key: cord-0691338-nkhpck9q
authors: Portales, Andrea Estefanía; Mustafá, Emilio Román; McCarthy, Clara Inés; Cornejo, María Paula; Couto, Paula Monserrat; Gironacci, Mariela Mercedes; Caramelo, Julio Javier; Perelló, Mario; Raingo, Jesica
title: ACE2 internalization induced by a SARS-CoV-2 recombinant protein is modulated by angiotensin II type 1 and bradykinin 2 receptors
date: 2022-01-14
journal: Life Sci
DOI: 10.1016/j.lfs.2021.120284
sha: a076a7ae6c8e06d53be6fcdf845050c35e351f06
doc_id: 691338
cord_uid: nkhpck9q

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. MATERIALS AND METHODS: To study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (Ca(V)2.2) currents by patch-clamp technique. KEY FINDINGS: RBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of Ca(V)2.2 currents. SIGNIFICANCE: Our work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.

Angiotensin-converting enzyme 2 (ACE2) is a key counterbalancing enzyme of the renin-angiotensin system (RAS) that catalyzes the conversion of the pressor hormone angiotensin II (Ang II) into the protective angiotensin 1-7 (Ang 1-7) [1] . Ang II acts through the angiotensin II type 1 receptor (AT1R) and causes vasoconstriction, apoptosis, inflammation and fibrosis. In contrast, Ang1-7 acts via the Mas receptor (MasR) and causes vasodilation as well as anti-inflammatory and anti-proliferative effects [2] [3] [4] . The interest on ACE2 research was renewed with the outbreak of the coronavirus infectious disease 2019 (COVID-19) since this cell surface enzyme was identified as the main receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entrance into host alveolar type II epithelial cells [5] [6] [7] . Similar to other viruses, SARS-CoV-2 takes advantage of the cellular endocytic machinery to initiate the infection [8] . The spike protein (S protein) anchored to the viral envelope binds to of GPCRs functionality by interaction with other receptors or membrane proteins, such as ACE2.

Here we explored the role of AT1R and B2R as well as the effect of their ligands on the internalization of GFP-tagged ACE2 (ACE2-GFP) induced by a recombinant RBD of SARS-CoV-2 S protein in a heterologous expression system. Moreover, we monitored changes in AT1R and B2R functionality upon ACE2 coexpression by assaying their effect on Ca V 2.2 currents. Our results unmask a role for AT1R and B2R on RBD-induced ACE2 internalization, and provide new insights into the modulation of SARS-CoV-2 infection with an ultimate therapeutic purpose.

We first assessed whether RBD induces internalization of ACE2 in our experimental model. We incubated HEK293T cells transfected with ACE2-GFP with recombinant RBD for 40 minutes and stained the plasma membrane with a live-cell labeling compound previously used by our group [52, 53, 55] . After the incubation, in RBD-treated cultures a fraction of cells showed clustered GFP signal at the plasma membrane or in intracellular compartments, whereas in vehicle-treated cultures cells only displayed GFP signal uniformly distributed at the plasma membrane (Fig. 1A ).

Next, we performed an immunostaining against RBD in HEK293T cells expressing ACE2-GFP incubated with RBD for 40 minutes to assess RBD internalization (Fig. 1B) .

