2.1.

Antibodies and Reagents

Monoclonal mouse Rab5 antibodies were purchased from BD Transduction Laboratories (Lexington, Kentucky); polyclonal rabbit anti-Rab5, polyclonal rabbit anti-Rab7, polyclonal rabbit anti-GFP, and normal mouse and rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California); polyclonal rabbit LAMP1 antibody was from Affinity Bioreagents, Inc. (Rockford, Illinois); monoclonal GFP antibody was purchased from Clontech (Mountain View, California); Alexa Fluor 546 anti-mouse, Alexa Fluor 633 anti-rabbit, Alexa Fluor 633 anti-mouse secondary antibodies, Alexa Fluor 546 phalloidin, and Alexa-555 protein labeling kits were purchased from Invitrogen (Eugene, Oregon). Monoclonal $\alpha$ -tubulin antibody and Ang II were purchased from Sigma (St. Louis, Missouri). Latrunculin A and nocodazole were purchased from Calbiochem (Gibbstown, New Jersey).

2.2.

Cell Culture and Transfection

HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Gaithersburg, Maryland), containing $4.5\mathrm{g}∕\mathrm{L}$ glucose, 10% fetal bovine serum, $2\mathrm{mM}$ L-glutamine, and $1\mathrm{mM}$ sodium pyruvate. Human ${\mathrm{AT}}_1\mathrm{R}$ -EGFP, or its empty vector pEGFP-N1,²⁵ was transfected into HEK293 cells using Lipofectamine^™ 2000 transfection reagents (Invitrogen), according to the manufacturer’s instructions, as previously described.²⁶

2.3.

Immunoprecipitation and Immunoblotting

HEK293 and ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells were sonicated in lysis buffer ( $20\mathrm{mM}$ Tris⋅HCl, pH $8.0∕1\mathrm{mM}$ $\mathrm{EDTA}∕1\mathrm{mM}$ $\mathrm{Na}{\mathrm{N}}_3∕2\mathrm{mM}$ $\mathrm{DTT}∕0.25\mathrm{M}$ sucrose), with $0.5\mathrm{mM}$ benzamidine hydrochloride and protease inhibitor cocktail (Thermo Scientific, Rockford, Illinois). The homogenates $\left(1\mathrm{mg}\right)$ were incubated (rocking, $4°\mathrm{C}$ , $4\mathrm{h}$ ) with $5\mu \mathrm{g}$ of anti-GFP rabbit IgG, $5\mu \mathrm{g}$ of anti-Rab5 rabbit IgG, or $6\mu \mathrm{g}$ of anti-Rab7 rabbit IgG in $0.5\mathrm{mL}$ of the lysis buffer with $20\mu \mathrm{M}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , 0.1% ovalbumin, $0.5\mathrm{mM}$ 4-(2-aminoethyl)benzene sulfonyl fluoride (AEBSF), 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Normal rabbit IgG was used as negative control. After the addition of $60\mu \mathrm{L}$ of a 50% slurry of protein G-Sepharose CL-4B (Amersham Pharmacia, Piscataway, New Jersey) in phosphate-buffered saline (PBS) and incubation at $4°\mathrm{C}$ overnight, the beads were washed three times with $1\mathrm{mL}$ of ice-cold PBS, containing $0.5\mathrm{mM}$ AEBSF. Proteins bound to beads were eluted and separated, as previously described.²²

2.4.

Confocal Immunofluorescence Microscopy

Cells on coverslips were fixed with 4% paraformaldehyde in PBS for $20\min$ at room temperature. After washing with PBS, the fixed cells were incubated overnight at $4°\mathrm{C}$ with $3\mu \mathrm{g}∕\mathrm{mL}$ Rab5 mAb, $5\mu \mathrm{g}∕\mathrm{mL}$ anti-Rab7 rabbit IgG, or $2\mu \mathrm{g}∕\mathrm{mL}$ anti-LAMP1 rabbit IgG. After washing, the coverslips were incubated with Alexa Fluor $-546$ or $-633$ secondary antibodies for $2\mathrm{h}$ at $4°\mathrm{C}$ . Coverslips were mounted in SlowFade mounting medium (Invitrogen, Eugene, Oregon) and sealed onto glass slides. Samples were imaged using an Olympus Fluoview FV300 laser scanning confocal microscope, equipped with a $60\times ∕1.4\mathrm{NA}$ oil-immersion objective. Quantitative analysis was performed using MetaMorph 6.1 (Molecular Devices, Downingtown, Pennsylvania). Background was subtracted from each image and then the images were thresholded to identify specific protein fluorescence. Percentage of overlap of integrated intensity measurement was determined for ${\mathrm{AT}}_1\mathrm{R}$ over Rab5, Rab7, or LAMP1.

