Functional Complexes of Angiotensin-Converting Enzyme 2 and Renin-Angiotensin System Receptors: Expression in Adult but Not Fetal Lung Tissue

International Journal of Molecular Sciences Article Functional Complexes of Angiotensin-Converting Enzyme 2 and Renin-Angiotensin System Receptors: Expression in Adult but Not Fetal Lung Tissue Rafael Franco 1,2,* , Alejandro Lillo 1, Rafael Rivas-Santisteban 1,2, Ana I. Rodríguez-Pérez 2,3, Irene Reyes-Resina 1,4, JoséL. Labandeira-García2,3 and Gemma Navarro 2,5,* 1Laboratory of Molecular Neurobiology, Department Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, 08007 Barcelona, Spain; [email protected] (A.L.); [email protected] (R.R.-S.); ire-r[email protected] (I.R.-R.) 2Network Center, Neurodegenerative Diseases (CiberNed), Spanish National Health Institute Carlos III, Valderrebollo 5, 28031 Madrid, Spain; anai.r[email protected] (A.I.R.-P.); [email protected] (J.L.L.-G.) 3Laboratory of Cellular and Molecular Neurobiology of Parkinson’s Disease, Research Center for Molecular Medicine and Chronic Diseases (CIMUS), Department of Morphological Sciences, IDIS, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain 4RG Neuroplasticity, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany 5 Department of Biochemistry and Physiology, School of Pharmacy and Food Science, University of Barcelona, 08007 Barcelona, Spain *Correspondence: [email protected] or [email protected] (R.F.); [email protected] or [email protected] (G.N.); Tel.: +34-934-021-208 (R.F.); +34-934-034-500 (G.N.) Received: 14 November 2020; Accepted: 11 December 2020; Published: 16 December 2020   Abstract: Angiotensin-converting enzyme 2 (ACE2) is a membrane peptidase and a component of the renin-angiotensin system (RAS) that has been found in cells of all organs, including the lungs. While ACE2 has been identified as the receptor for severe acute respiratory syndrome (SARS) coronaviruses, the mechanism underlying cell entry remains unknown. Human immunodeficiency virus infects target cells via CXC chemokine receptor 4 (CXCR4)-mediated endocytosis. Furthermore, CXCR4 interacts with dipeptidyl peptidase-4 (CD26/DPPIV), an enzyme that cleaves CXCL12/SDF-1, which is the chemokine that activates this receptor. By analogy, we hypothesized that ACE2 might also be capable of interactions with RAS-associated G-protein coupled receptors. Using resonance energy transfer and cAMP and mitogen-activated protein kinase signaling assays, we found that human ACE2 interacts with RAS-related receptors, namely the angiotensin II type 1 receptor (AT 1 R), the angiotensin II type 2 receptor (AT 2 R), and the MAS1 oncogene receptor (MasR). Although these interactions led to various alterations of signal transduction, but, more importantly, ligand binding to AT 1 R resulted in the downregulation of ACE2 cell surface expression, while ligand binding to AT 2 R, but not to MasR, resulted in upregulation of ACE2 cell surface expression. Proximity ligation assays performed in situ revealed macromolecular complexes containing ACE2 and AT 1 R, AT 2 R or MasR in adult but not fetal mouse lung tissue. These findings highlight the relevance of RAS in SARS-CoV-2 infection and the role of ACE2-containing complexes as potential therapeutic targets. Keywords: COVID-19; SARS-CoV-2 receptor; RAS; ACE2; angiotensin receptor; Mas receptor; lung 1. Introduction The current coronavirus disease-2019 (COVID-19) pandemic is the result of widespread infection with the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pathogen. The main cell Int. J. Mol. Sci. 2020,21, 9602; doi:10.3390/ijms21249602 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 9602 2 of 21 surface receptor for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2), which is an enzyme that catalyzes the conversion of angiotensin