transfected with luciferase constructs were stimulated in 96-well plates as indicated for 5 h, followed by lysis with the Dual Luciferase Reporter Assay System (Promega). Relative light units were detected using a Veritas Microplate luminometer (Turner Biosystems/Promega). Luciferase activity is reported relative to the non-stimulated group.
Chemical Cross-linking and Immunoprecipitation-Crosslinking has been described (17). Immunoprecipitation (IP) of samples was performed with 500 to 1000 g of protein and overnight incubation with 30 l of either anti-FLAG M2 or anti-HA affinity gel (Sigma).
Membrane Preparation and ␤AR Radioligand Binding-Membrane preparation and receptor binding were performed using [ 125 I]cyanopindolol as described previously (1,18,19). Membrane pellets were lysed for 1 h at 4°C with lysis buffer containing 1% dodecyl maltoside to maintain native oligomeric protein configurations (20,21) and underwent IP with rabbit anti-EGFR antibody or IgG control antibody (Upstate).
Statistical Analysis-Statistical tests were performed using either two-tailed unpaired t test or one-way analysis of variance with the Newman-Keuls multiple comparison post hoc test using Prism 5.0 software. p value (*, p Ͻ 0.05; †, p Ͻ 0.01, ‡, p Ͻ 0.001) of Ͻ0.05 was considered significant.
To determine whether cellular localization of ERK2-RFP in response to Dob and EGF stimulation correlated with ERK activation, we performed immunoblotting with an anti-phosphorylated ERK1/2 (P-ERK1/2) antibody. Dob and EGF each increased P-ERK1/2, effects that were significantly attenuated by EGFR inhibition (Fig. 1B). Although AG 1478 decreased Dob-induced P-ERK1/2 by ϳ70%, we tested whether ␤1ARmediated PKA signaling accounts for the residual Dob-induced P-ERK1/2 (Fig. 1C). In the presence of the PKA inhibitor H89, Dob-induced P-ERK1/2 levels were reduced by ϳ25% indicating that PKA-dependent signaling is responsible for only a small portion of P-ERK1/2 with the majority accounted for by ␤1AR-mediated EGFR transactivation. Additionally, cell fractionation experiments confirmed that Dob significantly induced only cytosolic ERK1/2 phosphorylation, whereas EGF significantly increased both cytosolic and nuclear ERK1/2 phosphorylation (Fig. 1D). Indeed, p75/p85 S6 kinase (S6K), a downstream target of cytosolic P-ERK, is phosphorylated in response to both catecholamine and EGF stimulation in an AG 1478-sensitive (sp) manner (Fig. 1E).
Consistent with the above data, Dob stimulation resulted in P-ERK2-RFP being targeted only to the cytosol ( Fig. 2A, panel  3), whereas EGF stimulation resulted in the targeting of P-ERK2-RFP to both cytosol and nucleus ( Fig. 2A, panel 5, arrow). Each of these responses was abrogated with the inclusion of AG 1478 ( Fig. 2A, panels 7 and 9). We then tested whether nuclear translocation of ERK1/2 would induce gene transcription as measured by Elk-1-Gal4 luciferase reporter activity. EGF stimulation resulted in a significant increase in the amount of Elk-1-Gal4-driven luciferase activity, indicative of increased ERK1/2 activity in the nucleus (Fig. 2B), which was blocked by AG 1478 pretreatment. Conversely, activation of Elk-1-Gal4 luciferase was not observed with Dob ( Fig. 2B) or ISO (Fig. 2D). To ensure catecholamine stimulation produced a ␤1AR-mediated signal in these assays, CREB-Gal4 luciferase activity was assessed, indicative of cAMP signaling downstream of ␤1AR activation. Using this system, both Dob ( Fig. 2C) and ISO (Fig. 2D) caused significant induction of CREB-Gal4-mediated luciferase activity. Therefore, whereas ␤1AR-mediated transactivation and direct EGF ligand stimulation each induce EGFR internalization and downstream phosphorylation of ERK, these stimuli result in differential intracellular targeting and function of ERK1/2.

