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AT1R-CB 1 R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II

2011; Springer Nature; Volume: 30; Issue: 12 Linguagem: Inglês

10.1038/emboj.2011.139

1460-2075

Raphaël Rozenfeld, Achla Gupta, Khatuna Gagnidze, Maribel P. Lim, Ivone Gomes, Dinah Lee-Ramos, Natalia Nieto, Lakshmi A. Devi,

Hormonal Regulation and Hypertension

Article3 May 2011free access AT1R–CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II Raphael Rozenfeld Raphael Rozenfeld Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Alcoholic Liver Disease Research Center, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Achla Gupta Achla Gupta Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Khatuna Gagnidze Khatuna Gagnidze Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Maribel P Lim Maribel P Lim Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Ivone Gomes Ivone Gomes Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Dinah Lee-Ramos Dinah Lee-Ramos Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Natalia Nieto Natalia Nieto Department of Medicine, New York Mount Sinai School of Medicine, New York, NY, USA Alcoholic Liver Disease Research Center, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Lakshmi A Devi Corresponding Author Lakshmi A Devi Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Raphael Rozenfeld Raphael Rozenfeld Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Alcoholic Liver Disease Research Center, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Achla Gupta Achla Gupta Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Khatuna Gagnidze Khatuna Gagnidze Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Maribel P Lim Maribel P Lim Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Ivone Gomes Ivone Gomes Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Dinah Lee-Ramos Dinah Lee-Ramos Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Natalia Nieto Natalia Nieto Department of Medicine, New York Mount Sinai School of Medicine, New York, NY, USA Alcoholic Liver Disease Research Center, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Lakshmi A Devi Corresponding Author Lakshmi A Devi Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Author Information Raphael Rozenfeld1,3, Achla Gupta1, Khatuna Gagnidze1, Maribel P Lim1, Ivone Gomes1, Dinah Lee-Ramos1, Natalia Nieto2,3 and Lakshmi A Devi 1 1Department of Pharmacology and Systems Therapeutics, New York Mount Sinai School of Medicine, New York, NY, USA 2Department of Medicine, New York Mount Sinai School of Medicine, New York, NY, USA 3Alcoholic Liver Disease Research Center, New York Mount Sinai School of Medicine, New York, NY, USA *Corresponding author. Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, 19-84 Annenberg Building, One Gustave L. Levy Place, New York, NY 10029, USA. Tel.: +1 212 241 8345; Fax: +1 212 996 7214; E-mail: [email protected] The EMBO Journal (2011)30:2350-2363https://doi.org/10.1038/emboj.2011.139 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 The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB1R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB1R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB1R is upregulated. We found a significant upregulation of AT1R–CB1R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB1R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology. Introduction The angiotensin II (Ang II) type 1 receptor (AT1R) is a G protein-coupled receptor (GPCR) that transduces the main physiological actions of the renin-angiotensin system in target cells. The major signalling events following agonist binding to this receptor are activation of phospholipase C via a Gαq protein, mobilization of calcium from intracellular stores, and activation of other signalling pathways such as the MAP kinase pathway that participates in the hypertrophic actions of Ang II (Clauser et al, 1996). AT1R is primarily involved in the control of blood pressure as demonstrated