II (Ang II) into angiotensin 1-7 (Ang 1-7). The link between ACE2 and SARS coronaviruses was discovered serendipitously [ 1 – 5 ]. ACE2 is a component of the renin-angiotensin system (RAS), which has been characterized extensively in the kidney and serves as the target of efficacious antihypertensive drugs. In addition to enzymes that process renin and angiotensin, components of the RAS include members of the G-protein-coupled receptor (GPCR) superfamily. Receptors for Ang II type 1 (AT 1 Rs) and Ang II type 2 (AT 2 Rs) share Ang II as an endogenous ligand. As noted, Ang II is also a substrate for enzymatic processing by ACE2 [ 6 ]. By contrast, the Mas1 oncogene receptor (MasR) interacts with Ang 1-7. Mas1, also known as the Mas-related proto-oncogene, is related to a putative ancestor gene identified in Saccharomyces cerevisiae that encodes mitochondrial assembly protein-1 [ 7 ]. Additional RAS receptors, the Mas-related GPCRs (Mrgprs), are also responsive to Ang 1-7 [ 8 – 10 ] and to another endogenous agonist that is an Ang 1-7 derivative, alamandine [6]. Coronaviruses and the human immunodeficiency virus (HIV), the latter pathogen recognized as the causative agent of acquired immunodeficiency syndrome (AIDS), have several common characteristics. Both RNA viruses contain nucleic acids enveloped within a membrane that contains host components and viral proteins that facilitate interactions with surface receptors on target cells. The most studied of the HIV subtypes, HIV-1, interacts with target cell surface receptors and co-receptors that are critical for entry into the host cell. HIV-1 entry requires interactions with the main receptor, CD4, and interactions with a GPCR co-receptor, most notably the CXC chemokine receptor, CXCR4 [ 11 – 17 ]. The chemokine CXCL12, also known as stromal-derived factor 1 (SDF-1), is the endogenous ligand of CXCR4. Interestingly, CXCL12/SDF-1 is degraded by dipeptidyl peptidase-4 (CD26/DPPIV). The actions of this enzyme serve to reduce the local concentration of CXCL12/SDF-1 and thereby protect the host cells from viral infection [ 18 , 19 ]. ACE2 and CD26/DPPIV are both proteases with several specific structural similarities. For example, both ACE2 and CD26/DPPIV are attached to cell membranes and can be removed and released into body fluids via a process known as shedding [ 20 , 21 ]. Furthermore, both enzymes are type I transmembrane proteins with a single transmembrane domain, a C-terminal domain facing the cytoplasm, and a large N-terminal extracellular domain that includes the catalytic site. Glycoprotein 120 kDa (gp120) found on the surface of HIV-1 virions interacts with CD26/DPPIV, which may interact with CXCR4. Among the findings that support our hypothesis, we previously characterized co-modulation of CXCR4 and CD26/DPPIV in human lymphocytes. We also found that the non-catalytic activating function of CD26/DPPIV was altered in the presence of gp120 via a mechanism that was dependent on the expression of both CD4 and CXCR4 [19,22]. Based on these findings, we hypothesized that the ACE2 may have the capacity to interact with receptors that are activated by its substrate, Ang II, and its product, Ang 1-7. Accordingly, this paper aimed at examining the physical and functional interactions of ACE2 with cell surface receptors for Ang II and Ang 1-7. We also performed experiments designed to detect enzyme-receptor complexes in lung tissue, which is the main portal of entry for SARS-CoV-2. 