Ligand Concentrations Matched for EGFR and ERK1/2 Activation
Maintain Differential ERK Targeting-HB-EGF is an endogenously expressed membrane protein known to be cleaved by matrix metalloproteinases in response to ␤AR stimulation (1,4,17). To test the effect of bypassing ␤1AR-mediated EGFR transactivation with exogenously added HB-EGF and EGF, we measured ERK1/2 activation and cellular targeting at various time points following HB-EGF, EGF, and Dob stimulation. In WT-␤1AR cells transiently transfected with FLAG-EGFR, Dob induced an early and significant increase in P-ERK1/2, peaking at 5 min and approaching basal levels by 60 min (Fig. 3A). Direct EGFR stimulation with HB-EGF or EGF induced rapid and significant P-ERK1/2 that peaked at 5 min and returned to ϳ 1 ⁄ 3 of maximum by 60 min (Fig. 3A). Confocal microscopy of Dob-stimulated WT-␤1AR cells transfected with EGFR-GFP and ERK2-RFP showed marked EGFR internalization after 10 min (Fig. 3B). Importantly, at no time point did Dob stimulation result in ERK2-RFP translocation to the nucleus, even with continued EGFR internalization (Fig. 3B). In contrast, both EGF and HB-EGF began to initiate EGFR internalization and ERK2-RFP translocation to the nucleus after 5 min of stimulation that persisted for 60 min (Fig. 3B). Consistent with our immunoblotting data, cell staining at various time points revealed peak ERK phosphorylation at 5 min in response to either Dob or EGF (Fig. 3C) and returning close to basal levels by 60 min.
We next compared the effects of increasing Dob, EGF, and HB-EGF concentrations on ERK1/2 phosphorylation (Fig.  4A). Analysis of the concentration-response curves revealed that stimulation with 1 M Dob, 0.1 nM EGF, and 0.01 nM HB-EGF resulted in a similar level of P-ERK1/2 (Fig. 4A). Although Dob did not induce nuclear ERK2-RFP trafficking even up to concentrations of 100 M, both 0.1 nM EGF and

␤1AR-EGFR Interaction Regulates ERK Trafficking
0.01 nM HB-EGF resulted in robust nuclear accumulation of ERK2-RFP (Fig. 4B). To assess the effects of increasing concentrations of Dob, EGF, and HB-EGF on EGFR-GFP internalization, we calculated the loss of EGFR-GFP from the plasma membrane in response to ligand stimulation (Fig.  4C). The concentrations of 0.1 nM EGF and 0.01 nM HB-EGF induced equivalent EGFR-GFP internalization as Dob at concentrations Ն1 M. Consistent with the internalization data, the level of P-EGFR in response to 1 M Dob was equivalent to that of 0.1 nM EGF indicating similar EGFR activation (Fig. 4D). To determine whether the concentrations of exogenous HB-EGF added in these experiments were within the range of endogenous HB-EGF shed in response to ␤1AR activation, we collected the media of WT-␤1AR cells stimulated with Dob for 5 min and measured the amount of HB-EGF released (Fig. 4E). Under non-stimulated conditions, cells released a basal level of 0.94 Ϯ 0.15 pM HB-EGF into the media. In contrast, the media of Dob-stimulated cells contained 3.54 Ϯ 0.13 pM HB-EGF. Thus, whereas Dob causes release of HB-EGF into the media that induces a similar level of EGFR internalization and P-ERK1/2 as 0.01 nM HB-EGF, differential mechanisms must be involved to account for the subsequent cytosolic retention of ERK.
