Competing G protein‐coupled receptor kinases balance G protein and β‐arrestin signaling
2012; Springer Nature; Volume: 8; Issue: 1 Linguagem: Inglês
10.1038/msb.2012.22
ISSN1744-4292
AutoresDomitille Heitzler, Guillaume Durand, Nathalie Gallay, Aurélien Rizk, Seungkirl Ahn, Ji‐Hee Kim, Jonathan D. Violin, Laurence Dupuy, Christophe Gauthier, Vincent Piketty, Pascale Crépieux, Anne Poupon, Frédérique Clément, François Fages, Robert J. Lefkowitz, Éric Reiter,
Tópico(s)Gene Regulatory Network Analysis
ResumoArticle26 June 2012Open Access Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling Domitille Heitzler Domitille Heitzler BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author Guillaume Durand Guillaume Durand BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Nathalie Gallay Nathalie Gallay BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Aurélien Rizk Aurélien Rizk Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author Seungkirl Ahn Seungkirl Ahn Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Jihee Kim Jihee Kim Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Jonathan D Violin Jonathan D Violin Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Laurence Dupuy Laurence Dupuy BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Christophe Gauthier Christophe Gauthier BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Vincent Piketty Vincent Piketty BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Pascale Crépieux Pascale Crépieux BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Anne Poupon Anne Poupon BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Frédérique Clément Frédérique Clément Sisyphe Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author François Fages François Fages Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author Robert J Lefkowitz Robert J Lefkowitz Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Department of Biochemistry, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Eric Reiter Corresponding Author Eric Reiter BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Domitille Heitzler Domitille Heitzler BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author Guillaume Durand Guillaume Durand BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Nathalie Gallay Nathalie Gallay BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Aurélien Rizk Aurélien Rizk Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author Seungkirl Ahn Seungkirl Ahn Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Jihee Kim Jihee Kim Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Jonathan D Violin Jonathan D Violin Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Laurence Dupuy Laurence Dupuy BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Christophe Gauthier Christophe Gauthier BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Vincent Piketty Vincent Piketty BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Pascale Crépieux Pascale Crépieux BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Anne Poupon Anne Poupon BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Frédérique Clément Frédérique Clément Sisyphe Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author François Fages François Fages Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France Search for more papers by this author Robert J Lefkowitz Robert J Lefkowitz Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Department of Biochemistry, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA Howard Hughes Medical Institute, Durham, NC, USA Search for more papers by this author Eric Reiter Corresponding Author Eric Reiter BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, UMR7247, Nouzilly, France Université François Rabelais, Tours, France IFCE, Nouzilly, France Search for more papers by this author Author Information Domitille Heitzler1,2,3,4,5,‡, Guillaume Durand1,2,3,4,‡, Nathalie Gallay1,2,3,4, Aurélien Rizk5, Seungkirl Ahn6, Jihee Kim6, Jonathan D Violin6, Laurence Dupuy1,2,3,4, Christophe Gauthier1,2,3,4, Vincent Piketty1,2,3,4, Pascale Crépieux1,2,3,4, Anne Poupon1,2,3,4, Frédérique Clément7, François Fages5, Robert J Lefkowitz6,8,9 and Eric Reiter 1,2,3,4 1BIOS Group, INRA, UMR85, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France 2CNRS, UMR7247, Nouzilly, France 3Université François Rabelais, Tours, France 4IFCE, Nouzilly, France 5Contraintes Team, INRIA Paris-Rocquencourt, Le Chesnay, France 6Department of Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA 7Sisyphe Team, INRIA Paris-Rocquencourt, Le Chesnay, France 8Department of Biochemistry, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC, USA 9Howard Hughes Medical Institute, Durham, NC, USA ‡These authors contributed equally to this work *Corresponding author. BIOS Group, UMR 7247, Unité Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France. Tel.:+33 