2. Results 2.1. Expression of ACE2 Downregulates AT1R-Mediated Signaling Induced by Ang II ACE2 has been identified as the main receptor for SARS coronaviruses. Its substrate, Ang II, is an endogenous agonist that activates the G-protein-coupled receptors (GPCRs) AT 1 R and AT 2 R. AT 1 R couples with the Gq protein; thus, activation by agonists increases the levels of inositol triphosphate and diacylglycerol and mobilizes intracellular calcium. In this first set of experiments, we aimed to determine whether the expression of ACE2 had any impact on AT 1 R-mediated signaling. Toward this end, we measured cytoplasmic Ca 2+ levels in a heterologous expression system using the calmodulin-derived Ca 2+ sensor, GCaMP6. Ang II at concentrations of 1 nM to 100 nM was added to HEK-293T cells that expressed both AT 1 R and GCaMP6. A fluorescent signal with a maximum of Int. J. Mol. Sci. 2020,21, 9602 3 of 21 9000 AU at 150 s was detected in response to the two highest concentrations of Ang II (Figure 4A). Interestingly, in a similar experiment targeting HEK-293T cells expressing AT 1 R and ACE2, a significant decrease in the maximum response was observed (6000 AU signal at the highest concentration of Ang II; Figure 4B). These results suggest that the expression of ACE2 may inhibit AT 1 R-mediated signaling. The possibility of functional selectivity and Gi coupling was discarded from experiments that evaluated intracellular cAMP levels in the presence or absence of forskolin. No effect on cAMP levels was identified in response to micromolar concentrations of Ang II. Moreover, the expression of ACE2 had no significant impact on these results (Figure 4C). Activation of GPCRs results in the engagement of the mitogen-activated protein kinase (MAPK) signaling pathway. As such, we measured extracellular signal-regulated kinase (ERK)1/2 phosphorylation in cells expressing AT1R and ACE2. While the addition of Ang II to HEK-293T cells expressing AT1R induced a 125% increase in ERK1/2 phosphorylation over baseline levels, the increase in phosphorylation observed in cells co-expressing AT 1 R and ACE2 was limited to 71% (Figure 4D). Similar results were obtained using dynamic mass redistribution (DMR) assays, which is a technique that can be used to measure changes in cytoskeletal structure in response to GPCR activation and the engagement of G-proteins. With this assay, we found that the expression of ACE2 resulted in a 30% decrease in the overall impact of Ang II at AT1R (Figure 4F,G). Finally, AT1R-mediated recruitment of β-arrestin was evaluated in cells that co-express β -arrestin II-RLuc and AT 1 R-YFP. The specific signal of 37 milliBioluminescence Resonance Energy Transfer (BRET) units (mBU) originally detected in cells expressing AT 1 R alone increased by 20 mBU in response to ACE2 co-expression (Figure 4E). These results suggest that the expression of ACE2 augments AT1R-mediated recruitment of β-arrestins. 2 Figure 1. Cont. Int. J. Mol. Sci. 2020,21, 9602 4 of 21 2 Figure 1. Impact of ACE2 on the functionality of AT 1 R. HEK-293T cells were transfected with either 0.4 µ g AT 1 R cDNA and 0.2 µ g ACE2-HA cDNA ( A , B , F , G ), 0.5 µ g AT 1 R-YFP cDNA, 0.2 µ g ACE2-HA cDNA, and 0.5 µ g β -arrestin II-RLuc cDNA ( C ), or 0.4 µ g AT 1 R cDNA, 0.2 µ g ACE2-HA cDNA, and 0.5 µ g cDNA encoding the Ca 2+ sensor, GCaMP6 ( D , E ). After 48 h of incubation, cells were treated with increasing concentrations of the AT 1 R agonist, Ang II. Cyclic AMP was measured after 15 min in response to pre-treatment with 0.5 µ M forskolin ( A ); as shown, this intervention resulted in approximately 4 nM cAMP, which corresponds to a 240% increase over baseline levels. Results from the evaluation of ERK1/2 phosphorylation ( B ), β -arrestin II recruitment ( C ) Ca 2+ levels ( D , E ), and DMR recordings ( F , G ) are presented as dose-response curves. Where indicated, cells were pre-treated with the selective AT 1 R receptor antagonist, candesartan (1 µ M), before challenge with the receptor agonist. Values shown are the mean ±SEM of 8 independent experiments each performed in triplicate. Taken together, our findings revealed that the expression of ACE2 results in a decrease in AT 1 R-mediated signaling in response to Ang II with a concomitant increase in the capacity for β-arrestin recruitment. 