␤1AR and EGFR Associate with Specificity and Their Association Is Differentially Regulated by Ligand Stimulation-To address the mechanism by which differential targeting of ERK in response to ligand may occur, we tested the possibility that ␤1AR and EGFR may interact and direct the cellular localization of ERK. Recently, a number of studies have reported an interaction between other 7TMRs and EGFR (5, 24 -28). In FLAG-␤1AR cells transfected with EGFR-GFP, IP of FLAG-␤1AR resulted in co-IP of EGFR (Fig. 5A). In contrast, EGFR-GFP overexpression in HEK 293 cells stably expressing the hemagglutinin-tagged angiotensin type 1 A receptor (HA-AT1 A R) did not result in the co-IP of EGFR with AT1 A R, even following angiotensin II stimulation (Fig. 5B). Angiotensin II stimulation did induce association between AT1 A R and ␤-ar-restin1/2, confirming receptor activation and ability to interact with predicted proteins. Thus, simple overexpression of another 7TMR (AT1 A R), which is known to transactivate EGFR (27,29,30), is insufficient to induce its interaction with EGFR in this system.

␤1AR-EGFR Interaction Regulates ERK Trafficking
To determine whether ␤1AR and EGFR interact at endogenous levels and in vivo, we performed co-IP experiments in U2S cells and human heart tissue, which basally express both the EGFR and ϳ35-60 fmol/mg protein of ␤ARs (Fig. 5C). Membrane preparations from both U2S cells and heart tissue were used to IP endogenous EGFR followed by radioligand binding with the highly specific ␤AR ligand [ 125 I]iodocyanopindolol. Nonspecific binding was determined by the separate addition of IgG followed by IP. Within the EGFR immunoprecipitates we detected a significant level of endogenous ␤AR compared with IgG control immunoprecipitates in both U2S cells (0.16 Ϯ 0.04 fmol of receptor, Fig. 5D) and human heart tissue (0.13 Ϯ 0.06 fmol of receptor, Fig. 5E). These experiments support our data that under basal conditions there is an interaction between ␤1ARs and EGFRs.
To determine the specificity of ␤1AR-EGFR interaction, we performed FRET experiments in HEK 293 cells stably expressing monomeric cyan fluorescent proteintagged ␤1AR (␤1AR-mCFP cells) and transiently expressing either EGFR-mYFP or myristoylated-palmitoylated mYFP (MyrPalm-mYFP). MyrPalm-mYFP is targeted to the plasma membrane (31), thereby providing a nonspecific membrane-bound FRET partner for ␤1AR-mCFP for comparison with EGFR-mYFP to determine specificity of interaction. As levels of Myr-Palm-mYFP and EGFR-mYFP increased, the amount of detectable FRET also increased, achieving maximal %FRET of ϳ50 and 20%, respectively (Fig. 5F). Because the maximal FRET efficiency between ␤1AR-mCFP and either mYFPtagged protein is influenced by both proximity and orientation of mCFP and mYFP, and dependent upon mYFP concentration, it does not necessarily represent the true amount of specific interaction between the partners. Therefore, we compared the relative affinities of EGFR-mYFP and MyrPalm-mYFP for ␤1AR-mCFP calculated via saturation binding analysis. A 5-fold greater affinity of EGFR for ␤1AR than that of MyrPalm-mYFP for ␤1AR was attained (Fig. 5G), indicating that the interaction of ␤1AR with EGFR has significantly higher specificity than with a general membrane-bound protein.
We next used FRET analysis to explore the possibility that agonist stimulation can regulate ␤1AR-mCFP-EGFR-mYFP association. Catecholamine stimulation (either Dob or ISO)

␤1AR-EGFR Interaction Regulates ERK Trafficking
induced a small reduction in FRET over time that was indistinguishable from non-stimulated cells (Fig. 5H). In contrast, EGF stimulation caused an immediate decrease in FRET signal that persisted. Nonlinear regression analysis of the data revealed approximately a 5-fold greater loss in FRET (%F max ) with EGF stimulation compared with Dob or ISO (Fig. 5I). Thus, EGF stimulation significantly disrupts ␤1AR-EGFR interaction, whereas catecholamine stimulation maintains receptor interaction.