2 47 42 77 83; Fax:+33 2 47 42 77 43; E-mail: [email protected] Molecular Systems Biology (2012)8:590https://doi.org/10.1038/msb.2012.22 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT1AR, and HEK293 cells expressing other 7TMRs. Synopsis The molecular mechanisms and hidden dynamics governing ERK activation by the angiotensin II type 1A receptor are studied and deciphered, revealing a signal balancing mechanism that is found to be relevant to a range of other seven transmembrane receptors. An ODE-based dynamical model of ERK activation by the prototypical angiotensin II type-1A seven transmembrane receptor has been built and validated. In order to deal with a limited number of experimental read-outs, unknown parameters have been inferred by simultaneously fitting control and perturbed conditions. In addition to its well-established function in G-protein uncoupling, G protein-coupled receptor kinase 2 has been shown to exert a strong negative effect on β-arrestin-dependent signaling and by doing so, to balance G-protein and β-arrestin signaling. This novel function of G protein-coupled receptor kinase 2 has also been evidenced in primary vascular smooth muscle cells naturally expressing the AT1AR and in HEK293 cells expressing other 7TMRs. Introduction Seven transmembrane receptors (7TMRs) regulate nearly every known physiologic processes in mammals. They represent the largest class of cell surface receptors and are the target of up to 40% of current pharmaceuticals (Ma and Zemmel, 2002). Signaling by 7TMRs is classically mediated by receptor coupling to heterotrimeric G proteins that activate a variety of effectors, including second messengers and the mitogen-activated protein kinase (MAPK) cascades (Reiter and Lefkowitz, 2006). G-protein coupling is rapidly hampered by a two step process, which begins with phosphorylation of the agonist-occupied receptor by G protein-coupled receptor kinases (GRKs; Lefkowitz, 1998). Cytoplasmic β-arrestins are subsequently recruited to the GRK-phosphorylated receptor and have key roles in both receptor desensitization and internalization (Goodman et al, 1996; Lefkowitz, 1998). Beside this classical paradigm, a growing body of evidence has revealed that β-arrestins also serve as signal transducers and adaptors (Lefkowitz and Shenoy, 2005; Reiter and Lefkowitz, 2006). So far, the best-characterized signaling mechanism to be stimulated by β-arrestins is the extracellular signal-regulated kinase (ERK) signaling pathway. β-Arrestins have been shown to scaffold components of this MAPK cascade (i.e., Raf-1, MEK1 and ERK) in complexes with receptors, leading to activation of ERK (DeFea et al, 2000; Luttrell et al, 2001; Ahn et al, 2003). Inhibition of β-arrestin 2, using specific small interfering RNA (siRNA), impairs the angiotensin II type 1A receptor (AT1AR)-stimulated ERK signaling, while β-arrestin 1 depletion enhances AT1AR-mediated ERK activation (Ahn et al, 2004b). Spatial distribution of ERK activated by the G protein- and the β-arrestin-dependent mechanisms is distinct: the G protein-dependent pathway triggers the nuclear translocation of pERK while the β-arrestin 2-activated ERK is confined to the cytoplasm (DeFea et al, 2000; Luttrell et al, 2001; Ahn et al, 2004a). The two pathways also have distinct activation kinetics: the rapid and transient G protein-dependent activation and the slower but persistent β-arrestin 2-mediated activation (Ahn et al, 2004a). Interestingly, transient and sustained ERK activation have been shown to regulate cell fates such as growth and differentiation (Sasagawa et al, 2005). In addition, the β-arrestin 2-dependent ERK pathway can be activated independently of G proteins as shown using the (DRY/AAY) mutant of AT1AR or a modified angiotensin II peptide (SII) (Wei et al, 2003). Noteworthy, different GRK subtypes have been shown to have specialized regulatory functions for the G protein and β-arrestin-dependent signaling mechanisms. Activation of the β-arrestin 2-dependent ERK pathway by the AT1AR (Kim et al, 2005), V2 vasopressin (Ren et al, 2005), β2 adrenergic (Shenoy et al, 2006) and follicle-stimulating hormone (FSHR) (Kara et al, 2006) receptors specifically requires GRK5 and GRK6 action. Second messenger generation by both V2 vasopressin (Ren et al, 2005) and H1 histamine (Iwata et al, 2005) receptors is negatively regulated by GRK2 but unaffected by GRK5 or 6. A number of studies have provided mathematical models of ERK activation by EGF (Kholodenko et al, 1999; Orton et al, 2005; Borisov et al, 2009; Kholodenko et al, 2010), including the dynamics of transient and sustained ERK activation (Sasagawa et al, 2005; Nakakuki et al, 2010). ERK response to a G protein-coupled 7TMR in yeast and mammals (Hao et al, 2007; Csercsik et al, 2008) as well as the role of GRK in