2.2. Expression of ACE2 Downregulates AT2R-Mediated Signaling Induced by Ang II Signaling assays were performed to assess the impact of ACE2 on AT 2 R function. As AT 2 R couples with Gi, we first determined intracellular cAMP levels in cells treated with forskolin. Findings shown in Figure 2A reveal that the addition of the selective AT 2 R agonist, CGP-42112A (CGP), resulted in a 73% reduction in cAMP levels. This strong effect was markedly attenuated in cells that co-expressed ACE2 (only 12% reduction in cAMP levels). While similar results were obtained in DMR assays (Figure 2F,G), our findings revealed qualitative differences with respect to the engagement of the MAPK signaling pathway. Of note, we found that ERK1/2 phosphorylation following activation of AT 2 R was increased from 90% to 232% in response to ACE2 (Figure 2B). These results indicate that ACE2 expression potentiates the link between AT2R and MAPK signaling. Int. J. Mol. Sci. 2020,21, 9602 5 of 21 Figure 2. Impact of ACE2 on the functionality of AT 2 R. HEK-293T cells were transfected with either 0.3 µ g AT 2 R cDNA and 0.2 µ g ACE2-HA cDNA ( A , B , F , G ), 0.4 µ g AT 2 R-YFP cDNA, 0.2 µ g ACE2-HA cDNA, and 0.5 µ g β -arrestin II-RLuc cDNA ( C ), or 0.3 µ g AT 2 R cDNA, 0.2 µ g ACE2-HA cDNA, and 0.5 µ g cDNA encoding the Ca 2+ sensor, GCaMP6 ( D , E ). After 48 h of incubation, cells were treated with increasing concentrations of the selective AT 2 R agonist, CGP. Cyclic AMP was measured after 15 min in response to pre-treatment with 0.5 µ M forskolin ( A ); see also Legend to Figure 4. Results from the evaluation of ERK1/2 phosphorylation ( B ), β -arrestin II recruitment ( C ), Ca 2+ levels ( D , E ), and DMR recordings ( F , G ) are presented as dose-response curves. Where indicated, cells were pre-treated with selective AT 2 R receptor antagonist, PD123319 (PD; 1 µ M), before challenge with the receptor agonist. Values are the mean ±SEM of 8 independent experiments each performed in triplicate. Int. J. Mol. Sci. 2020,21, 9602 6 of 21 By contrast, expression of ACE2 had a less profound impact on β -arrestin recruitment via AT 2 R when compared to responses mediated by AT 1 R. BRET max was 22 mBU in cells expressing AT 2 R; this response increased to 29 mBU when ACE2 was also expressed (Figure 2C). As anticipated from findings of AT 2 R coupling with Gi and not to Gq, Ang II-mediated activation of this receptor-induced minimal mobilization of intracellular Ca 2+ . This response was completely abolished in the presence of ACE2 (Figure 2D,E). 2.3. Expression of ACE2 Potentiates MasR-Mediated Signaling Assays analogous to those described in earlier sections were performed to determine the impact of ACE2 on the functionality of MasR. While the recruitment of β -arrestin in response to receptor activation with its ligand, Ang 1-7, was not affected by co-expression of ACE2, expression of ACE2 resulted in the enhancement of all other signals transduced via this receptor. This was observed in assays targeting cAMP and ERK1/2 phosphorylation and was notably strong in DMR readouts (Figure 3). As such, not only does the catalytic activity of ACE2 generate the endogenous agonist for this receptor, our results reveal that the expression of this enzyme may increase the signaling output from MasR via an enzyme-independent mechanism. We confirmed that MasR activation resulted in no changes in cytoplasmic Ca2+levels, regardless of the presence or absence of ACE2 (Figure 3D,E). Figure 3. Cont. Int. J. Mol. Sci. 2020,21, 9602 7 of 21 Figure 3. Impact of ACE2 on the functionality of MasR. HEK-293T cells were transfected with 0.5 µ g MasR cDNA and 0.2 µ g