in numerous animal models (Ito et al, 1995; Sugaya et al, 1995; Le et al, 2003). Using functional complementation experiments, AT1R was among the first class A GPCR to be proposed to function as a dimer (Monnot et al, 1996), and formation of AT1R heteromers was later found to be involved in pathology (Barki-Harrington, 2004). For instance, the contribution of AT1R to specific forms of hypertension has been shown to be regulated by interactions with B2 bradykinin receptor (AbdAlla et al, 2001), and recently, physical interactions with the apelin receptor was proposed to regulate the atherosclerotic effect of Ang II (Chun et al, 2008), suggesting that heteromerization of AT1R with other GPCRs contributes to the pathophysiological effects of Ang II. In addition to its role in blood pressure regulation, AT1R hypertrophic properties contribute to the development of fibrosis in a number of organs. However, the molecular or cellular events that cause AT1R to become profibrogenic remain elusive. Type I cannabinoid receptor (CB1R) is widely expressed in the brain and essentially absent in peripheral tissues under normal conditions. The development of an antibody specifically recognizing the dimeric CB1R demonstrated that this receptor exists as homodimers (Wager-Miller et al, 2002). In addition, CB1R has been shown to form heteromers with a number of other GPCRs, leading to an alteration in receptor coupling as in the case of CB1R–D2 dopamine receptor (Kearn et al, 2005), trafficking, as in the case of CB1R–orexin-1 receptor (Ellis et al, 2006), and signalling, as in the case of CB1R–A2aR (Carriba et al, 2007). CB1R is upregulated in some peripheral tissues during chronic diseases, such as liver fibrosis. CB1R exhibits marginal expression in the normal liver but a robust expression in fibrotic liver, predominantly in activated hepatic stellate cells (HSCs) (Teixeira-Clerc et al, 2006); these cells are primarily responsible for the fibrogenic response in the liver (Friedman, 2008). HSCs also express AT1R (Pereira et al, 2009), and both AT1R and CB1R antagonists exhibit anti-fibrotic properties (Teixeira-Clerc et al, 2006; Schuppan and Afdhal, 2008). Here, we propose that enhanced CB1R expression in activated HSCs could affect AT1R properties and contribute to the profibrogenic effect of Ang II. Given the implication of heteromerization in the pathophysiological function of both CB1R and AT1R, it was of interest to determine whether such a AT1R–CB1R heteromer exists and could be selectively targeted pharmacologically, or if cross-talk between AT1R and CB1R could result from heteromerization-independent functional interactions, as previously reported for other Gαq- and Gαi-coupled receptor pairs (Rives et al, 2009). We first probed the interaction between AT1R and CB1R in recombinant systems using biophysical and biochemical methods and found that the two receptors associate. This association leads to changes in coupling and enhanced signalling by AT1R that could be blocked by heteromer-selective antibodies. We also found that the AT1R-mediated signalling is controlled by CB1R. For example, basal endocannabinoid tone enhances Ang II-mediated signalling and CB1R antagonists block AT1R-mediated signalling. We then examined the consequences of AT1R–CB1R interaction on Ang II-mediated profibrogenic responses in activated HSCs. We show that AT1R interacts with CB1R to form heteromers in these cells, and this facilitates the profibrogenic effect of Ang II. These results provide evidence for the contribution of heteromer-directed signal specificity in pathology. Results Ang II-mediated ERK phosphorylation is altered in the presence of CB1R We first examined the effect of CB1R coexpression on AT1R signalling. We took advantage of Neuro2A cells, a neuroblastoma cell line that contains endogenous CB1R to examine functional interactions of CB1R with AT1R. For this, we generated stable cell lines expressing an N-terminally Flag-tagged AT1R (Neuro2A-AT1R), and explored