␤1AR-EGFR Interaction Regulates ERK Trafficking
To explore the possible requirement of ␤-arrestin in mediating ␤1AR-EGFR trafficking after catecholamine stimulation, we assessed the ability of WT-␤1AR and GRK Ϫ ␤1AR cells to recruit ␤-arrestin. Stimulation with catecholamine induced a significant rapid increase in ␤-arrestin recruitment to the WT-␤1AR, peaking at 5 min and remaining elevated up to 30 min post-stimulation (Fig. 6C). Importantly, stimulation with EGF in WT-␤1AR cells or with ISO in GRK Ϫ ␤1AR cells did not result in ␤-arrestin association suggesting that ␤-arrestin recruitment to the receptor complex is required for their continued association in the presence of catecholamine.
Furthermore, we investigated the co-localization of ␤1AR and EGFR in the presence of CTL versus ␤arr1/2 siRNA via confocal microscopy. Cells transfected with either siRNA maintained ␤1AR and EGFR co-localization at the cell surface in a nonstimulated state (Fig. 7B, panels 1, 2, 5, and 6). Treatment with ISO caused the formation of internalized puncta containing both ␤1AR and EGFR in CTL siRNAtreated cells (Fig. 7B, panels 3 and 4, arrowheads), but only ␤1AR in ␤arr1/2 siRNA-treated cells (Fig. 7B, panels 7 and 8,  arrowhead). EGF stimulation induced internalization of only EGFR-GFP in both CTL and ␤arr1/2 siRNA-treated cells (not shown). These data demonstrate that whereas the basal association of receptors at the cell surface does not require ␤-arr1/2, following catecholamine stimulation endogenous ␤-arr1/2 recruitment is necessary to maintain ␤1AR-EGFR interaction and co-internalization.

DISCUSSION
Although 7TMR-mediated transactivation of EGFR has been shown to induce phosphorylation of ERK1/2, the mechanisms responsible for this effect vary as widely as the 7TMRs that transactivate EGFR (17,24,28,34,35). Previously, we have shown a role for ␤-arrestin in the regulation of ␤1AR-mediated transactivation of EGFR (1,17) and in this study we show that ␤1AR and EGFR interact as a complex whose continued association following agonist stimulation is dependent upon GRK phosphorylation sites in the C-terminal tail of ␤1AR, and on the recruitment of ␤-arrestin. We identify that ␤1AR-mediated EGFR transactivation leads to differential intracellular trafficking of ERK1/2 compared with direct ligand stimulation of EGFR. Each stimulus induces EGFR activation and internalization, but despite this common feature, the biological consequences of these distinct stimuli are divergent. Others have reported that ␤-arrestin functions as a scaffold for ERK and their upstream kinases, thus providing a pool of ␤-arrestin-bound ERK1/2 in the cytosol (36 -38). Thus, we propose that upon cate- FIGURE 7. ␤-Arrestin-mediated retention of ␤1AR-EGFR interaction following catecholamine stimulation. A, ␤1AR-mCFP cells transiently transfected with EGFR-mYFP and either CTL or ␤arr1/2 siRNA underwent stimulation with Dob (1 M) after 5 min of baseline FRET was obtained. Addition of Dob did not significantly reduce %FRET in the presence of CTL siRNA, but did cause a marked reduction in %FRET in cells transfected with ␤arr1/2 siRNA (Ͼ90% ␤arr1/2 knockdown as determined by immunoblotting (IB)). Data represent the mean Ϯ S.E. from 10 independent experiments. B, WT-␤1AR cells were transfected with either CTL or ␤arr1/2 siRNA and EGFR-GFP (shown as merged