Artigo Acesso aberto Revisado por pares

β-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling

2009; Springer Nature; Volume: 28; Issue: 18 Linguagem: Inglês

10.1038/emboj.2009.215

ISSN

1460-2075

Autores

Gaël Barthet, Gaëlle Carrat, Elizabeth Cassier, Breann L. Barker, Florence Gaven, Marion Pillot, Bérénice Framery, Lucie P. Pellissier, J Augier, Dong Soo Kang, Sylvie Claeysen, Éric Reiter, Jean‐Louis Banères, Jeffrey Benovic, Philippe Marin, Joël Bockaert, Aline Dumuis,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Article6 August 2009free access β-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling Gaël Barthet Gaël Barthet Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Gaëlle Carrat Gaëlle Carrat Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Elizabeth Cassier Elizabeth Cassier Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Breann Barker Breann Barker Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Florence Gaven Florence Gaven Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Marion Pillot Marion Pillot Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Bérénice Framery Bérénice Framery Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Lucie P Pellissier Lucie P Pellissier Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Julie Augier Julie Augier Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Dong Soo Kang Dong Soo Kang Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Sylvie Claeysen Sylvie Claeysen Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Eric Reiter Eric Reiter INRA, GMR6175 Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, Nouzilly, France University of Tours, Nouzilly, France Search for more papers by this author Jean-Louis Banères Jean-Louis Banères Institut des Biomolécules Max Mousseron, CNRS UMR5247, Montpellier, France Search for more papers by this author Jeffrey L Benovic Jeffrey L Benovic Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Philippe Marin Philippe Marin Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Joël Bockaert Corresponding Author Joël Bockaert Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Aline Dumuis Corresponding Author Aline Dumuis Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Gaël Barthet Gaël Barthet Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Gaëlle Carrat Gaëlle Carrat Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Elizabeth Cassier Elizabeth Cassier Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Breann Barker Breann Barker Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Florence Gaven Florence Gaven Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Marion Pillot Marion Pillot Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Bérénice Framery Bérénice Framery Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Lucie P Pellissier Lucie P Pellissier Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Julie Augier Julie Augier Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Dong Soo Kang Dong Soo Kang Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Sylvie Claeysen Sylvie Claeysen Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Eric Reiter Eric Reiter INRA, GMR6175 Physiologie de la Reproduction et des Comportements, Nouzilly, France CNRS, Nouzilly, France University of Tours, Nouzilly, France Search for more papers by this author Jean-Louis Banères Jean-Louis Banères Institut des Biomolécules Max Mousseron, CNRS UMR5247, Montpellier, France Search for more papers by this author Jeffrey L Benovic Jeffrey L Benovic Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Philippe Marin Philippe Marin Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Joël Bockaert Corresponding Author Joël Bockaert Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Aline Dumuis Corresponding Author Aline Dumuis Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France INSERM, Montpellier, France Search for more papers by this author Author Information Gaël Barthet1,2,‡, Gaëlle Carrat1,2,‡, Elizabeth Cassier1,2, Breann Barker3, Florence Gaven1,2, Marion Pillot1,2, Bérénice Framery1,2, Lucie P Pellissier1,2, Julie Augier1,2, Dong Soo Kang3, Sylvie Claeysen1,2, Eric Reiter4,5,6, Jean-Louis Banères7, Jeffrey L Benovic3, Philippe Marin1,2, Joël Bockaert 1,2 and Aline Dumuis 1,2 1Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS, Montpellier, France 2INSERM, Montpellier, France 3Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA 4INRA, GMR6175 Physiologie de la Reproduction et des Comportements, Nouzilly, France 5CNRS, Nouzilly, France 6University of Tours, Nouzilly, France 7Institut des Biomolécules Max Mousseron, CNRS UMR5247, Montpellier, France ‡These authors contributed equally to this work *Corresponding authors. Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier Cedex 5, F-34094, France. Tel.: +33 467 14 29 30; Fax: +33 467 54 24 32; E-mail: [email protected] or Tel.: +33 467 14 29 34; Fax: +33 467 54 24 32; E-mail: [email protected] The EMBO Journal (2009)28:2706-2718https://doi.org/10.1038/emboj.2009.215 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein-independent 5-HT4 receptor (5-HT4R)-operated Src/ERK (extracellular signal-regulated kinase) pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor' C-terminus in both human embryonic kidney (HEK)-293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser412) bound to the receptor. Phosphorylated β-arrestin1 in turn prevented activation of Src constitutively bound to 5-HT4Rs, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that β-arrestin1 phosphorylation by GRK5 regulates G protein-independent signalling. Introduction G protein-coupled receptor (GPCRs) have been first characterized for their ability to activate G proteins and to engage G protein-dependent signalling pathways. The desensitization process, which controls the duration and the intensity of these G protein-mediated signals, has been extensively characterized during the last two decades. It requires GPCR kinase (GRK)-dependent phosphorylation of GPCRs, their uncoupling from the G protein, and subsequent recruitment of β-arrestins (β-arrs). This latter event promotes receptor endocytosis, precluding further G protein activation. Recently, additional signalling pathways have been discovered in addition to the classical G protein-dependent signalling responsible for second messenger generation. For instance, activation of the extracellular signal-regulated kinase (ERK) (Shenoy et al, 2006), Src (Heuss et al, 1999; Barthet et al, 2007; Sun et al, 2007) and phospholipase D pathways (Cao et al, 1996) by ligands of certain GPCRs occurs independently of G protein activation and the generation of second messengers (see, for reviews, Heuss and Gerber, 2000; Bockaert et al, 2004a; Kim et al, 2005; Lefkowitz and Shenoy, 2005; Premont and Gainetdinov, 2007). The regulation and desensitization of G protein-independent signalling pathways are poorly characterized. In particular, a fundamental issue is to determine whether the desensitization of G protein-dependent and -independent signals are governed by common or distinct molecular mechanisms, and whether key molecules involved in the desensitization of G protein-dependent signalling such as GRKs and β-arrs are also important for G protein-independent signalling desensitization. A few reports have described a role of GRKs in the regulation of G protein-independent ERK signalling. GRK2, the major GRK involved in recruitment of β-arrs by GPCRs, is also able to inhibit G protein-independent, β-arr-dependent ERK activation by angiotensin II type 1A (AT1A), V2 vasopressin (V2) and follicle-stimulating hormone (FSH) receptors (Hunton et al, 2005; Kim et al, 2005; Ren et al, 2005; Kara et al, 2006). In contrast, GRK5/6 promote rather than inhibit G protein-independent, β-arr-mediated ERK activation by AT1A, V2, β2-adrenergic (β2-AR) and FSH receptors (Kim et al, 2005; Ren et al, 2005; Kara et al, 2006; Shenoy et al, 2006). In addition to their role in desensitization of G protein-dependent signals, both β-arr1 and β-arr2 can serve as platforms for the recruitment of specific signalling proteins. For instance, they have been identified as essential components involved in the activation of the ERK signalling pathway mediated by several GPCRs (DeWire et al, 2007). However, for certain GPCRs such as AT1A receptor, β-arr1 can act as a dominant-negative inhibitor of β-arr2-dependent, receptor-operated ERK activation (Ahn et al, 2004a, 2004b). The 5–HT4R is a GPCR, which signals through both G protein-dependent and -independent pathways. The major G protein-dependent pathway engaged by 5–HT4R is a Gs/cAMP/PKA pathway, which is desensitized by GRK2 (Barthet et al, 2005). We have recently described a G protein-independent pathway activated by this receptor (the Src/ERK