ACE2-HA cDNA ( A , B , F , G ), 0.6 µ g MasR-YFP cDNA, 0.2 µ g ACE2-HA cDNA, and 0.5 µ g encoding β -arrestin II-RLuc cDNA ( C ), or 0.5 µ g MasR cDNA, 0.2 µ g ACE2-HA cDNA, and 0.5 µ g cDNA encoding the Ca 2+ sensor, GCaMP6 ( D , E ). After 48 h of incubation, cells were treated with increasing concentrations of the selective MasR agonist, Ang 1-7. Cyclic AMP was measured after 15 min in response to pre-treatment with 0.5 µ M forskolin ( A ); see also Legend to Figure 4. Results from the evaluation of ERK1/2 phosphorylation (B), β -arrestin II recruitment ( C ), Ca 2+ levels ( D , E ), and DMR recordings ( F , G ) are presented as dose-response curves. Where indicated, cells were pre-treated with the selective MasR receptor antagonist, A779 (1 µ M), before challenge with the receptor agonist. Values presented are the mean ±SEM of 6 independent experiments each performed in triplicate. 2.4. ACE2 Interacts Directly with AT1R, AT2R, and MasR Naïve HEK-293T cells do not express functionally significant concentrations of RAS components. As such, we hypothesize that outcomes associated with ACE2 expression may be a direct result of receptor-enzyme interactions. This hypothesis was tested using BRET assays. First, HEK-293T cells were transfected with a constant amount of cDNA encoding AT 1 R-RLuc and increasing amounts of cDNA encoding ACE2-eGFP. Findings shown in Figure 4A include a saturable BRET curve which is indicative of direct interactions between AT 1 R and ACE2 (BRET max =120 ± 20 mBU and BRET 50 =16 ± 4). Similar assays were performed in cells pretreated for 10 min with Ang II or with the selective AT 1 R antagonist, candesartan. While a significant decrease in BRET signal was detected after challenging with the agonist ( BRETmax =90 ±10 mBU and BRET 50 =21 ± 5), these parameters did not undergo significant change in response to treatment with a receptor antagonist, candesartan (BRET max =12 ± 20 mBU and BRET 50 =15 ± 5; Figure 4A). These results may be explained by conformational changes that alter the distance between BRET donor and BRET acceptor. Another possibility is internalization, disassembly and recycling in response to agonist challenge. The latter process would lead to reduce the number of AT1R-ACE2 complexes. Figure 4. Cont. Int. J. Mol. Sci. 2020,21, 9602 8 of 21 Figure 4. Interactions of RAS receptors and ACE2 as assessed by Bioluminescence Resonance Energy Transfer (BRET) assays. BRET assays were performed in HEK-293T cells transfected with constant amounts of cDNA encoding AT 1 R-RLuc (0.5 µ g) ( A ), AT 2 R-RLuc (0.4 µ g) ( B ), MasR-RLuc (0.6 µ g) ( C ), or GHS-R1a-RLuc (0.3 µ g; negative control) ( D ) together with increasing amounts of cDNA encoding ACE2-eGFP (0.1 to 1 µ g). Cells were treated (red symbols) or not (black symbols) for 25 min with selective antagonists (candesartan for AT 1 R, PD123319 for AT 2 R or A779 for MasR, both at 1 µ M; red symbols) or selective agonists (Ang II for AT 1 R, CGP for AT 2 R or Ang 1-7 for MasR, all at 100 nM; green symbols). Values correspond to experimental points from 6 independent experiments each performed in quadruplicate. BRET 50 and BRET max values were calculated by non-linear regression using Prism GraphPad software; specific parameters are as described in the text. Potential AT 2 R-ACE2 interactions were examined in experiments performed in HEK-293T cells transfected with a constant amount of cDNA encoding AT 2 R-RLuc and increasing amounts of cDNA encoding ACE2-eGFP. The saturable BRET curve (BRET max =510 ± 20 mBU and BRET50 =11 ±2 ) revealed the formation of AT 2 R-ACE2 complexes in the co-transfected HEK-293T cells. However, we observed a significant increase in the height at saturation (BRET max =750 ± 70 mBU and BRET50 =31 ±6 ) in co-transfected cells that were challenged with the AT 2 R