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. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Hypertension. 2011 Jun 13;58(2):176–181. doi: 10.1161/HYPERTENSIONAHA.111.173344

Angiotensin-(1-7) Induces Mas Receptor Internalization

Mariela M Gironacci 1, Hugo P Adamo 1, Gerardo Corradi 1, Robson A Santos 1, Pablo Ortiz 1, Oscar A Carretero 1
PMCID: PMC3141282  NIHMSID: NIHMS305555  PMID: 21670420

Abstract

Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor (R) associated with cardiac, renal and cerebral protective responses. Physiological evidence suggests that Mas R undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating R activity is unknown. We investigated the hypothesis that Mas R desensitizes and internalizes upon stimulation with Ang-(1-7). For this purpose, we generated a chimera between the Mas R and the fluorescent protein YFP (MasR-YFP). MasR-YFP transfected HEK 293T cells were incubated with Ang-(1-7) and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 min. Following the time course of [125I]Ang-(1-7) endocytosis we observed that half of MasR-YFP underwent endocytosis after 10 min and this was blocked by a Mas R antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1 and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin-1 suggesting that at some point MasR-YFP traverses caveolin-1 positive compartments. In conclusion, Mas R undergoes endocytosis upon stimulation with Ang-(1-7) and this event may explain the desensitization of Mas R responsiveness. In this way, Mas R activity and density may be tightly controlled by the cell.

Keywords: receptor internalization, desensitization, angiotensin-(1-7), Mas receptor, trafficking

Introduction

The renin angiotensin system (RAS) consists of 2 distinct and counterregulatory axes. The classic angiotensin converting enzyme (ACE)/angiotensin (Ang) II/AT1 receptor (R) axis is responsible for the vasoconstrictive, proliferative, hypertensive, and fibrotic actions of the RAS. Its hyperactivity is associated with hypertension and cardiovascular diseases such as cardiac hypertrophy, heart failure, stroke, coronary artery disease, and end-stage renal disease. This axis is the primary target for the antihypertensive therapy.1 The ACE2/Ang-(1-7)/Mas R axis constitutes an alternative axis that represents an intrinsic mechanism to induce vasoprotective actions by counterregulating the ACE/AngII/AT1R axis, thus inducing many beneficial effects in cardiovascular diseases. This vasoprotective axis of the RAS could be targeted for novel therapeutics strategies.1,2

Ang-(1-7) is the endogenous ligand for the G protein-coupled receptor (GPCR) Mas.3 Prolonged stimulation of Mas R with Ang-(1-7) or with high concentrations of the ligand caused an attenuation of R responsiveness,4-6 suggesting receptor desensitization. Receptor desensitization represents an important physiological “feedback” mechanism that protects against both acute and chronic R overstimulation and is the consequence of a combination of different mechanisms.7 These mechanisms include the uncoupling of the R from heterotrimeric G proteins in response to R phosphorylation followed by the internalization of cell surface receptors to intracellular membranous compartments. Once internalized, the R may be recycled back to the cell surface in a resensitized state competent for signaling, or may be sorted to lysosomes or proteasome for degradation, a process important for signal termination.7-9 Thus, R trafficking has critical function in signal termination and propagation as well as receptor resensitization. The rates of GPCR internalization, recycling and lysosomal sorting differ widely among receptors, suggesting that different mechanisms control trafficking of distinct R.8 Thus, the spatial and temporal control of GPCRs determines the specificity of receptor-mediated signal transduction among the distinct downstream effectors and the ultimate cellular response.

The underlying molecular mechanism of Mas R desensitization is unknown. In this study we investigated the early fate of Mas R following agonist exposure. To better image receptor trafficking we generated a chimera between the Mas R and the fluorescent protein YFP (MasR-YFP).