Consistent with our previous observations, in vehicle-treated cultures we only found cells with GFP signal uniformly distributed at the plasma membrane, whereas in RBDtreated cultures we mostly found cells with GFP signal clustered in spots in the plasma membrane or inside the cell. As expected, red fluorescent signal indicating RBD immunoreactivity was only observed in transfected cells (ACE2-GFP positive) of RBDtreated cultures. Importantly, RBD-immunoreactive signal was distributed in discrete clusters at the plasma membrane and the intracellular compartment and colocalized with GFP signal, suggesting an RBD-ACE2 joint internalization. These observations indicate that RBD induces ACE2-GFP internalization in our experimental system. follows: GFP uniformly distributed in the plasma membrane (PMU GFP), GFP clustered in the plasma membrane (PMC GFP), and GFP clustered in intracellular compartments with or without clustered GFP in the plasma membrane (Internalized GFP). In vehicletreated cultures, all cells corresponded to the PMU GFP group, whereas in RBDtreated cultures a significant number of cells with PMC GFP or internalized GFP was observed (Fig. 2B) . After 5 and 10 minutes of RBD incubation, the majority of the cells (~65-70%) showed PMC GFP signal, with a small proportion showing internalized GFP at 10 minutes. Notably, the percentage of cells displaying internalized GFP increased (~60%) and the percentage of cells with PMC GFP (~30%) decreased at 40 minutes of incubation. In order to statistically compare this observation, we arranged the different GFP signal patterns into degrees of internalization (PMU GFP-degree 0, PMC GFPdegree 1, and Internalized GFP-degree 2) and tested the association between RBD incubation time and the frequency of cells with different ACE2-GFP internalization degree. We found a strong positive association between these two variables (χ 2 = 190.03, P chi < 0.0001; Somers´ D = 0.57, directional coefficient for the association between RBD incubation time and ACE2-GFP internalization degree). These results suggest that RBD induces ACE2 internalization in a time-dependent manner, and allow us to propose that the different patterns of GFP signal discriminated in our system correspond to different degrees of ACE2-GFP internalization.

In order to test if AT1R affects RBD-induced ACE2 internalization, we transfected HEK293T cells with ACE2-GFP alone or with ACE2-GFP plus AT1R and incubated them with RBD for 40 minutes (Fig. 3A) . We found that the coexpression of AT1R failed to change the distribution of cells with different GFP signal patterns in both vehicle-and RBD-treated conditions in comparison with cultures only expressing ACE2-GFP. To assess the effect of AT1R ligands on RBD-induced ACE2-GFP internalization, we next incubated cells coexpressing ACE2-GFP and AT1R with vehicle or RBD plus vehicle, an AT1R agonist (Ang II) or an AT1R antagonist internalization degree. In particular, we considered AT1R activity levels as 0 in telmisartan-coincubated cells, 1 in vehicle-coincubated cells and 2 in Ang IIcoincubated cells, and the internalization degrees as done above (PMU GFP-degree 0, PMC GFP-degree 1, and Internalized GFP-degree 2). We found a significant positive correlation between AT1R activity levels and the frequency of cells displaying the different ACE2-GFP internalization degrees (χ 2 = 17.89, P chi < 0.01; Somers´ D = 0.24).

These data indicate that RBD induced-ACE2 internalization depends on AT1R activity.

In order to test if B2R affects RBD-induced ACE2 internalization, we incubated HEK293T cells transfected with ACE2-GFP alone or plus B2R with RBD for 40 minutes (Fig. 4) . In RBD-treated cultures, we found that B2R coexpression dramatically reduced 

Increasing evidence indicates that AT1R and B2R form heteromers whose properties differ from the individual receptors [36, 43, 61, 62] . To test if AT1R and B2R coexpression affects RBD-induced ACE2-GFP internalization, we incubated HEK293T cells co-transfected with ACE2-GFP and AT1R or ACE2-GFP, AT1R and B2R with increase in the frequency of cells with internalized GFP concomitant with a decrease in the frequency of cells with PMC GFP compared with the other conditions indicating a partial rescue of RBD-induced ACE2-GFP internalization. Taken together, these experiments suggest that Ang II has a pro-internalizing effect that overcomes B2R inhibitory action on RBD-induced ACE2 internalization.