the desensitization, internalization and recycling of the β2 adrenergic receptor (β2AR) (Violin et al, 2008; Vayttaden et al, 2010) have also been modeled. In addition, a number of other studies have modeled different aspects of 7TMRs signaling, including calcium signaling (see Linderman, 2009 for a recent review). However, no mathematical model has been developed to address GRK-mediated regulation of ERK activation by a 7TMR. In this study, we used experimental and computational modeling approaches to decipher the molecular mechanisms governing ERK activation by the AT1AR in human embryonic kidney (HEK)293 cells. Results Construction of a minimal model for ERK activation by the AT1AR Although some aspects of ERK activation by the AT1AR are understood, the mechanisms controlling the balance and distinct kinetic properties of the G protein- and β-arrestin-dependent pathways activating ERK remain largely unknown. To address this question, we have developed a deterministic dynamical model made of a system of ordinary differential equations (ODE). Previous attempts to consider all distinct biochemical species and interactions led to combinatorial complexity associated with a dramatic increase in the number of unknown parameters and, as a consequence, were impractical for dynamical simulation (Borisov et al, 2005; Hlavacek et al, 2006; Birtwistle et al, 2007). Therefore, we chose to construct a minimal model encompassing the G protein- and β-arrestin-dependent transduction mechanisms while keeping key molecules and experimentally measurable variables. To build the structure of the model, we established and iteratively refined a simplified molecular interaction network capturing the available knowledge and experimental data. We generated an initial version of the minimal model amenable to numerical simulations and optimization of the unknown parameters by training with experimental data. Importantly, we found out that with the initial network structure, parameter estimation did not lead to simulations satisfyingly fitting experimental data. This was not surprising since, as is generally the case for biological systems, the knowledge available on the signaling network was incomplete and a number of competing hypotheses existed. We took advantage of this situation by exploring a variety of model structures to minimize discrepancies between the simulated model and the experimental data. The model structure was reassessed back and forth until a satisfactory solution was reached. Importantly, the main structural predictions that arose during this process were experimentally validated (see below). The final model resulting from this iterative process is a network of molecular interactions, which can be formally described either using a diagrammatic notation such as Systems Biology Graphical Notation (SBGN) (Figure 1; Kitano et al, 2005) or equivalently by a set of reaction rules written in the Systems Biology Markup Language (SBML) or in BIOCHAM syntax (Supplementary Figure S1). Figure 1.Model for ERK activation by AT1AR. Red-colored arrow indicates the starting point of the activation process (i.e., ligand binding to the receptor). Green-colored species correspond to experimental read-outs. Targeted experimental perturbations are indicated in red. Blue-colored reactions and species correspond to new features of the model as compared with classical views in GPCR transduction. Hypothesis made during the modeling process are numbered and circled in blue (1, co-existence of two distinct phosphorylated forms of the receptor, HRP1 and HRP2; 2, reversibility of β-arrestin-dependent ERK phosphorylation; 3, β-arrestin-dependent ERK phosphorylation undergoes enzymatic amplification; 4, non-phosphorylated ligand-bound receptor still has the ability to recruit β-arrestin 2 and to induce ERK phosphorylation; and 5, differential phosphorylation of the ligand-bound receptor by GRK2/3 versus GRK5/6 leading to HRP1 and HRP2). Cell Designer has been used to represent the topology of the network in Systems Biology Graphical Notation (SBGN). The following semantic is used: state transition (); proteins (); active protein (); simple molecule (); receptor (); catalysis (); association (); dissociation (); phosphorylation (); Boolean logic gate OR (). Complexes are surrounded by a box. Source data for Figure 1 [msb201222-sup-0001-SourceData-S1.zip] Download figure Download PowerPoint For simplification purpose, only one cellular compartment (volume=1 pL) was considered. In addition, neither degradation nor synthesis of the different molecular species was taken into account, and we used mass action laws to model the dynamics of all reactions. This led to 11 kinetic reactions, depending on 26 kinetic rates (Supplementary Figure S2), and 7 conservation laws (Supplementary Figure S3). Classical G-protein coupling