2.5.

FLIM System

The FLIM system was described in our previous report.²⁷ Briefly, we used a Nikon TE300 epi-fluorescence microscope equipped with a Plan Fluor $60\times ∕1.2\mathrm{NA}$ water-immersion IR objective. The microscope was coupled to a Biorad Radiance 2100 confocal/multiphoton system with the LaserSharp2000 software. A $10\text{-}\mathrm{W}$ Verdi pumped, tunable $(700–1000\mathrm{nm})$ mode-locked ultrafast $\left(78\mathrm{MHz}\right)$ pulsed $\left(150\mathrm{fs}\right)$ laser (Mira 900, Coherent, Inc., Santa Clara, California) was tuned to the $870\text{-}\mathrm{nm}$ wavelength to excite the donor fluorophore (EGFP). Emitted photons were collected by a fast photomultiplier tube with a $500–550\text{-}\mathrm{nm}$ bandpass emission filter. The lifetime photomultiplier tube was coupled in the connection arm of the external detector with a response time of $\sim 150\mathrm{ps}$ (PMC-100-0, Becker & Hickl GmbH, Berlin, Germany). The use of the time-correlated single-photon-counting module board (SPC-150, Becker & Hickl GmbH, Berlin, Germany) with a minimum temporal resolution of $\sim 40\mathrm{ps}$ allowed pixel-by-pixel registration of the accumulated photons. A fluorescence decay histogram of photons at different emission times relative to the laser excitation pulse was generated from the distribution of interpulse intervals at each pixel of the image.

2.6.

FLIM Data Acquisition

Polyclonal rabbit Rab5, Rab7, and LAMP1 IgG were digested by papain and separated by Sephadex G-100, and then conjugated with Alexa 555, according to the manufacturer’s instructions (Alexa Fluor 555 protein labeling kits, Invitrogen, Eugene, Oregon). ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells were treated with vehicle, Ang II ( $100\mathrm{nM}$ , 5 and $30\min$ ) without or with pretreatment with latrunculin A $\left(300\mathrm{nM}\right)$ , an actin polymerization inhibitor²³ or nocodazole $\left(10\mu \mathrm{M}\right)$ , a microtubule polymerization inhibitor²⁴ and fixed with 4% paraformaldehyde in PBS for $20\min$ at room temperature. After washing with PBS, cells on coverslips were incubated overnight at $4°\mathrm{C}$ with Alexa Fluor 555-conjugated Fab fragment (with a 3:1 of dye: Fab ratio) of polyclonal rabbit Rab5, Rab7, or LAMP1. Donor fluorophores were continuously scanned at the $870\text{-}\mathrm{nm}$ wavelength with appropriate excitation average power for $\sim 90\mathrm{s}$ to accumulate enough photon counts for fluorescence lifetime analysis. The same settings were used for samples in the presence or absence of the acceptor fluorophores (Alexa 555-labeled Rab5, Rab7, or LAMP1). In additional experiments, the DPSS $561\text{-}\mathrm{nm}$ laser, equipped on a ZEISS 510 Meta microscope system, was employed to photobleach the acceptor fluorophores (at least 50% intensity) in the region of interest or whole visual fields. A series of pre- and postbleaching FLIM data sets were acquired using the FLIM system, as described above. The comparison of the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetimes obtained before and after photobleaching the acceptor fluorophores was determined by Student’s t-test from 74–89 cells from three independent sets of experiments.

2.7.

FLIM Data Analysis

The data analysis was performed using the SPCImage software (V2.9, Becker & Hickl GmbH, Berlin, Germany), which also provides an estimate of the instrument response function of the FLIM system. The fluorescence lifetimes of the donor fluorophores were obtained with single or double exponential fitting on a pixel-by-pixel basis based on a weighted least-squares numerical approach as previously described.^{20, 27} The unquenched donor lifetime was obtained by fitting the donor-alone data with the single exponential decay. The double exponential fittings were employed to extract the quenched lifetime of the donor fluorophores, in the presence of the acceptor fluorophores. The fitting significance was judged based on the ${\chi }^2$ value and visually comparing the fitting curve with the raw data points. The average lifetime $\left({\tau }_m\right)$ is used for comparison of a change in lifetime.

3.1.