the modulation of AT1R function by CB1R. Stimulation with Ang II led to a rapid, robust, and dose-dependent increase in pERK levels (Figure 1A; Supplementary Figure S1A). This was blocked by pretreatment with the specific AT1R antagonist Losartan, and was dependent on AT1R since wild-type (non-transfected) Neuro2A cells did not respond to Ang II (Supplementary Figure S1A). We examined if the presence of CB1R contributed to the response of AT1R to Ang II stimulation, by directly altering CB1R expression. RNAi-mediated CB1R downregulation (see Figure 1B) led to a dramatic decrease (by ∼50%) in Ang II-mediated signalling (Figure 1A; Supplementary Figure S1A). Dose response experiments indicated that CB1R affects AT1R signalling by increasing the efficacy of Ang II (Figure 1A). Figure 1.CB1R modulates AT1R signalling. (A) Neuro2A-AT1R cells transfected or not with a siRNA to CB1R were stimulated with increasing concentrations of Ang II for 3 min. Data represent mean±s.e.m. (n=3–5). ***P<0.001. (B) Representative western blot analysis of Neuro2A-AT1R cells transfected with control or CB1R-targeting siRNA and probed for the levels of CB1R (∼70% decrease with CB1R siRNA), AT1R (no change) and calnexin as a loading control. Mean±s.e.m. densitometry from three independent transfections are indicated below the western blot. ***P<0.001. (C) Neuro2A-AT1R cells starved for 4 h, were stimulated with increasing concentrations of Ang II for 3 min, in the presence of the CB1R antagonist (SR141716; 1 μM) or in the presence of a non-signalling dose of the CB1R agonist (Hu210; 0.1 nM). Data represent mean±s.e.m. (n=3–5). **P<0.01; ***P<0.001. (D) Phospho-ERK levels after Ang II stimulation were examined in Neuro2A-AT1R cells, after treatment with the diacylglycerol lipase inhibitor THL (1 μM; 2 h pretreatment) alone or together with Hu210 (0.1 nM); or, (E) with increasing concentrations of 2-AG. Data are expressed as the mean±s.e.m. (n=3–4). **P<0.01; ***P 70% Ang II response (Figure 1C) while the CB1R agonist Hu210 potentiated this response (Figure 1C). Since CB1R antagonist blocked AT1R signalling, we hypothesized that in the absence of exogenous CB1R ligand, CB1R-mediated increase in Ang II efficacy could be the result of activation of CB1R by endocannabinoids. We tested this possibility by inhibiting the production of the endocannabinoid 2-arachidonoyl glycerol (2-AG). Blocking the enzyme, diacylglycerol lipase (DAGL), responsible for 2-AG production, with the DAGL inhibitor THL, led to a complete blockade of Ang II response (Figure 1D). We also examined if the addition of exogenous CB1R agonists (Hu210 or 2-AG) could reverse the effect of THL treatment. In the presence of THL, addition of Hu210 or 2-AG was able to restore Ang II-mediated signalling (Figure 1E). The effect of cannabinoid ligands on Ang II-mediated ERK phosphorylation was not observed upon RNAi-mediated downregulation of CB1R (Supplementary Figure S1B). The potentiating effect of CB1R activity on Ang II-mediated signalling was not restricted to ERK signalling, since other signalling events such as Ang II-induced p38 and JNK phosphorylation were also attenuated by downregulating CB1R (Supplementary Figure S2A). Blockade of Ang II-mediated ERK phosphorylation by SR141716 or by THL could also be seen in HEK293 cells coexpressing AT1R and CB1R, but not in cells expressing AT1R alone (Supplementary Figure S2B). Together, these data indicate that AT1R signalling is controlled by CB1R activity. In the absence of exogenous cannabinoid ligands, endocannabinoid-mediated basal CB1R activation is sufficient to allow and potentiate AT1R signalling. AT1R physically interacts with CB1R Using a variety of approaches, we examined if the functional interaction between CB1R and AT1R was the consequence of physical interaction (heteromerization) between these two receptors. First, we carried out co-immunoprecipitation experiments and detected interaction between CB1R and AT1R only in cells coexpressing the two receptors (Figure 2A). This interaction was also supported by colocalization of Flag–AT1R with CB1R in Neuro2A-AT1R cells (Figure 2B). Since individually expressed AT1R is at the plasma membrane and CB1R is in intracellular compartments (Pignatari et al, 2006; Rozenfeld and Devi, 2008), we examined changes in CB1R and AT1R localization upon coexpression of the receptors in HEK293 cells (Supplementary Figure S2C). We find that when coexpressed with AT1R, CB1R was found primarily at the plasma membrane colocalized with AT1R (Supplementary Figure S2D), supporting an association between the two receptors since such a change in receptor localization has been previously reported to be due to receptor heteromerization (Ellis et al, 2006). Figure 2.Interaction between AT1R and CB1R. (A) Association of AT1R and CB1R in Neuro2A-AT1R. Lysates from Neuro2A and Neuro2A-AT1R were subjected to immunoprecipitation using a protein A agarose-coupled anti-CB1R antibody (1 μg), and to western blotting analysis with an anti-AT1R antibody (1:200). AT1R is detected in the CB1R immunoprecipitate from Neuro2A-AT1R. (B) Immunofluorescence and confocal microscopy analysis of Neuro2A cells expressing endogenous CB1R and of Neuro2A cells stably expressing Flag–AT1R. Cells grown on coverslips were fixed with 4% PFA, permeabilized in 0.1% Triton X-100, and stained with primary rabbit polyclonal anti-CB1R (1:500) and mouse monoclonal M2 anti-Flag (1:1000) antibodies. After fluorescent secondary antibody staining, the coverslips were mounted with mowiol. Slides were examined with a Leica SP5 confocal microscope. (C) Detection of AT1R–CB1R heteromers by BRET in living HEK293 cells. BRET experiments were carried out using C-terminally Renilla luciferase-tagged CB1R, and eGFP-tagged AT1R (∼400–500 fmol receptor/mg protein). BRET ratio was measured in cells expressing the indicated constructs. To assess the specificity of interaction, BRET ratios were measured in cells coexpressing increasing concentrations of untagged AT1R, in cells coexpressing untagged endothelin converting enzyme-2, or in cells coexpressing MOR–Luc with AT1R–eGFP. In addition, BRET saturation curve was generated (insert). HEK-293 cells were co-transfected with a constant DNA concentration of CB1R–Rluc and increasing DNA concentrations of AT1R–eGFP. Curves were fitted using a non-linear regression equation assuming a single binding site (GraphPad Prism). Results are mean values±s.e.m. (n=3 experiments). ***P<0.001; NS, non-significant, versus CB1R–Luc/AT1R–eGFP. (D) Detection of AT1R–CB1R heteromers with heteromer-selective monoclonal antibodies. Receptor abundance was determined in Neuro2A, Neuro2A-AT1R, and Neuro2A-AT1R cells where CB1R was downregulated by RNAi (Neuro2A-AT1R siCB1R) with a monoclonal antibody to AT1R–CB1R, or polyclonal antibodies to AT1R or CB1R by ELISA. Results are mean values±s.e.m. (n=3 experiments). ***P<0.001; NS, non-significant, versus Neuro2A-AT1R. (E) Inhibition of AT1R–CB1R signalling by the AT1R–CB1R heteromer antibody. Neuro2A-AT1R and Neuro2A-AT1R cells where CB1R was downregulated by RNAi (Neuro2A-AT1R/siCB1R) were incubated with increasing concentrations of the monoclonal anti-AT1R–CB1R antibody (hydridoma supernatant, +, 1:20 v/v; ++, 1:10 v/v; +++, 1:5 v/v; ++++, 2:5 v/v) for 30 min, and then were stimulated with 10 nM Ang II for 3 min. Cell lysates and media were subjected to western blotting analysis using antibodies to pERK and ERK (1:1000) (lysate) and anti-mouse IgG (media). Imaging and quantification were carried out using the Odyssey Imaging system (Li-Core Biosciences). Results are mean values±s.e.m. (n=4 experiments). ***P<0.001; NS, non-significant, versus the corresponding Ang II treatment. (F) [35S]GTPγS-binding assay. Membranes from Neuro2A-AT1R cells were treated with increasing concentrations of the AT1R agonist Ang II, in the absence or presence of the CB1R antagonist PF514273 (1 μM). [35S]GTPγS binding was measured as described in 'Materials and methods'. Results are