Methods

Materials

Fetal bovine serum, penicilin-streptomycin, Lipofectamine 2000, goat anti-mouse antibody coupled to Alexa 594 and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, CA, USA). Bovine seroalbumin (BSA), paraformaldehyde, phosphate buffered saline (PBS) and the protease inhibitors cocktail were from Sigma Chemical Co. (St. Louis, MO, USA). Mouse anti-GFP monoclonal antibody was from Clontech. Mouse anti-AP50, anti Rab5, anti-caveolin-1 (Cav-1) or anti-early endosome antigen 1 (EEA1) antibodies were purchased from BD Biosciences. [3H]arachidonic acid was from Perkin Elmer, Boston, MA. Ang-(1-7) and [D-Ala7]-Ang-(1-7) were synthesized in our laboratory by the Merrifield solid-phase procedure, as previously described.10 Peptide purity (> 97%) was confirmed by matrix assisted laser desorption mass spectrometry. All other chemicals were analytical grade reagents of the highest purity available.

DNA construction

The yellow fluorescent protein (YFP) cDNA was attached to the carboxy-terminus end of the cDNA encoding the Mas receptor by the megaprimer method11 into XhoI-ApaI sites of pEYFP-N1 plasmid (Clontech). The construct was verified by DNA sequencing.

Cell culture and transfection

Human embryonic kidney (HEK) 293T cells were grown in DMEM high glucose supplemented with 10% heat inactivated fetal bovine serum and penicilin-streptomycin at 37 °C in a humidified atmosphere at 95% air and 5% CO2. Cells were transiently transfected using Lipofectamine 2000 according to instructions of the manufacturer and were used 36 h post-transfection.

MasR-YFP expression

The expression of Mas R fused to YFP in transfected HEK 293T cells was evaluated by Western-blot as previously described10. MasR-YFP expression was evaluated with a mouse anti-GFP monoclonal antibody (dilution 1/1000).

MasR-YFP expression was also evaluated by laser scanning confocal microscopy (Olympus Fluoview FV1000, Japan).

[125I]Ang-(1-7) binding studies

Thirty six hours post-transfection, cells on 12-well plates were rinsed two times with DMEM and equilibrated on ice with incubation buffer (DMEM containing 0.2% BSA and a protease inhibitors cocktail, pH: 7.4) for 30 min. Subsequently, the plates were incubated at 4°C for 60 min with incubation buffer containing 0.5 nmol/L [125I]Ang-(1-7) (labeled in our laboratory as previously described12). Incubation was stopped by rinsing the cells three times with ice-cold PBS. Cells were solubilized by incubation with 0.1 mol/L NaOH for 60 min and the radioactivity was measured. Nonspecific binding was determined in the presence of 10 μmol/L unlabeled Ang-(1-7), which was no higher than 15 %. Specific binding was calculated by the subtraction of nonspecific binding from total binding. Competition binding experiments were performed and Ki was calculated using Graphpad Prism (Graphpad Software Inc., San Diego, CA).

Arachidonic acid (AA) release

Twenty four hours posttransfection cells were labeled with [3H]AA (0.2 μCi/well) for 16 h as described previously3. Then, cells were washed with DMEM containing 2% BSA and incubated with Ang-(1-7) during different times at 37 °C. Radioactivity in the supernatant was measured. For total cellular radioactivity, cells in each well were solubilized with 1 mol/L NaOH and counted. [3H]AA released into the medium was expressed as percent of the total cellular radioactivity and referred to as fractional release.

Receptor internalization assay

It was measured according to the method described by Thomas et al.13 Briefly, thirty six hours posttansfection, cells on 12-well plates were rinsed two times with DMEM, preincubated with incubation buffer (DMEM containing 0.2% BSA and a protease inhibitors cocktail, pH: 7.4) for 30 min and then incubated with 0.5 nmol/L [125I]Ang-(1-7) (labeled in our laboratory as previously described12 during different times. After two washes with ice-cold PBS, surface-bound [125I]Ang-(1-7) associated with non-internalized receptors was separated by treating the cells with an ice-cold acid solution (0.2 mol/L acetic acid, 0.5 mol/L NaCl, pH:5.5) for 5 min on ice and radioactivity was determined (acid-sensitive fraction). Cells, which contain the internalized [125I]Ang-(1-7), were lysed by incubation with 0.1 mol/L NaOH for 60 min and the radioactivity was measured (acid-insensitive fraction). The index of receptor internalization was determined as acid-insensitive cpm as a percentage of the total binding (acid-sensitive + acid-insensitive). Non-receptor-mediated [125I]Ang-(1-7) and surface binding was measured in the presence of 10 μmol/L unlabeled Ang-(1-7) and subtracted from total binding to calculate the specific values. Curve fitting was performed with Graphpad Prism (Graphpad Software Inc., San Diego, CA).