6. ACE2-GFP coexpression modifies AT1R-, but not B2R-ligand evoked inhibition of Ca V 2.2 currents.

Since AT1R and B2R modulated RBD-induced ACE2-GFP internalization, we then explored if conversely ACE2-GFP affects AT1R and B2R ligand-evoked action on Ca V 2.2, a well-established GPCRs target [46] . First, we tested the effect of ACE2-GFP coexpression on BK-evoked B2R activity in HEK293T cells expressing Ca V 2.2 (plus its auxiliary subunits) and B2R in presence or absence of ACE2-GFP. As shown before [50] , BK-evoked B2R activity inhibited Ca V 2.2 currents. Similar percentages of Ca V 2.2 current inhibition were detected in cells coexpressing ACE2-GFP in presence of BK ( Fig. 6A) . Interestingly, AT1R co-expression reduced BK-induced Ca V 2.2 current inhibition, and such effect was restored to control levels (+ B2R) in cells coexpressing B2R, AT1R and ACE2-GFP. Next, in order to test the effect of ACE2-GFP coexpression on Ang II-evoked AT1R activity we measured calcium currents in HEK293T cells expressing Ca V 2.2 (plus its auxiliary subunits) and AT1R in presence or absence of ACE2-GFP. As previously shown [49, 57] , Ang II-evoked AT1R activity inhibited Ca V 2.2 currents (Fig. 6B) . Notably, the coexpression of AT1R with either ACE2-GFP, B2R or ACE2-GFP plus B2R decreased Ang II-mediated Ca V 2.2 currents inhibition. Thus, ACE2-GFP coexpression impacts on Ang-evoked AT1R action on Ca V 2.2 currents but does not affect BK-evoked B2R effect on these currents.

To study if ACE2-GFP coexpression modifies the ligand-independent activities of B2R and AT1R, we then assessed Ca V 2.2 currents in HEK293T cells expressing B2R, AT1R or both receptors in presence or absence of ACE2-GFP. We found that ACE2-GFP coexpression did not change basal Ca V 2.2 macroscopic currents in AT1Rexpressing cells but significantly reduced them in B2R-expressing cells, in comparison with cells expressing only the respective GPCR ( Fig. 7A and B) . Strikingly, ACE2-GFP coexpression increased the Ca V 2.2 currents in HEK293T cells expressing B2R and AT1R (Fig. 7C) . Thus, ACE2-GFP coexpression has opposite effects on basal Ca V 2.2 currents in B2R and B2R-AT1R expressing cells.

Journal Pre-proof DISCUSSION ACE2 is a transmembrane enzyme important for the regulation of the RAS that has regained interest because it was recently identified as the receptor for SARS-CoV-2. Thus, the study of the factors and mechanisms that regulate ACE2 internalization would provide new insights into the modulation of coronavirus infection. Here, we not only confirmed that the RBD of SARS-CoV-2 S protein induces ACE2 internalization, but also demonstrated for the first time that this process is modulated by GPCRs involved in the RAS. Using the AT1R agonist Ang II and the AT1R antagonist telmisartan, we revealed that the activity of AT1R positively correlates with the degree of RBD-induced ACE2 internalization. We also uncover a novel and strong inhibitory effect of B2R on RBD-induced ACE2 internalization that persists in the presence of AT1R and is mildly modified by its endogenous ligand BK. Notably, the inhibitory action of B2R coexpression on ACE2 internalization was reversed by Ang II in the presence of AT1R, which positions this hormone as a pro-internalizing factor. Complementary, we found that ACE2 modifies the capability of AT1R and B2R to inhibit Ca V 2.2 calcium currents likely due to an interaction with these receptors.