Signal transduction is initiated by hormone (i.e., angiotensin) binding to the receptor. At the angiotensin concentration used in our experiments (i.e., 100 nM), 99% of the receptor is ligand bound. Therefore, for simplification purpose, we aggregated the hormone binding process in a single variable called HR. The hormone-receptor complex then couples to heterotrimeric G protein, αq subunit is activated (G_a) by exchanging GDP with GTP and is subsequently released (Northup et al, 1980; Samama et al, 1993; Lefkowitz, 1998). Since this process is rapid, transient and dependent upon receptor activation, in the model we expressed the whole heterotrimeric G-protein activation/deactivation process as G being reversibly activated in G_a under the catalytic control of HR. GRK induces phosphorylation of the receptor thereby leading to the formation of phosphorylated receptor HRP1 (Lefkowitz, 1998). However, the phosphorylation only partially quenches G-protein activation (Benovic et al, 1987). Consequently, HRP1 also catalyzes G_a formation in the model. It is also well documented that 7TMRs induce G-protein activation constitutively (Samama et al, 1993). We took this process into account in the model by adding the activation of G into G_a in a parallel reaction independent of either HR or HRP1 catalysis. Desensitization of G-protein activation and recycling of the receptor Complete desensitization of G-protein activation is achieved upon β-arrestin binding to the GRK-phosphorylated receptor (Benovic et al, 1987; Lefkowitz, 1998). In the model, HRP1 interacts and forms a complex with either β-arrestin 1 (barr1) or β-arrestin 2 (barr2). Those two complexes are internalized; the receptor is then either degraded or dephosphorylated and recycled back at the plasma membrane (Lefkowitz and Whalen, 2004). As explained above, we did not consider synthesis/degradation in the model. The multiple dephosphorylation/recycling steps were shortened in one single irreversible reaction departing from each complex and providing barr1, barr2 and HR to the system. G protein-dependent signaling to ERK Once activated, Gαq protein induces a second messenger signaling cascade sequentially involving PLC activation, IP3 and DAG accumulation from PIP2, calcium release and PKC activation (Milligan and Kostenis, 2006). Since we were able to measure DAG accumulation and PKC activity, we included these variables in the model: all the reactions leading to DAG activation/synthesis are modeled as one reaction transforming PIP2 into DAG catalyzed by G_a (Figure 1). In turn, DAG catalyzes PKC activation in PKC_a. Both DAG accumulation and PKC activation have been shown to be deactivated through complex mechanisms (Newton, 1995), accordingly both reactions were made reversible in the model. Activated PKC triggers the activation of many downstream targets including Ras and the ERK MAPK signaling module: Raf, MEK, ERK (Wei et al, 2003; Luttrell and Gesty-Palmer, 2010). ERK phosphorylated through G protein-dependent mechanisms was noted GpERK in the model. Intermediate reaction was omitted in the model, and the whole process represented as one single reaction: PKC_a catalyzes GpERK formation from ERK. This reaction was made reversible to account for the activity of dual specificity ERK phosphatases (Dickinson and Keyse, 2006). β-Arrestin-dependent signaling to ERK β-Arrestin 2 contributes to ERK phosphorylation, independently of G proteins, through the formation of a multiprotein complex with the GRK-phosphorylated receptor, Raf and Mek (DeFea et al, 2000; Luttrell et al, 2001; DeWire et al, 2007). Since this mechanism is known to be differentially affected by GRK2/3 or GRK5/6 depletion (Kim et al, 2005), we made the assumption that the phosphorylated form of the receptor engaged into β-arrestin-dependent ERK phosphorylation was distinct from the desensitized form. In addition, β-arrestin 2 depletion has been reported to negatively impact β-arrestin-dependent ERK signaling while β-arrestin 1 removal leads to increased signaling by this pathway (Ahn et al, 2004b). Consistently, in the model, a distinct GRK-phosphorylated form of the receptor, HRP2, associates with β-arrestin 2 (barr2) to form the complex HRP2barr2 (Figure 1, hypothesis 1). HRP2barr2 then promotes the reversible formation of β-arrestin-dependent phosphorylated ERK (bpERK) from the cellular pool of ERK (here again Raf and MEK were omitted in the model). GpERK and bpERK were distinguished in the model since it is well documented that they exhibit distinct kinetics, subcellular localization and downstream targets (Ahn et al, 2004a; DeWire et al, 2007; Luttrell and Gesty-Palmer, 2010). During the parameter optimization process (see below), we found that the β-arrestin-dependent pathway for ERK activation needed to be negatively regulated to fit the biological data. As