pathway) whose desensitization mechanism is unknown (Barthet et al, 2007). Here, we investigated the regulation of the G protein-independent signalling of 5–HT4R by GRKs. We showed that the G protein-independent Src/ERK activation by 5–HT4R was inhibited by GRK5. This negative regulation was specific of the G protein-independent Src/ERK pathway, as GRK5 only played a marginal role in 5–HT4Rs/Gs uncoupling (Barthet et al, 2005). Moreover, we demonstrated that GRK5 inhibited 5–HT4R-mediated Src/ERK activation through direct phosphorylation of β-arr1 bound to a S/T cluster located within its C-terminal domain (C-t). Results Inhibition of the 5-HT4R-stimulated G protein-independent Src/ERK pathway by GRK5 We earlier identified GRK2 as the major GRK responsible for the desensitization of the 5–HT4R-mediated Gs pathway (Barthet et al, 2005). Here, we have searched for a GRK able to inhibit the 5-HT4R-mediated G protein-independent Src/ERK pathway without affecting the 5-HT4R-mediated Gs pathway. We focused on GRK5 because its membrane recruitment at the plasma membrane does not depend on G proteins. Co-expression of GRK5 with 5–HT4R in human embryonic kidney (HEK)-293 cells did not significantly reduce cAMP formation evoked by 5-HT (Supplementary Figure S1A). In contrast, GRK5 expression strongly attenuated phosphorylation of ERK induced by 5- and 30-min exposures to 10 μM 5-HT (Figure 1; Supplementary Figure S1B). Moreover, GRK5 expression prevented 5-HT-induced activation of Src, as assessed by its phosphorylation on Tyr416 (p-Y416-Src, Figure 1). These inhibitory effects depended on GRK5 activity as expression of a kinase-dead GRK5 mutant (K215R) did not reduce receptor-mediated phosphorylation of Src and ERK (Figure 1). In contrast, expression of GRK5 (K215R) enhanced ERK phosphorylation (+30±11%, n=6, Figure 1B). This observation likely reflects a dominant negative effect of overexpressed GRK5 (K215R) on the inhibition of the ERK pathway elicited by endogenous GRK5. Figure 1.GRK5 suppresses the 5-HT4R-mediated activation of the Src/ERK pathway. (A) HEK-293 cells transiently expressing 5-HT4Rs were co-transfected or not with GRK5 or the kinase-dead mutant GRK5 (K215R) and serum starved before 5-HT (10 μM) exposure for the indicated time. HEK-293 cells were lysed in SDS sample buffer, subjected to SDS–PAGE. ERK and Src activation were analysed by western blotting using antibodies against phospho-Thr202/Tyr204-ERK1/2 (p-ERK) and phospho-Tyr416-Src (p-Y416-Src). Total ERK1/2 and total Src were revealed on the same blot with polyclonal antibody recognizing ERK and Src independently of their phosphorylation sites (not shown). Note that total ERK and Src were not affected in all experiments. The data are representative of a series of blots performed in the same conditions. (B) P-ERK and p-Y416-Src expressed as percentage of maximal 5-HT stimulation±s.e.m., represented by densitometric quantification of western blot performed from four different experiments. *P<0.05 or **P<0.01 versus corresponding values measured in cells transfected with WT 5-HT4R alone. Download figure Download PowerPoint Role of a S/T cluster within the 5-HT4R C-terminus on GRK5-mediated inhibition of the receptor-operated ERK pathway The 5-HT4R C-t encompasses several S/T residues, which are potential GRK5 phosphorylation sites. We first examined the possible role of these residues in GRK5-mediated inhibition of ERK by generating several 5-HT4R truncated mutants (Figure 2A). GRK5 failed to inhibit ERK phosphorylation induced by 5-HT in cells expressing either the Δ329 mutant lacking the entire C-terminus or the Δ346 mutant lacking a S/T cluster (residues 347–355, Figure 2A). Deletion of the S/T cluster likewise prevented inhibition, by GRK5, of Src phosphorylation elicited by 5-HT (Figure 2B). GRK5 still inhibited phosphorylation of ERK induced by 5-HT in cells expressing either the truncated receptor lacking its PDZ ligand (ΔSCF, Figure 2A) with a putative phosphorylated site (S385) or the Δ358 mutant comprising the S/T cluster and lacking the last four scattered S/T residues, but not in cells expressing the corresponding truncated receptor in which all S/T residues of the cluster have been mutated into alanine (Δ358-Ala, Figure 2A). Figure 2.A Serine/Threonine cluster within the 5-HT4R C-t is essential for inhibition, by GRK5, of receptor operated ERK signalling. (A) Topology of 