Current results indicate that RBD rapidly induces ACE2-GFP internalization in a time-dependent fashion. ACE2 internalization is induced by SARS-CoV-2 and other related viruses such as HCoV-NL63 and SARS-CoV [7, 9, 63, 64] . RBD is the minimal receptor-binding domain of the S protein from SARS-CoV-2 and its predecessor SARS-CoV, and has been previously used as a model to study viral infections [65] . In cell cultures incubated with RBD, we observed clear signs of ACE2-GFP internalization with cells displaying clustered GFP signal at the cytoplasm or the plasma membrane that colocalized with RBD signal. A similar ACE2 signal pattern denoting internalization has been previously shown by other authors studying SARS-CoV infections. In those reports, nearly all HEK293T cells expressing ACE2-GFP incubated for 3 hours with a recombinant RBD, S protein, or lentiviruses pseudotyped with S protein exhibited punctuated cytoplasmic GFP signal [65, 66] . However, cells with clustered GFP signal at the plasma membrane (PMC GFP) were not reported presumably due to the longer incubation time tested (3 hours) [65, 66] in comparison with ours (40 min), which may allow a complete internalization of ACE2. We hypothesize that the prevalence of cells with PMC GFP at short time points indicates an initial stage in the internalization of the ACE2-RBD complex. Further supporting this notion, a similar distribution of clustered signal at the plasma membrane was shown for other membrane proteins endocyted via clathrin-mediated pathways [67] and upon inhibition of membrane receptors internalization [68] . Notably, the kinetics of ACE2-GFP internalization observed in the J o u r n a l P r e -p r o o f Journal Pre-proof current study is in agreement with recent observations showing SARS-CoV-2 S protein uptake as early as 5 minutes, and higher uptake at 30 minutes of exposure [10] .

Here we reveal a novel inhibitory action of B2R on RBD-induced ACE2-GFP internalization. Previous studies have reported effects of B2R retaining receptors at the cell membrane by heteromerization. In this regard, B2R coexpression was shown to delay Ang1-7-induced sequestration of MasR from the plasma membrane [69] , and to stabilize B1R at the cell surface in the absence of its ligand [70] . Thus, it is likely that B2R prevents RBD-induced ACE2-GFP internalization due to a direct interaction with ACE2. Further supporting this possibility, we found that ACE2 coexpression alters basal calcium currents in cells expressing B2R alone or plus AT1R. Besides, the formation of complexes between ACE2 and other receptors of the RAS (AT1R, AT2R, MasR) has been extensively demonstrated [21, 22, 69] . Interestingly, we also observed that neither AT1R coexpression, a GPCR known to heteromerize with B2R [43, 61, 62] , nor BK application were able to unlock the B2R inhibition of ACE2 internalization suggesting that the functional crosstalk between B2R and ACE2 is strong. In the context of SARS-CoV-2 infection, our findings indicate that B2R is a protective factor that acts interrupting or delaying viral entry. Further protective effects of B2R might emerge from the retention of ACE2 on the plasma membrane that could avoid Ang II accumulation and promote the Ang 1-7/MasR axis counteracting Ang II detrimental effects in the infection and inflammation [16] .

We found that RBD-induced ACE2-GFP internalization is modulated by AT1R.

Previous studies have shown that Ang II triggers AT1R and ACE2 co-internalization [22] , and ACE2 coexpression downregulates Ang II induced-AT1R signaling [21] as a consequence of an interaction between ACE2 and AT1R. Further supporting this, in our electrophysiological experiments ACE2 coexpression dramatically impaired Ang IIinduced AT1R inhibitory action on Ca V 2.2 calcium currents. Thus, it is likely that Ang II enhances ACE2 internalization trough the AT1R-ACE2 complex increasing the percentage of cells with internalized ACE2-GFP. Conversely, telmisartan binding to AT1R would modify the conformation of the ACE2-AT1R complex and prevent the interaction of RBD with ACE2 leading to a reduction in the percentage of cells with internalized ACE2-GFP. Interestingly, Ang II not only increased ACE2-GPF internalization in presence of AT1R, but also in cells coexpressing AT1R and B2R, unlocking the strong B2R inhibition of RBD-induced ACE2 internalization. This observation indicates a stronger influence of AT1R on ACE2 than B2R. In summary, our findings show that Ang II is a potent pro-internalizing factor that might promote SARS-CoV-2 entry, while telmisartan has opposite effects and might act as an infection Acute respiratory distress syndrome is a hallmark of severe cases of and is the result of a systemic inflammatory response known as the cytokine storm [71] . It has been suggested that accumulation of Ang II due to the reduction in plasma membrane ACE2 during SARS-CoV-2 infection contributes to such pro-inflammatory state [1, 13, 18] . AT1R blockers have been proposed to avoid the detrimental effects of Ang II accumulation [19, 20] . In this regard, in a recent clinical trial in COVID-19 patients telmisartan showed potent anti-inflammatory actions as well as a reduction in the morbidity and mortality of the disease [72] . The modulation of RBD-induced ACE2 internalization by AT1R ligands described here demonstrates an effect of these compounds in the infection per se, besides the previously shown in the inflammatory state of patients suffering from COVID-19. This suggests a dual role of AT1R ligands in the disease: Ang II might act as a pro-inflammatory and pro-internalizing factor, whereas telmisartan might act as an anti-inflammatory and an infection-delaying agent.