shown in Supplementary Figure S4, when rate constants k17 (parameter 39 controlling HRP2 dephosphorylation), k24 (parameter 46 controlling HRP2barr2 dissociation) or k25 (parameter 47 controlling bpERK dephosphorylation) are set to 0, total pERK formation remains stable after reaching its maximum. In the experimental data, however, pERK formation clearly decreases between 5 and 90 min. As a consequence, we made the reaction leading to bpERK formation reversible (Figure 1, hypothesis 2). In addition, based on the actual quantities of receptors (0.08 μmol l−1; Ahn et al, 2004a; Kim et al, 2005) of β-arrestin 2 (0.483 μmol l−1; Ahn et al, 2004b) and of phosphorylated ERK (1.265 μmol l−1; Dupuy et al, 2009) measured in HEK293 cells, ERK phosphorylation within the β-arrestin scaffold could not work with a 1:1 stoichiometry. Therefore, we hypothesized the existence of an enzymatic amplification within the β-arrestin scaffold. Using a recently validated reverse phase protein array method (Dupuy et al, 2009), we were able to experimentally test this hypothesis. We measured the molar quantities of phosphorylated MEK and of phosphorylated ERK in the whole cell as well as within the β-arrestin 2 scaffold (i.e., cells treated with a PKC inhibitor (Ro-31-8425) to disrupt GpERK) (Supplementary Figure S5). In all cases, we found substantially more phosphorylated ERK than phosphorylated MEK (5- to 36-fold, depending on the conditions). With these data in hand, we switched from the equimolar scaffold representation to enzymatic catalysis of bpERK formation (Figure 1, hypothesis 3). Since it is well documented that ERK activated by the β-arrestin-dependent mechanism accumulates within cytosolic vesicles, which also contain the receptors and β-arrestins (Ahn et al, 2004a; DeWire et al, 2007), our current data suggest that ERK leave the β-arrestin scaffold once they are phosphorylated but remain trapped in the vesicles through a mechanism yet to be identified. GRK-independent β-arrestin signaling Although GRK phosphorylation is well known to significantly enhance β-arrestins' affinity for the receptor (Lefkowitz, 1998), the main driving force for β-arrestin recruitment and activation is the agonist-induced transconformation of the receptor itself (Gurevich and Benovic, 1993; Lefkowitz and Shenoy, 2005). Moreover, DeWire et al (2007) have reported that AT1ARΔ324 with a truncated C-terminal tail is still capable of recruiting β-arrestins (albeit weakly) and inducing ERK phosphorylation in a β-arrestin-dependent manner. With that in mind, we made the assumption that β-arrestin 2 could be active even in the absence of GRK phosphorylation. We tested this hypothesis by comparing wild-type AT1AR (WT) with a mutant (13A) in which all the 13 serines and threonines present in the C-tail of the receptor were replaced by alanines (Figure 2A). When expressed in HEK293 cells at similar levels, the two receptors behaved differently. In control conditions, the 13A triggered significantly more ERK phosphorylation than the WT, which possibly reflects the lack of GRK-mediated desensitization (Figure 2B; Supplementary Figure S6). As expected, siRNA-mediated β-arrestin 2 knock-down led to a dramatic decrease in ERK phosphorylation by the WT, especially at late time points (Figure 2C; Supplementary Figure S6A and B). Importantly, using the 13A, β-arrestin 2 depletion also led to a significant inhibition of the late ERK response, strongly supporting our initial assumption that the non-phosphorylated receptor could still activate bpERK (Figure 2D; Supplementary Figure S6C). As a consequence, we added a reversible complexation reaction between HR and barr2 to form HRbarr2, which catalyzes bpERK formation (Figure 1, hypothesis 4). Figure 2.Role of the AT1AR C-tail phosphorylation in ERK activation. HEK293 cells were transfected with either WT or a mutant (13A) AT1AR. The indicated siRNAs were transfected simultaneously with the receptors. Values in graphs are expressed as percent of control (CTL) stimulated for 5 min with AngII (100 nM). (A) C-terminal sequence of the WT (Ser and Thr residues in blue) and 13A (mutated residues in red) AT1AR. (B) Time course of pERK response by WT versus 13A. (C, D) Effect of β-arrestin 2 depletion on pERK response by WT and 13A, respectively. (E, G) Effect of GRK2 depletion on pERK response by WT and 13A respectively. (F, H) Effect of GRK5 depletion on pERK response by WT and 13A, respectively. Mean±s.e.m. from at least four independent experiments with *P<0.05; **P<0.01; ***P<0.001 and NS, not significant, as compared with controls. Download figure Download PowerPoint GRK isoforms act differentially at the receptor Upon receptor activation, GRK2 and GRK3 are translocated to the plasma membrane where they form a complex with the free
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