5-HT4R C-t domain. The successive points of truncation are illustrated represented on the left with an arrow. HEK-293 cells were transiently transfected with WT 5-HT4R or the corresponding truncated or mutated receptors (Δ329, Δ346, Δ358, Δ358Ala) in combination with or without GRK5. Identical expression levels of the transfected constructs were controlled by ELISA. Cells expressing the indicated receptors were treated or not with 10 μM 5-HT for 5 min. ERK activation was analysed by immunoblotting with p-ERK1/2 antibody. (B) HEK-293 cells were either transfected with a plasmid encoding Myc-tagged WT 5-HT4R or Myc-tagged Δ346 alone or co-transfected with GRK5. They were challenged with 10 μM 5-HT for 5 min. Total lysates were analysed by sequential immunoblotting, using p-Y416-Src, p-ERK1/2 antibodies. Download figure Download PowerPoint Collectively, these results indicate that the S/T cluster (residues 347–355), which was previously shown to bind to β-arr (Barthet et al, 2005), constitutes a key molecular determinant implicated in the regulation of ERK activation by GRK5. Further supporting its essential role in the regulation by GRK5 of the ERK pathway, progressive deletion of the S/T residues within the cluster concomitantly reduced the GRK5-mediated inhibition of ERK phosphorylation (Supplementary Figure S2). We then analysed agonist-dependent phosphorylation sites on the receptor by tandem mass spectrometry (MS/MS). HEK-293 cells expressing HA-tagged 5-HT4R and GRK5 constructs were treated or not with 5-HT for 10 min. Receptors were then immunoprecipitated on anti-HA agarose beads. Immunoprecipitated receptors were resolved by SDS–PAGE and digested in-gel with trypsin. In control cells (not treated with 5-HT), analysis of duplicate samples revealed the presence of a non-phosphorylated form of the peptide comprising the S/T cluster (R336-R359). In contrast, several versions of the peptide were identified in cells exposed to 5-HT: the non-phosphorylated one and three phosphorylated forms with one, two and three phosphates attached, respectively. Loss of phosphate on fragmentation indicated phosphorylation of S354 in the mono-phosphorylated peptide (Figure 3B) and the presence of an additional phosphorylated residue within the S347TTT350 motif in the peptide with two phosphates attached (Figure 3C). The tri-phosphorylated peptide incorporated an additional phosphate within the S347TTT350 motif (not illustrated). These results indicated sequential phosphorylation of the peptide, first on S354 and then in the S347TTT350 motif. Figure 3.Analysis of 5-HT-dependent phosphorylation of 5-HT4R by tandem mass spectrometry. HEK-293 cells co-transfected with HA-tagged 5-HT4R and GRK5 constructs were treated with 5-HT (10 μM, 10 min). Immunoprecipitated receptors were digested with trypsin and peptides were analysed by nano-LC FT MS/MS. MS/MS spectra resulting from higher energy collisional dissociation (HCD) fragmentation of the non-phosphosphorylated, monophosphorylated and bi-phosphorylated versions of the R336-R359 peptide are depicted in (A), (B) and (C) respectively. The three peptides have respective mascot scores of 66, 65 and 59. (C) The bracket indicates that one residue of the S347TTT350 motif is phosphorylated. Its exact position could not be determined by MS/MS. Download figure Download PowerPoint Physical association of GRK5 with 5-HT4Rs, a necessary step in GRK5-mediated inhibition of 5-HT4R-operated ERK signal We next determined whether GRK5 physically interacted with 5-HT4R. GRK5/5-HT4R interaction was first investigated in an in vitro binding assay using purified GRK5 and recombinant S-tagged receptors immobilized on a S-protein agarose column (Baneres et al, 2005). 5-HT induced association of GRK5 with 5-HT4R, as assessed by the lack of GRK5 detection in the flow-through fraction when 5-HT was present (FT, Figure 4A). Moreover, a greater amount of GRK5 was co-eluted with the receptor compared with sample not treated with 5-HT (E, Figure 4A). In contrast, leukotriene B4 failed to induce association of GRK5 with immobilized, recombinant BLT1 receptors (Figure 4A), indicating that GRK5 does not interact with every GPCR on activation by its cognate agonist. Figure 4.A direct interaction between GRK5 and 5-HT4R is necessary for ERK pathway regulation. (A) Recombinant purified His-tag 5-HT4R obtained as described earlier (Baneres et al, 2005) were