mean values±s.e.m. (n=3 experiments). ***P<0.001. (G) Reciprocal regulation of CB1R signalling by AT1R. Neuro2A or Neuro2A-AT1R cells were incubated in the presence of increasing concentrations of Hu210 for 5 min, in the absence or presence or 0.01 nM Ang II. Data represent mean±s.e.m. (n=3). ***P<0.001. Download figure Download PowerPoint We then used bioluminescence resonance energy transfer (BRET) technology that allows the detection of energy transfer between one receptor bearing the BRET donor (Renilla luciferase) and the second receptor bearing the acceptor (green fluorescent protein) when the two receptors are in close proximity ( 80%) only in cells coexpressing CB1R and AT1R (Figure 3D and F), but not in cells expressing AT1R alone, that is, upon downregulation of CB1R expression (Figure 3E and F). These results are consistent with the notion that in the presence of CB1R, AT1R couples to Gαi for signalling to the ERK pathway. We assessed the specificity of this switch in coupling of AT1R by examining the sensitivity to PTX of Ang II-mediated ERK phosphorylation, when AT1R was coexpressed with other Gαi-coupled receptors, namely MOR or DOR. We found that PTX prevents Ang II-mediated ERK phosphorylation, only when AT1R is coexpressed with CB1R, but not when expressed alone or with the other Gαi-coupled receptors (Supplementary Figure S4E), supporting the specificity of the switch in G protein coupling to the AT1R–CB1R heteromer. Figure 3.Switch in AT1R G protein coupling to the ERK pathway within the AT1R–CB1R heteromer. Neuro2A-AT1R cells were starved for 4 h and the levels of pERK were measured after various treatments (see below), after 3 min stimulation with 10 nM Ang II. (A) Phospho-ERK levels after Ang II stimulation were examined in Neuro2A-AT1R cells, after transfection with a Gαq dominant-negative construct (DN Gαq, see insert). (B) Phospho-ERK levels after Ang II stimulation were examined in Neuro2A-AT1R cells transfected with a siRNA to CB1R, after transfection with or without a Gαq dominant-negative construct. (C) Data are expressed as the mean±s.e.m. (n=4 independent experiments). *P<0.05; ***P<0.001. (D) Phospho-ERK levels after Ang II stimulation were examined in Neuro2A-AT1R cells after incubation with pertussis toxin (PTX; 15 ng/ml for 16 h). (E) Phospho-ERK levels after Ang II stimulation were examined in Neuro2A-AT1R cells transfected with a siRNA to CB1R, after incubation with PTX. (F) Data are expressed as the mean±s.e.m. (n=4 independent experiments). *P<0.05; ***P<0.001. Download figure Download PowerPoint We then examined if AT1R coupling to Gαi led to inhibition of cAMP production by Ang II treatment. In Neuro2A-AT1R cells, Ang II stimulation led to a dose-dependent inhibition of cAMP production, supporting a coupling to Gαi. This effect was blocked by co-treatment with the CB1R antagonist PF514273 and was markedly decreased upon RNAi-mediated downregulation of CB1R (Figure 4A and C). This regulation of cAMP production by heteromerization was also reciprocal, since the presence of unstimulated AT1R impaired the inhibition of cAMP production induced by the CB1R agonist Hu210 (Figure 4B and C). These experiments confirm that in the context of the AT1R–CB1R heteromer, AT1R couples to Gαi, and that the basal activity of CB1R is required for this pathway. Figure 4.AT1R couples to Gαi within the AT1R–CB1R heteromer. (A) Neuro2A-AT1R cells transfected or not with a siRNA to CB1R (siCB1R) in 24-well plates were incubated with increasing concentrations of Ang II in the absence or presence of the CB1R antagonist PF514273 (1 μM) or (B) Neuro2A and Neuro2A-AT1R cells in 24-well plates were incubated with increasing concentrations of Hu210 at 37°C for 15 min in the cAMP treatment buffer (0.5 mM isobutylmethylxanthine and 10 μM forskolin in Krebs-Ringer-HEPES buffer). After terminating the reaction by heating at 95°C, cAMP concentrations were determined as described in 'Materials and methods'. Data are expressed as the mean±s.e.m.