In another set of experiments, transfected cells were incubated with the Mas R antagonist [D-Ala7]-Ang-(1-7) (100 nmol/L) during 15 min and then internalized [125I]Ang-(1-7) was determined as described.

MasR-YFP trafficking

Trafficking pathway was evaluated by colocalization between MasR-YFP and endocytosis markers signals by immunocytochemistry. Briefly, thirty six hours posttansfection, cells were incubated with 10 μmol/L Ang-(1-7) (synthetized in our lab by the Merrifield method as previously described10) for 15 min at 37 °C. After two washes with PBS, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS and incubated in blocking solution (PBS/0.2% Triton X-100/3% BSA) for 30 min at room temperature. Cells were then incubated with different primary antibodies (anti-AP50, anti Rab5, anti-Cav-1 or anti-EEA1 mouse monoclonal antibodies, diluted 1:150 in blocking solution) overnight at 4 °C. The samples were rinsed twice in PBS/0.2% Triton X-100, and exposed to the secondary antibody (goat anti-mouse antibody coupled to Alexa 594, dilution 1:600 in blocking solution) for 2h at room temperature.

Samples were mounted and imaged using an Olympus Fluoview FV1000 spectral laser scanning confocal microscope with a 60× oil immersion lens using dual excitation (473 nm for YFP and 559 nm for Alexa 594). Due to the spectral properties of the scan head, fluorescence emission was collected between 520 and 550 nm for YFP and 600-660 nm for Alexa 594. Images were obtained using sequential scanning for each channel to eliminate the cross-talk of chromophores. Quantitative colocalization was estimated by Pearson's correlation coefficient and overlap coefficient according to Manders14, which were calculated using Image-Pro Plus software (MediaCybernetics Inc.). Negative controls consisted of mocked transfected cells treated with blocking solution in the absence of the primary antibody. Some images (Figure 3) were acquired using a laser scanning confocal system (Visitech International, England) mounted on a Nikon TE2000-eclipse microscope, 100× 1.3 NA lens, and visualized at 514 nm excitation, 550 LP emission. Identical laser, slit, and acquisition settings were used to obtain all images.

Figure 3. Agonist-induced subcellular distribution of MasR-YFP.

Figure 3

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Subcellular localization of the MasR-YFP fusion protein in HEK 293T transfected cells and stimulated with 1 μmol/L Ang-(1-7) for the defined time periods. Scale bar = 10 μm.

Data analysis

All average results are presented as mean ± SEM. One-way ANOVA computation combined with the Bonferroni test was used to analyze data with unequal variance between each group. A probability level of 0.05 was considered significant.

Results

Characterization of MasR-YFP

To better image Mas R trafficking we generated the chimera C-terminally YFP tagged Mas R. Since the fusion of YFP to the C-terminal of the R may alter its correct folding and hence its functionality, we first characterized the C-terminally tagged MasR-YFP. HEK293T cells transfected with the DNA coding for the chimera showed that MasR-YFP was found predominantly in the plasma membrane (Figure 1A). However, intracellular localization of the fusion protein was also detectable, especially in cells showing higher levels of R expression. In some cells, the MasR-YFP was present around the nucleus, presumably in the endoplasmic reticulum, and probably represents newly synthesized molecules passing through the secretory pathway, but intranuclear localization of the receptor was not observed.

Figure 1. Characterization of MasR-YFP.