The pro-internalizing action of Ang II found here extends the knowledge about the mechanisms underlying the occurrence of hypertension as a risk factor for the severity of COVID-19 [28] [29] [30] 73] . On the other hand, the anti-internalizing action of telmisartan supports the use of AT1R blockers to avoid or counteract Ang II detrimental effects and could explain in part the reduced severity and mortality of the disease in hypertensive patients chronically treated with AT1R [74] [75] [76] .

In summary, our study demonstrates that AT1R and B2R modulate the internalization of ACE2 induced by a SARS-CoV-2 surface protein, and inversely, ACE2 modifies AT1R and B2R functionality. Altogether, the current findings contribute to understand the role of RAS components in the susceptibility to SARS-CoV-2 infection and in the severity of COVID-19, and pave the way through the development of drugs aimed to limit infections caused by viruses using ACE2 as membrane receptor. 

The RBD of spike protein 1 from SARS-CoV-2 was synthesized as described before [77] . Angiotensin II was purchased from Sigma-Aldrich (A9525). Bradykinin was with CsOH). The test-pulse protocol consisted in square pulses applied from -100 to +10 mV for 30 ms every 10 s All recordings were obtained at room temperature (~ 24 °C).

Data of GFP patterns abundance from imaging experiments were represented in Graph Pad Prism (GraphPad Software, Inc.) and analyzed in IBM SPSS Statistics 26 (IBM Corp., Armonk, N.Y., SA). For the analysis, data were distributed in contingency tables and analyzed by the Chi-square (χ 2 ) test to determine the association between variables. The association was considered statistically significant when the P corresponding to the χ 2 value (P chi ) was lower than 0.05. To determine statistical significance of the difference between the observed and expected frequencies of GFP patterns abundance in each condition an adjusted standardized residual (z-score) was calculated for each cell of the contingency table and transformed into a P value. The abundance of a GFP pattern was considered statistically different from the expected frequency when its calculated P value was lower than the P corrected for type I error (P corr ) [79, 80] , and was indicated with an asterisk in the graph. The expected GFP pattern frequencies were displayed in the corresponding graph. In cases with a significant association between two ordinal variables, the Somers´ D coefficient was Data were expressed as mean ± standard error of the mean (SEM). 

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We thank the Profesional de Apoyo Principal Dr. Silvia Susana Rodriguez (Comisión de Investigaciones Científicas, Provincia de Buenos Aires CIC-PBA) for her excellent technical work.

This work was supported by grants from the Fondo para la Investigación Científica y Tecnológica (FONCyT, PICT2017-0602 to J.R. and PICT2016-1084 to M.P). A.E.P was also supported by FONCyT and E.R.M., and J.R. were supported by (CONICET).

The authors declare that there are no conflicts of interest.

The datasets that support the current study are available from the corresponding author upon request.