agonist, CGP (Figure 4B). These results may be explained by conformational changes that reduce the distance between the BRET donor and BRET acceptor. The findings might also be explained by an increase in the number of receptor-enzyme complexes. Challenge with the AT 2 R antagonist, PD123319, led to a significant decrease in the BRET signal when compared to results from untreated cells (BRET max =400 ± 20 mBU and BRET50 =12 ±2). Finally, we addressed the possibility of physical interactions between MasR and ACE2. BRET assays confirmed these interactions. However, we observed no responses to treatment with MasR agonists or antagonists (Figure 4C). Parameters defining this interaction were: BRET max =362 ± 21 mBU and BRET 50 =10 ± 2 in the absence of receptor activation, BRET max =420 ± 60 mBU and BRET50 =16 ±5 in cells treated with receptor agonist, and BRET max =370 ± 40 mBU and BRET 50 =12 ± 4 in cells treated with receptor antagonist. A non-specific linear signal was obtained in HEK-293T cells that were transfected with the cDNA encoding GHS-R1a-RLuc (negative control) together with increasing amounts of ACE2-eGFP (Figure 4D). 2.5. Cell Surface Expression of ACE2 Following Activation of AT1R and AT2R Our findings revealed that ACE2 was capable of functionally interact with RAS receptors. We also discovered that direct interactions between ACE2 and AT 1 R and AT 2 R underwent a quantitative change in response to receptor activation. As such, our next aim was to assess cell surface expression of ACE2 following RAS receptor activation. Cell surface expression of ACE2 was first assessed by immunocytochemistry assays targeting HEK-293T cells that express this enzyme together with AT 1 R, AT 2 R, or MasR. As shown in Figure 5A, ACE2 and the three receptors were all detected at the plasma membrane and were also associated with Int. J. Mol. Sci. 2020,21, 9602 9 of 21 intracellular structures in the cytoplasm. Dual localization was also observed in cells treated with selective agonists. Increased green fluorescence documenting ACE2 immunoreactivity in the cytoplasm was detected in AT 1 R and ACE2-expressing cells that were treated with Ang II. These results suggested that activation of AT 1 R results in decreased expression of ACE2 at the plasma membrane (Figure 5A). By contrast, no significant changes in ACE2 immunoreactivity were detected when cells expressing AT 2 R or MasR were treated with their respective receptor agonists. 1 Figure 5. Cont. Int. J. Mol. Sci. 2020,21, 9602 16 of 21 4.11. Immunoblotting To determine levels of immunoreactive AT 1 R, AT 2 R, MasR, and ACE2-HA expression in transfected HEK-293T cells, equivalent amounts of cell protein (10 µ g) were separated by denaturing 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF)-fluorescence membranes. Membranes were treated overnight at 4 ◦ C with a mixture of a mouse antiβ -tubulin antibody (1:2000; Sigma-Aldrich), a rabbit anti-ACE2 antibody (1:1000; Cat# ab108252, RRID:AB_10864415, Abcam, Cambridge, UK) and a mouse monoclonal anti-AT 1 R antibody (1:1000; Cat# sc-515884, RRID:AB_2801404, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit monoclonal anti-AT 2 R antibody (1:1000; Cat# ab92445, RRID:AB_10561969, Abcam, Cambridge, UK) and a mouse monoclonal anti-MasR antibody (1:1000; Cat# sc-390453, RRID:AB_2801406, Santa Cruz Biotechnology). Cells were subsequently treated with a mixture of IRDye 800-conjugated anti-mouse antibody (1:10,000; #A9044 from Sigma-Aldrich) and IRDye 680-conjugated anti-rabbit antibody (1:10,000; #926-68071 from LICOR Biosciences, Lincoln, NE, USA) for 2 h at room temperature. Bands were scanned using the Odyssey infrared scanner (LI-COR Biotechnology, Lincoln, NE, USA). Band densities were quantified using the scanner software and receptor level was normalized for differences in loading via normalization to tubulin protein band intensities. 