Figure 1

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A, Expression and cellular localization of the MasR-YFP fusion protein in transfected HEK 293T cells. B, Immunoblot of extracts of HEK 293T cells transfected with empty vector (lane 1), the DNA encoding MasR-YFP (lanes 2 and 3) or YFP (lane 4). C, Specific binding of [125I]Ang-(1-7) to MasR-YFP transfected cells in the presence of Ang-(1-7) or the Mas R antagonist (D-Ala-Ang-(1-7). Values are mean±SE.

MasR-YFP expression was also investigated by Western-blot. Figure 1B shows that transfected cells expressed a protein with a molecular weight corresponding to the MasR-YFP chimera (68 kDa).

The functional integrity of the YFP tagged receptor was investigated by Ang-(1-7) binding. As shown in Figure 1C, increasing concentrations of unlabeled Ang-(1-7) as well as the Mas receptor antagonist [D-Ala7]-Ang-(1-7) displaced the binding of [125I]Ang-(1-7) to transfected cells (Ki: 4.92±0.12 ×10-8 mol/L for Ang-(1-7) and 4.96 ±0.18×10-9 mol/L for D-Ala7]-Ang-(1-7)). These results demonstrate that MasR-YFP attained a correct folding in the plasma membrane and binds its physiological ligand as well as its antagonist. Cells transfected with the empty vector showed undetectable Ang-(1-7) binding (total binding: 480±68 cpm vs nonspecific binding 395±87 cpm).

Receptor functionality and coupling were evaluated by measuring Mas R signaling. Mas R activation is coupled to AA release.3,5 To determine the functionality of MasR-YFP, HEK 293T cells transfected with the MasR-YFP construct and labeled with [3H]AA were exposed to increasing concentration of Ang-(1-7). As shown in Figure 2, 100 nmol/L Ang-(1-7) caused an increase in [3H]AA release in MasR-YFP transfected cells. A higher concentration (1 μmol/L) produced a smaller response, suggesting receptor desensitization. When cells were incubated with Ang-(1-7) for a prolonged period, a decrease in the Ang-(1-7)-induced [3H]AA release was observed (Figure 2), suggesting receptor desensitization. Cells transfected with the empty vector (mock) showed no changes in [3H]AA release upon Ang-(1-7) stimulation (Figure 2).

Figure 2.

Figure 2

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A, [3H]AA release from mocked transfected cells or cells transfected with the DNA coding for MasR-YFP in the absence (C) and presence of Ang-(1-7) during 15 min. Results are presented as the percentage of the response detected in control, taking them as 100%. Values are mean ± SEM. * P < 0.05 compared with control. B, [3H]AA release from MasR-YFP transfected cells in the presence of 100 nmol/L or 1 μmol/L Ang-(1-7) (Ang) during 10, 20 and 30 min. Results are presented as the percentage of the response detected in control, taking them as 100%. Values are mean ± SEM. * P < 0.05 compared with control.

Collectively, these data show that MasR-YFP is properly expressed and fully active. Furthermore, our results show that higher concentrations or longer times of stimulation with Ang-(1-7) caused a decrease in its response (AA release) demonstrating receptor desensitization.

Ang-(1-7) induces MasR-YFP internalization

To investigate whether Ang-(1-7) induces Mas receptor internalization, MasR-YFP transfected cells were incubated with 1 μmol/L Ang-(1-7) during different times and the relative cellular distribution of MasR-YFP was observed by laser scanning confocal microscopy. In resting cells, MasR-YFP was mostly localized on the cell membrane and in the endoplasmic reticulum. Treatment of cells with Ang-(1-7) induced redistribution in fluorescence after 5 min stimulation changing the localization of MasR-YFP to intracellular vesicles of various sizes (Figure 3), suggesting internalization of the receptor upon agonist stimulation.

The capacity of MasR-YFP to internalize in response to Ang-(1-7) was determined by following the time course of [125I]Ang-(1-7) endocytosis. Half of MasR-YFP underwent endocytosis after 10 min, and this internalization was partially blocked by the Mas receptor antagonist, [D-Ala7]-Ang-(1-7) (Figure 4).