images of EGFR-GFP and Texas Red-labeled ␤1AR). ␤1AR and EGFR-GFP co-localized at the plasma membrane in nonstimulated cells (panels 1, 2, 5, and 6). Treatment with ISO (1 M, 20 min) caused formation of puncta containing both ␤1AR and EGFR in CTL siRNA-treated cells (panels 3 and 4, arrowheads), but only ␤1AR in ␤arr1/2 siRNA-treated cells (panels 7 and 8, arrowheads). Scale bars ϭ 10 m, upper panels; 1 m, lower panels. Images shown are representative of at least four independent experiments. C, WT-␤1AR cells transiently transfected with EGFR-GFP and either CTL or ␤arr1/2 siRNA underwent IP with FLAG-M2-agarose gel. In CTL-treated cells, EGFR-GFP associated with WT-␤1AR in the presence or absence of ISO (1 M, 5 min), whereas ␤-arr1/2 and P-ERK1/2 were detected in the IP only following ISO stimulation. In ␤arr1/2 siRNA-treated cells (ϳ90% knockdown), both EGFR-GFP and P-ERK1/2 association with ␤1AR was reduced following agonist stimulation. As summarized in the histograms, ISO-induced ␤1AR-EGFR association and ␤1AR-P-ERK1/2 interaction were significantly reduced in the presence of ␤arr1/2 siRNA versus CTL siRNA. Data represent the mean Ϯ S.E. from five independent experiments. ‫,ء‬ p Ͻ 0.05; †, p Ͻ 0.01. D, proposed mechanism of regulation of ␤1AR-EGFR interaction via transactivation. ␤1AR and EGFR associate at the plasma membrane. Catecholamine stimulation induces GRK-mediated phosphorylation of the cytoplasmic tail of ␤1AR and recruitment of ␤arr1/2 bound to ERK. Transactivation of EGFR, phosphorylation of ERK, and internalization of the ␤1AR-EGFR complex occurs in a ␤-arr-dependent manner. ␤-arr1/2 dissociates from the receptor complex and P-ERK is restricted to the cytosol (green arrow) and does not translocate to the nucleus (capped red arrow). E, direct ligand stimulation of EGFR with EGF does not initiate GRK or ␤-arr1/2 signaling, but induces dissociation of EGFR from ␤1AR, recruitment and phosphorylation of non-␤-arr1/2-bound ERK, and internalization of EGFR away from the plasma membrane. P-ERK is targeted throughout the cytosol and the nucleus (green arrows). Yellow circles indicate phosphorylation.
cholamine stimulation, GRK-mediated phosphorylation of the cytoplasmic tail of ␤1AR favors recruitment of ␤arr1/2bound ERK1/2 over free cytosolic ERK1/2. ␤-Arrestin induces transactivation of EGFR, which allows for phosphorylation of the ␤-arrestin-recruited ERK1/2. Following ␤-arrestin-mediated internalization of the ␤1AR-EGFR complex, ␤-arrestin dissociates from the receptor complex and restricts bound P-ERK1/2 to the cytosol (Fig. 7D). Whether ERK1/2 phosphorylation is dependent on ␤1AR-EGFR internalization, or occurs simultaneously but independent of receptor internalization, is not known. Direct ligand stimulation of EGFR with EGF, however, does not initiate GRK or ␤-arrestin signaling, but induces dissociation of EGFR from ␤1AR, recruitment and phosphorylation of non-␤-arr1/2bound ERK1/2, and internalization of EGFR away from the plasma membrane. This pool of P-ERK1/2 is targeted throughout both the cytosol and nucleus (Fig. 7E). These findings provide new insight into the mechanism of ␤-arrestin-mediated signaling following catecholamine stimulation, which not only induces EGFR transactivation, but also induces internalization of a ␤1AR-EGFR complex and directs intracellular ERK1/2 targeting.