4.12. Biotinylation Experiments Cell surface proteins were biotinylated as previously described [ 55 , 56 ] using HEK-293T cells that transiently express AT 1 R-RLuc, AT 2 R-RLuc, or MasR-RLuc in the presence or the absence of ACE2-HA. Before initiation of the experiment, eGFP fluorescence was adjusted to 10,000 fluorescence units and receptor-RLuc to 100,000 bioluminescent units. Briefly, cells were washed three times with borate buffer (10 mM H 3 BO 3 , pH 8.8 with 150 mM NaCl) and incubated with 50 µ g/mL sulfo-NHS-LC-biotin (ThermoFisher Scientific, Halethorpe, MD, USA) in borate buffer for 5 min at room temperature. Cells were then washed three times in borate buffer and again incubated with 50 µ g/mL sulfo-NHS-LC-biotin in borate buffer for 10 min at room temperature. This was followed by the addition of 13 mM NH 4 Cl for 5 min to quench the remaining biotin. Cells were washed in PBS, disrupted using a polytron (3 strokes at 10 s each), and centrifuged at 16,000 × gfor 30 min. The pellet was solubilized in an ice-cold RIPA buffer (50 mM Tris–HCl, 1% Triton X-100, 0.2% SDS, 100 mM NaCl , 1 mM EDTA, 0.5% sodium deoxycholate) for 30 min and centrifuged at 16,000 × gfor 20 min. The supernatant was incubated with 80 µ l streptavidin-agarose beads (Sigma-Aldrich) for 1 h with constant rotation at 4 ◦ C. Beads were washed three times with ice-cold lysis buffer and aspirated to dryness using a 28-gauge needle. Subsequently, 50 µ l of SDS–PAGE sample buffer (8 M urea, 2% SDS, 100 mM dithiothreitol, 375 mM Tris, pH 6.8) was added to each sample. Proteins were dissociated by heating to 37 ◦C for 2 h, resolved by SDS-PAGE (10% gels), and immunoblotted as described above. 4.13. In Situ Proximity Ligation Assays (PLA) Lungs from adult and 19-day-old fetal CD-1 mice were fixed in 4% paraformaldehyde for 1 day followed by processing with decreasing concentrations of sucrose. Tissue samples were cut in 30 µ m-thick sections in a cryostat (Leica CM3050S), mounted on coverslips, and frozen. Frozen tissue samples were washed with PBS containing 20 mM glycine and permeabilized by incubation for 30 min in PBS-glycine containing 0.05% Triton X-100. Tissue sections were incubated for 1 h at 37 ◦ C with blocking solution followed by specific antibodies, including mouse anti-AT 1 R (1:100; Cat# sc-515884, RRID:AB_2801404, Santa Cruz Biotechnology), rabbit anti-AT 2 R (1:100; Cat# ab92445, RRID:AB_10561969, Abcam), mouse anti-MasR (1:100; Cat# sc-390453, RRID:AB_2801406, Santa Cruz Biotechnology), and rabbit anti-ACE2 (1:100; Cat# ab108252, RRID:AB_10864415, Abcam). These samples were processed using PLA probes that detect rabbit and mouse antibodies (Duolink II PLA probe anti-Rabbit plus and Duolink II PLA probe anti-Mouse minus). Nuclei were stained with Hoechst (1/200; Sigma-Aldrich) and mounted in 30% Mowiol (Calbiochem). Samples were observed Int. J. Mol. Sci. 2020,21, 9602 17 of 21 using a Zeiss 880 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an apochromatic 63 × oil-immersion objective (N.A. 1.4), and 405 nm and 561 nm laser lines. For each field of view, a stack of two channels (one per staining) and three to four Z stacks with a step size of 1 µ m were acquired. Duolink Image took software was used to identify cells with one or more red spots vs. total cells (cell count determined by the presence of a single blue-stained nucleus). The ratio r was determined as the number of red spots per cell in all red spot-containing cells. This analysis was performed in a blinded fashion (i.e., the observer did not know which sample was undergoing processing and the analyzer did not know whether the results came from adult or fetal mice. 4.14. Validation of Antibody Specificity Despite the excellent performance of