Figure 4. Agonist-induced internalization of MasR-YFP.

Figure 4

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MasR-YFP transfected cells were incubated with [125I]Ang-(1-7) in the absence (circles) or presence of the Mas R antagonist [D-Ala7]-Ang-(1-7) (squares) and internalized [125I]Ang-(1-7) was evaluated as described in Methods. An index of internalization was calculated by expressing the internalized radioactivity (acid-resistant) as a percentage of the total binding (acid-resistant plus acid-susceptible). Data are means±SE for four independent determinations.

MasR-YFP trafficking

The Rab family of small GTPases is integral in determining the fate of a GPCR.15 Different Rab GTPase family members selectively associate with specific intracellular structures including both recycling and sorting endosomes, where they mediate multiple steps of vesicular membrane trafficking including vesicle budding, docking and fusion.15 Rab 5 plays a central role in endocytosis via clathrin-coated pits and subsequent fusion of vesicles with early endosomes.15 To investigate whether MasR-YFP was targeted to early endosome, MasR-YFP transfected cells were stimulated with Ang-(1-7) during 15 min and then co-localization of MasR-YFP with Rab5 was investigated. A fraction of MasR-YFP colocalized with Rab5 (Figure 5A) showing that the R was internalized and targeted to early endosomes. In addition to Rab5, early endosomes contain Rab5 effectors and regulator proteins, including EEA1.9 To provide further confirmation that MasR-YFP is targeted to early endosome, colocalization studies between MasR-YFP and the EEA1 marker protein after agonist stimulation was performed. Figure 5B shows that MasR-YFP also colocalized with EEA1, confirming that MasR-YFP is targeted to early endosomes after Ang-(1-7) stimulation.

Figure 5. MasR-YFP trafficking.

Figure 5

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Colocalization of MasR-YFP (green) and early endosomes markers (Rab 5 and EEA1), clathrin-coated pits (AP50) or caveolin-1 (caveolae marker) (red) in MasR-YFP transfected HEK293T cells stimulated with Ang-(1-7) during 15 min. Three independent experiments were analyzed. See text for analysis. Scale bar = 5 μm.

Most GPCR are endocyted through clathrin-mediated endocytosis.8,9,16 However, some GPCRs preferentially localize to and/or internalize via specialized lipid raft/caveolae microdomains of the plasma membrane.9,17,18 Caveolae are Cav-1-enriched smooth invaginations of the plasma membrane that form a subdomain of lipid rafts and represent a non-clathrin internalization pathway. To begin studying the mechanisms by which Mas R is internalized, MasR-YFP transfected cells were stimulated with Ang-(1-7) and then co-localization of MasR-YFP with markers of the clathrin or caveolin pathway were investigated. We evaluated whether MasR-YFP is endocyted through clathrin-coated pits by analyzing its colocalization with the adaptor protein complex 2 (AP-2, μ or AP50), which is an essential component of the clathrin-coated vesicle machinery.9 Upon Ang-(1-7) stimulation, a fraction of MasR-YFP colocalized with AP-50 (Figure 5C), which suggests that MasR-YFP is internalized via a clathrin-mediated pathway.

To further study whether the caveolae-dependent pathway was involved in the endocytosis of MasR-YFP, colocalization with Cav-1 was evaluated. After Ang-(1-7) stimulation, colocalization of MasR-YFP and Cav-1 specific signal was observed (Figure 5D).

Altogether, these results indicate that MasR-YFP is internalized into early endosomes via a clathrin-dependent pathway. However co-localization with Cav-1 suggests that at some point in the receptor trafficking, MasR-YFP traverses Cav-1 positive compartments.

