Luminal ANG II is internalized as a complex with AT1R/AT2R heterodimers to target endoplasmic reticulum in LLC-PK1 cells

Abstract

ANG II has many biological effects in renal physiology, particularly in Ca²⁺ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT₁ and AT₂ receptors (AT₁R and AT₂R) to stimulate sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT₁R/AT₂R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK₁ cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT₁R/AT₂R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of β-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of β-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT₁R, AT₂R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT₁/AT₂ complex to target ER, where it might trigger intracellular Ca²⁺ responses.

Keywords: ANG II-AT1R/AT2R complex; ANG II-AT1R/AT2R internalization; LLC-PK1 luminal membranes; endoplasmic reticulum; microtubule-dependent clathrin-independent endocytosis.

Fig. 1.

ANG II is internalized through the luminal membranes of LLC-PK₁ cells with AT₁R/AT₂R heterodimers. A: cells incubated with FAM-ANG II (0.1 μM) for 30 min at 37°C. B–D: cells treated with losartan (1 μM) (LOS) or PD123319 (1 μM) (PD), or both antagonists for 20 min before FAM-ANG II incubation. Arrows indicate ANG II intracellular localization (A, D) or plasma membrane retention (B, C). E: negative control (cells only, no FAM-ANG II).

Fig. 2.

HPLC quantification of ANG II in culture medium containing LLC-PK₁ cells preincubated or not with losartan (0.1 nM), PD123319 (0.1 μM), or with both antagonists, as shown on the abscissa, for 15 min at 37°C before adding 8 µM ANG II. The assayed material was further incubated for 2 h at 37°C. Data bars indicate means ± SE (n = 3−7). The empty bar corresponds to ANG II at zero time. Different lowercase letters above the bars indicate statistical difference between the mean values (P < 0.05).

Fig. 3.

ANG II-induced stimulation of SERCA activity from LLC-PK₁ cells is preserved when the AT₁R and AT₂R antagonists are both added. Cells were preincubated with losartan (0.1 nM) (Los) or PD123319 (0.1 μM) (PD), or with both antagonists, as shown on the abscissa, for 20 min before adding 0.1 nM ANG II. The cells were further incubated at 37°C for 30 min; longer times imply loss of activation, as described in Ref. . Data bars indicate means ± SE (n = 3–7). Different lowercase letters above the bars indicate statistical difference between the mean values (P < 0.05). Following the Committee on Publication Ethics (COPE) guidelines, we disclose that we have previously reported internal controls using losartan or PD123319 alone (19).

Fig. 4.

Internalization of ANG II-AT₁R/AT₂R complex is a microtubule-dependent and clathrin-independent endocytic pathway. The empty column in each panel represents medium ANG II before HPLC development (zero time). A: gray bar shows that 50% of the ANG II incubated for 2 h at 37°C was internalized, while the black bar shows binding of ANG II to the luminal membranes after 2 h at 4°C, since low temperature prevents internalization (27). B: cells incubated with 1 µM colchicine (microtubule polymerization inhibitor) for 2 h before 8 µM ANG II was added followed by incubation for 2 h at 37°C. C: cells incubated with 30 µM Pitstop 2 (a clathrin inhibitor) for 30 min before 8 µM ANG II was added and incubated for 2 h at 37°C. After the last incubation, culture media were carefully collected and analyzed by HPLC. The graphs represent ANG II in the culture media. Data bars indicate means ± SE (n = 3 or 4). Different lowercase letters above the bars indicate statistical differences among the mean values (P < 0.05).

Fig. 5.

ANG II stimulates β-arrestin phosphorylation in LLC-PK₁ cells. A: cells incubated with or without 1 μM ANG II for 30 min. Cell lysates (50 and 100 μg protein) were Western blotted using a polyclonal anti-p-β-arrestin antibody (top blot). After being stripped, membranes were probed with monoclonal anti-β-actin (bottom blot). B: densitometric representation of the immunoreactive signal ratio p-β-arrestin/β-actin. Data bars indicate means ± SE using different cell lysate preparations (n = 7 or 8). Different lowercase letters indicate statistical differences (P < 0.05).

Fig. 6.

ANG II internalized via luminal membrane of LLC-PK₁ is localized in the ER. LLC-PK₁ cells were incubated with ANG II-Alexa Fluor 488 (0.3 μM) for 1 h at 37°C and then with ER Tracker Red (0.1 μM) for 15 min at 37°C. Living cell fluorescent imaging using confocal microscopy was obtained for ANG II-Alexa Fluor 488 (A, D), ER Tracker (B, E), and merge (C, F). A: negative control for ANG II (cells only). B, C: cells incubated with only ER Tracker, i.e., without ANG II.

Fig. 7.

AT₁R and AT₂R and PKCα content increased in the ER of LLC-PK₁ cells after ANG II-AT₁R/AT₂R internalization. A: cell fractions p1−p6 obtained by differential centrifugation were Western blotted using monoclonal anti-Na⁺/K⁺-ATPase and anti-SERCA antibodies (upper and lower blots, respectively). B: cells incubated with or without 0.1 nM ANG II for 30 min, as indicated on the abscissa. Cell fractions p3−p6 were Western blotted using monoclonal anti-AT₁R, polyclonal anti-AT₂R, monoclonal anti-SERCA, and polyclonal anti-PKCα antibodies. C: densitometric representation of the immunoreactive signal ratio primary antibodies (AT₁R, AT₂R, or PKCα) and SERCA antibody in p5. Data bars indicate means ± SE (n = 2 or 3). *Statistically different from the control without ANG II (P < 0.05).

Fig. 8.

Specificity of the ANG II AT₁R and AT₂R antibodies. ER-enriched membrane preparations were probed against AT₁R monoclonal (A) or AT₂R polyclonal (B) antibodies with or without preincubation with recombinant AT₁R or AT₂R protein (as indicated). Preincubation of each antibody with the corresponding immunogenic peptide decreased the immunosignal, whereas preincubation with the other recombinant protein did not. 38 kDa corresponds to standard molecular weight used in all conditions.