these antibodies in different laboratories [ 57 – 60 ], the specificity of antibodies directed against angiotensin receptors is always subject to question. As such, we performed a series of experiments in which the anti-Ang II receptor antibodies were tested against naïve HEK-293T cells and against cells that express either AT 1 R or AT 2 R. Signal detected from anti-AT 1 R antibody binding was negligible in both naïve and AT 2 R-expressing HEK-293T cells. Similarly, the signal detected from anti-AT 2 R antibody binding was negligible in both naïve and AT 1 R-expressing cells (Supplementary Figure S2). These results are consistent with previous studies that addressed the specificity of antibodies used to detect AT1Rs in mitochondria [61]. 4.15. Data Analysis Data were obtained from at least five independent experiments and are presented as the mean ±standard error of the mean (SEM). Two-group comparisons were performed using unpaired Student’s t-tests. Multiple comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test or two-way ANOVA followed by Tukey’s post hoc test. The normality of populations and homogeneity of variances were tested before performing ANOVA. Post hoc tests were run only in the cases in which F achieved p<0.05 and in which there was no significant variance with respect to homogeneity. Statistical analysis was undertaken only when each group size was at least n=5, with n representing the number of independent variables. Technical replicates were not treated as independent variables. Unequal group sizes were due to (a) different sources due to the wide variety of experimental approaches, (b) the need to increase the “n” to ensure data reliability in some of the assays, (c) animal availability, and/or (d) economy of resources as directed by the 3Rs (Replacement, Reduction, and Refinement) rule that governs experimentation with animals. Differences were considered significant when p ≤ 0.05. Statistical analyses were carried out with GraphPad Prism software version 5 (San Diego, CA, USA; (RRID: SCR_002798)). Outlier tests were not used; all data points (representing the means of technical replicates) were used for analysis. The data and statistical analysis comply with the recommendations detailed elsewhere [49]. 4.16. Nomenclature of Targets and Ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY [ 62 ] and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 [6]. Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/24/9602/s1, Figure S1: Expansion of the image areas in Figure 5and position of the MW markers corresponding to immunoblots in Figure 5of the main paper (panels refer to those in Figure 5). Figure S2: Antibody specificity control assays. Author Contributions: G.N., J.L.L.-G. and R.F. designed and supervised the project. G.N. did all experiments except for those involving proximity ligation assays. A.L. and R.R.-S. helped with the preparation and cell transfection for the signaling assays. A.L., R.R.-S., A.I.R.-P. and I.R.-R. isolated tissue from fetuses and adult mice, performed PLA assays, and analyzed the results in a blinded fashion. R.F. and G.N. actively participated in writing and editing the manuscript. All authors have edited the paper and have received a copy of the final version. All authors have read and agreed to the published version of the manuscript. Int. J. Mol. Sci. 2020,21, 9602 18 of 21 Funding: The Molecular Neurobiology Laboratory of the University of Barcelona is considered a research group of excellence by the Regional Catalonian Government (grup consolidat #2017 SGR 1497). This organization does not provide specific funding for personnel, equipment, or reagents nor any payment for services. Conflicts of Interest: The authors declare no conflict of interest. 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