Discussion

The endocytic pathway tightly controls the activity of GPCR. Ligand-induced endocytosis can drive R into divergent lysosomal and recycling pathways, producing essentially opposite effects on the strength and duration of cellular signaling, allowing for the fine-tuning of signal magnitude and duration.9,19 Our study shows that Ang-(1-7) induces Mas R internalization, a process that is involved in the feedback desensitization of GPCR responsiveness that protects against R overstimulation.7 This event may explain the decrease in Ang-(1-7) response in releasing AA from tissue lipids and in stimulating 6-keto-prostaglandin F1α production in rabbit aortic smooth muscle cells,5 phosphatydylcholine biosynthesis in the rat renal cortex4 and the attenuation in responsiveness in the Ang-(1-7)-induced proliferation of endothelial progenitor cells from sham or infarcted rodents.6

GPCR desensitization also acts to filter information from multiple receptor inputs into an integrated and meaningful biological signal. Present results show that MasR-YFPs are internalized together with Ang-(1-7). This event may be important for directing Ang-(1-7) to certain cellular locations for full expression of its biological response, as it happens with Ang II at the renal tissue.20,21 Renal AT1 receptors are responsible for internalizing Ang II, and the presence of substantial Ang II in endosomes in both control and Ang II-infused hypertensive rats supports their internalization into a protected compartment that prevents degradation of some of the internalized Ang II.20,21 In this way, the internalized Ang II activates various signaling pathways, contributing to fibrogenic proliferative responses while also migrating to the nucleus to exert transcriptional effects.21

The fact that a fraction of 20% of the binding of radiolabeled Ang-(1-7) was not completely displaced by Ang-(1-7) or its antagonist or that its endocytosis was not completely prevented by the Mas R antagonist suggests that some of the Ang-(1-7) may be bound to some AT1 or AT2 R or an unknown R or be internalized by another mechanism. For instance, Gonzalez-Villalobos et al22 have shown that the scavenger receptor megalin binds and internalizes Ang-(1-7).

GPCR endocytosis, in addition to playing a role in receptor desensitization, has been shown to have other important functions in regulating and even promoting GPCR signaling.19,23 Recent studies indicate that GPCRs can continue signaling after internalization together with their agonists.19,23 GPCR endocytosis appears to be required for efficient mitogen activated protein kinase (MAPK) signaling by certain GPCRs.19 Growing evidence shows that caveolae is not only involved in endocytosis but also functions as cell surface signal transduction domain by affecting both signaling selectivity and coupling efficacy,17,18 as it happens for the AT1 R.24,25 After agonist binding, the AT1R moves to caveolae and this event is involved in the reactive oxygen species-dependent AT1 R signaling regulating vascular smooth muscle cells hypertrophy.24,25 It is increasingly evident that endocytosis and signaling are not only connected but likely inextricably intertwined. Our present results show that Mas R is internalized upon ligand stimulation into early endosomes via a clathrin-dependent pathway; however, co-localization with Cav-1 suggests that at some point in the receptor trafficking MasR-YFP traverses Cav-1 positive compartments (present results). The relative contribution of both clathrin- and caveolae-dependent pathways in Mas R desensitization deserves further future investigation.

Perspectives

Through desensitization and internalization Mas R activity and density may be tightly controlled by the cell. The broad data available reveal a degree of specificity and plasticity in the cellular regulation of GPCRs by endocytic membrane trafficking. We showed that Mas R is internalized upon ligand stimulation into early endosomes via a clathrin-dependent pathway; however, at some point in the receptor trafficking MasR-YFP traverses Cav-1 positive compartments. To our knowledge this is the first report on Mas R regulation and it opens the way for a better understanding of Mas R biochemistry. Within the cardiovascular system, regulation of GPCR endocytosis and trafficking is of fundamental importance both for physiological homeostasis and molecular response to physiological perturbation.16,19 Many studies suggest that the endocytic trafficking of GPCR is highly controlled and has profound functional consequences in vivo.16,19 Elucidating those mechanisms will grow our understanding of GPCR pharmacology and function, and open new opportunities for the development of strategies to therapeutically manipulate GPCR function in diseases associated with altered GPCR signaling, such as hypertension and congestive heart failure.26,27

Acknowledgments

Sources of Funding: This work was supported by UBACyT B121, CONICET PIP 112-200801-02376 and NIH HL28982.

Footnotes

Disclosures: None

References

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