incubated with purified full-length GRK5 (Cell Signalling Technology) in the presence or absence of 5-HT (10 μM). The western blot analysis with the antibody against GRK5 or anti-His-tag (receptor) showed the fractions eluted from the S-agarose column. Fraction FT: in flow-through fractions the unbound proteins were detected. Fraction E: the receptor immobilized on the column and its tightly associated proteins were recovered in this fraction E (see Materials and methods). His-Tag-BLT1, which did not interact with GRK5, is used as control (right part). − and + indicate the absence or the presence of the agonists, 5-HT for 5-HT4R at 10 μM and LTB4 at 10 μM for BLT1R. The control has been carried out only in the presence of the agonist LTB4. (B) HEK-293 cells were either transfected with a plasmid encoding Myc-tagged WT 5-HT4R or different Myc-tagged mutants, co-transfected with GRK5. DDE330−332 indicated by grey circles in Figure 2A, represent the putative binding site of GRK5. Cells co-expressing the indicated receptors and GRK5 were challenged with 10 μM 5-HT for 5 min. Receptors were immunoprecipitated using polyclonal-anti-Myc antibody. Co-precipitated GRK5 was analysed by western blotting by using the antibody against GRK5 a gift from Dr RJ Lefkowitz (Duke University Medical Center, Durham, NC). Immunoprecipitated proteins were analysed by western blotting using the monoclonal anti-Myc antibody. On the left part, inputs represent 5% of the total protein amount used in immunoprecipitation. Quantification of GRK5 bound to the receptor was performed by densitometry using the NIH Image Sofware. Data are means±s.e.m. of results obtained in four independent experiments. **P<0.01 versus corresponding values measured in experiments performed from cells expressing WT 5-HT4Rs. (C) The 5-HT4R C-t peptide (Inh-Pep) reduced the association of GRK5 with the receptor. The sequence (330 to 346) located on 5-HT4R-C-t (see Figure 2A) (DDERYKRPPILGQTVPC) fused to the transduction domain of TAT protein (YGRKKRRQRRR) was used as inhibitor peptide. HEK-293 cells co-transfected with Myc-tagged-WT 5-HT4R and GRK5 were treated with either 10 μM of TAT-Inh-Pep or TAT-Ctrl (Ctrl-Pep) (the C-t residues of 5-HT2c-C-t VNPSSVVSERISSV fused with TAT protein (YGRKKRRQRRR) for 1 h with 10 μM 5-HT for 5 min. Receptors were immunoprecipitated using the polyclonal anti-Myc antibody. Immunoprecipitated receptor was detected with the monoclonal anti-Myc antibody. Co-precipitated GRK5 was analysed by western blotting using the anti-GRK5 antibody. Intensities of the bands in immunoblots were measured by densitometry analysis using Image J software. Data are means±s.e.m. of results obtained in four independent experiments. **P<0.01 versus corresponding values obtained from cells treated with the control peptide (Ctrl-Pep). (D) The 5-HT4R C-t peptide (Inh-Pep) increased the 5-HT4R induced ERK signalling. HEK-293 cells were pre-incubated for 1 h with either 10 μM TAT-peptides (Inh-Pep) or a control peptide (see C) before the 5-min challenge (10 μM 5-HT). Cells were then lysed in SDS sample buffer and subjected to SDS–PAGE. ERK activation was analysed by western blot using the polyclonal antibody against phospho-Thr202/Tyr204-ERK1/2. Illustrated data are representative of four blots performed in different sets of cultured cells. The histogram is the means± of densitometric quantification of four western blots using Image J software. Download figure Download PowerPoint These results indicate that 5-HT promotes direct interaction of GRK5 with 5-HT4Rs. We then explored the molecular determinants involved in the interaction of GRK5 with 5-HT4Rs in HEK-293 cells by co-immunoprecipitation. GRK5 co-immunoprecipitated with 5-HT4R and the amount of GRK5 was enhanced on 5-HT exposure (Figure 4B). GRK5 did not co-immunoprecipitate with the Δ329 truncation mutant lacking the entire C-terminus in the presence of 5-HT (Supplementary Figure S3), whereas the Δ346 mutant that lacks the S/T (347–355) cluster phosphorylated by GRK5 (Supplementary Figure S2) still interacted with GRK5 (Figure 4B). Thus, GRK5 can bind to 5-HT4Rs without the presence of the S/T cluster substrates. However, note that 5-HT did not stimulate GRK5 binding to the Δ346 mutant. An earlier study has shown that acidic residues located upstream GRK phosphorylation sites participate in the recruitment of GRKs by several GPCRs (Berrada et al, 2000). A series of acidic residues located in the juxtamembrane region of 5-HT4R C-t (D330DE332) was likewise critical for GRK5 recruitment by 5-HT4Rs. Indeed, mutation of these residues into alanine in WT and Δ346 receptors strongly reduced GRK5 recruitment (Figure 4B). Collectively, these results identified the proximal region of the 5-HT4R C-t (amino-acids 330–346) as the GRK5 binding sequence and the D330DE332 motif as the most important determinant. To further explore the role of GRK5/5-HT4R interaction, we synthesized an interfering peptide (Inh-Pep for inhibitor peptide) comprising the GRK5 binding site (residues 330–345, Figure 2A). The peptide was N-terminally fused to the transduction domain of the TAT protein from HIV Type 1 to allow its intracellular delivery (Aarts et al, 2002). Treatment of HEK-293 cells co-transfected with 5-HT4R and GRK5 with Inh-Pep (10 μM, 1 h) reduced 5-HT4R/GRK5 co-immunoprecipitation (Figure 4C). In non-GRK5 transfected cells, Inh-Pep increased by 50% the 5-HT4R-stimulated ERK phosphorylation compared with the 5-HT4R-stimulated ERK phosphorylation measured in cells treated with a control TAT-derived peptide (including the 5-HT2C receptor C-t) (Figure 4D). This effect likely reflected competition of Inh-Pep with endogenously GRK5 and reversal of its inhibitory effect. β-arrestin1 is essential for GRK5-mediated inhibition of the 5-HT4R-operated Src/ERK pathway Our data indicated that association of GRK5 with the proximal region of 5-HT4R C-t (sequence 330–346) was required but was not sufficient. Phosphorylation of S/T residues within the S/T cluster (residues 347–355) was also essential. As this cluster is known to bind to β-arrs (Barthet et al, 2005), we explored a possible role of β-arrs in the GRK5 effect. WT receptor interacted with both endogenous β-arr1 and β-arr2 (Figure 5A). This association was increased by 5-HT (Figure 5A). The Δ346 mutant slightly interacted with endogenous β-arr2, but not with β-arr1. Furthermore, the association of β-arr2 with the mutant was not increased on 5-HT exposure (Figure 5A; Supplementary Figure S4). Transfection of HEK-293 cells with siRNA directed against β-arr1, which strongly decreased β-arr1 expression compared with control siRNA-transfected cells, markedly impaired GRK5-mediated inhibition of the 5–HT4R-operated activation of the ERK pathway (Figure 5B and C). To further confirm that β-arr1 was essential for GRK5-mediated inhibition of ERK signalling, we transfected mouse embryonic fibroblasts (MEFs) lacking β-arr1 and β-arr2 (β-arr1/2−/−) with 5–HT4R. As these cells endogenously express various 5-HTRs, 5-HT4Rs were stimulated by BIMU8 (a selective 5–HT4R agonist) instead of 5-HT. Overexpression of GRK5 did not inhibit the 5-HT4R-mediated activation of the ERK pathway in β-arr1/2−/− MEF cells (Figure 6). This inhibition was rescued by co-transfecting cells with β-arr1 and to a much lesser extent with β-arr2. Figure 5.Role of β-arrestins in the inhibition, by GRK5, of the 5-HT4R-activated Src/ERK pathway (A) Activated WT 5-HT4R and Δ346 mutant differentially interact with endogenous β-arr1. HEK-293 cells co-transfected with either Myc-tagged Δ346 or Myc-tagged WT 5-HT4R and GRK5 were challenged with 10 μM 5-HT for 5 min. Receptors were immunoprecipitated using the polyclonal anti-Myc antibody. Co-precipitated β-arr 1/2 were analysed by western blotting by using the anti-β-arr A1CT antibody, which equally recognizes β-arr1 and 2, a gift from Dr RJ Lefkowitz (Duke University Medical Center, Durham, NC). (B) Down-regulation of β-arr1 expression, by siRNA, enhances inhibition by GRK5 of the 5-HT4R-activated Src/ERK pathway. HEK-293 cells expressing Myc-tagged-WT were transfected with either control or β-arr1-specific siRNAs. At 72 h after transfection, cells were stimulated 5 or 30 min before lysis. P-ERK and β-arr contents were analysed by sequential western blotting with p-ERK, and β-arr antibodies. (C) Quantification of ERK1/2 phosphorylation (top) and β-arr1 (bottom) was performed by densitometry using the Image J software. Data are means±s.e.m. of results obtained in four independent experiments. **P<0.01 versus corresponding values measured in cells transfected with control siRNA. Download figure Download PowerPoint Figure 6.GRK5 fails to inhibit 5-HT4R-mediated Src/ERK pathway in MEF β-arr1−/

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