EGFR is known to undergo rapid internalization and targeting for recycling or degradation following stimulation with EGF in a concentration-dependent manner (8,41,42). Catecholamine stimulation of ␤ARs induces a ␤-arrestin-associated internalization that leads to receptor recycling (11). Our data demonstrate co-localization of ␤1AR, EGFR, and ␤-arrestin following adrenergic stimulation that maintains cytosolic ERK1/2 signaling. Interestingly, even low concentrations of EGF or HB-EGF, which produced similar P-ERK1/2 levels as ␤1AR stimulation, did not lead to cytosolic-restricted ERK1/2. Therefore, ␤-arrestin recruitment to the receptor complex is necessary to induce EGFR transactivation, as we previously reported (1,17), and to subsequently promote the maintenance of the receptor complex and cytosolic retention of ERK1/2. Indeed, our data demonstrate that an internalization-deficient ␤-arrestin mutant (RRK/Q), or the absence of ␤-arrestins altogether (siRNA knockdown), decreases ␤1AR-EGFR association by ϳ80% following catecholamine stimulation and significantly reduces interaction with phosphorylated ERK1/2 by ϳ70%. Thus, ␤-arrestin recruitment is essential not only for the induction of ␤1AR-mediated EGFR transactivation but also for directing ␤1AR-EGFR internalization and P-ERK1/2 trafficking in the cell. Conversely, direct ligand stimulation of EGFR does not recruit ␤-arrestins, leading to rapid dissociation of the receptor complex, internalization of EGFR alone, and simultaneous cytosolic and nuclear targeting of P-ERK1/2. Proximal to ␤-arrestin signaling, we also show a role for GRK phosphorylation sites in the regulation of ␤1AR-EGFR interaction, suggesting the C-terminal conformation of ␤1AR is important in allowing its interaction with EGFR.
ERK1 and Ϫ2 lack both NLS and NES motifs, but do typically traffic into the nucleus following mitogenic stimulation via regulatory proteins (43). Indeed, several studies have shown that ␤-arrestins act as scaffolds for the components of ERK1/2 signaling following stimulation of various 7TMRs, including the AT1 A R and ␤2AR, and that stronger receptor-␤-arrestin interactions usually lead to cytosolic retention of ERK1/2 (11,12). In this study we show the cytosolic retention of ERK1/2 in response to ␤1AR stimulation is ␤-arrestin-dependent despite ␤ARs classically having a lower affinity for ␤-arrestin (11). This may be reflective of either a small pool of ␤-arrestin-bound ERK1/2 available for phosphorylation via ␤1AR-mediated EGFR transactivation or the potential association of a protein phosphatase in the ␤1AR-␤-arrestin1/2-EGFR-ERK1/2 complex, because ␤-arrestins have been shown to interact with several phosphatases (44).
The spectrum of 7TMRs that induce EGFR transactivation is an ever-expanding cohort that highlights the importance of EGFR as an alternative 7TMR signaling pathway beyond the classical G protein-dependent paradigm (1-7, 24 -30, 35, 45-50). Although a number of 7TMRs have been shown to induce transactivation of EGFR, only a handful have been demonstrated to interact with EGFR, although the mechanism(s) of these associations have not been elucidated (5, 24 -28). In our study we demonstrate a ligand-dependent, dynamic interaction between ␤1AR and EGFR using overexpression systems and show that this interaction occurs at endogenous receptor levels in cells and human heart tissue. Moreover, the recruitment of endogenous ␤-arr1/2 directs internalization of the ␤1AR-EGFR complex and is essential for their continued association upon catecholamine stimulation, because direct EGFR stimulation does not recruit ␤-arrestin and leads to the disruption of the receptor complex. Thus, our study provides new mechanistic insights into the regulation of this receptor complex by ␤-arrestin and the impact of this regulation on intracellular ERK1/2 targeting and activity. We propose that this dynamic ␤-arrestin-regulated receptor-receptor interaction may be a mechanism by which the ␤1AR can regulate EGFR signaling to exert differential cellular effects. Furthermore, we believe our findings may provide an attractive explanation for the cardiopro-