Cellular Localization of Human ${\mathrm{AT}}_1\mathrm{R}$ and Rab Proteins

Consistent with previous observations,^{18, 26} ${\mathrm{AT}}_1\mathrm{R}$ tagged with EGFP was observed primarily at the plasma membrane, but also in the intracellular compartments and cytoplasm in HEK293 cells, under basal conditions [Fig. 1 ]. Endogenous Rab5 was observed in the cytoplasm as punctuate vesicles of different sizes and intracellular membrane networks; slight staining of the plasma membrane was also observed [Fig. 1]. Endogenous Rab7 was scattered throughout the cytoplasm in punctuate vesicles of varying sizes, some of which were observed in perinuclear areas [Fig. 1]. Minimal colocalization was observed between ${\mathrm{AT}}_1\mathrm{R}$ and nuclei [Fig. 1]. However, some colocalization between ${\mathrm{AT}}_1\mathrm{R}$ and Rab5 [Fig. 1], and between ${\mathrm{AT}}_1\mathrm{R}$ and Rab7 [Fig. 1] was observed (see Fig. 3 for quantification).

Fig. 1

Subcellular distributions of (a) ${\mathrm{AT}}_1\mathrm{R}$ , (b) Rab5, and (c) Rab7 in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. The ${\mathrm{AT}}_1\mathrm{R}$ , tagged with EGFP at its C-terminus, is localized mainly at the plasma membrane. Both Rab5-Alexa 546 and Rab7-Alexa 633 are seen as punctuate structures throughout the cytoplasm. (d) overlay of ${\mathrm{AT}}_1\mathrm{R}$ with DAPI (blue) nuclear stain, (e) overlay of (a) ( ${\mathrm{AT}}_1\mathrm{R}$ -EGFP, green) and (b) (Rab5-Alexa 546, red), and (f) overlay of (a) and (c) (Rab7-Alexa 633, blue). Bar, $10\mu \mathrm{m}$ .

Fig. 3

Quantitative analysis of the colocalizations of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP with Rab5-Alexa 546, Rab7-Alexa 633, and LAMP1-Alexa 633 in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells were treated with vehicle (basal) or with Ang II $\left(100\mathrm{nM}\right)$ for 5 or $30\min$ , as indicated. Quantitative analysis was carried out using MetaMorph 6.1 (Molecular Device, Downingtown, Pennsylvania), as described in section 2. Data are $\text{mean}\pm \mathrm{S.E.}$ , $n=8–11$ cells per group. ANOVA, Student-Newman-Keuls test. ∗, $P<0.05$ versus Basal; #, $P<0.05$ versus Ang II $5\min$ treatment.

As shown in Fig. 2 , ${\mathrm{AT}}_1\mathrm{R}$ , as expected, internalized on activation with Ang II $\left(100\mathrm{nM}\right)$ . Endocytosis was more obvious with the $30\text{-}\min$ than with the $5\text{-}\min$ treatment [Figs. 2 and 2]. Ang II treatment did not induce any obvious change in the subcellular localization of Rab5 [Fig. 2 versus Fig. 1] and Rab7 [Fig. 2 versus Fig. 1]. However, the relationship between ${\mathrm{AT}}_1\mathrm{R}$ and these Rab proteins was changed dramatically compared to the basal state (Fig. 3 ). Colocalization was observed between ${\mathrm{AT}}_1\mathrm{R}$ and Rab5 at $5\min$ [Fig. 2]. At this time point, the colocalization between ${\mathrm{AT}}_1\mathrm{R}$ and Rab7 and between ${\mathrm{AT}}_1\mathrm{R}$ and LAMP1 was modest [Figs. 2, 3, 7], close to the basal state [Figs. 1 and 3]. When the duration of the Ang II treatment was increased to $30\min$ , ${\mathrm{AT}}_1\mathrm{R}$ was observed to colocalize with both Rab5 [Figs. 2 and 3] and Rab7 [Figs. 2 and 3]. There were a few vesicles showing the colocalization of the three proteins [Fig. 2].

Fig. 2

Internalization of ${\mathrm{AT}}_1\mathrm{R}$ induced by Ang II $\left(100\mathrm{nM}\right)$ treatment for (a–f) $5\min$ and (g–l) $30\min$ in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. Ang II induced the cellular internalization of (a,g) ${\mathrm{AT}}_1\mathrm{R}$ , but not (b,h) Rab5 or (c,i) Rab7. (d,j) overlays of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP (green) and Rab5-Alexa 546 (red); (e,k) overlays of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP (green) and Rab7-Alexa 633 (blue), and (f,l) overlays of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP (green), Rab5-Alexa 546 (red), and Rab7-Alexa 633 (blue). Bar, $10\mu \mathrm{m}$ .

Fig. 7

Subcellular distribution of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP and LAMP1-Alexa 633 in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. Cells were treated with Ang II $\left(100\mathrm{nM}\right)$ , at the indicated time periods, as described in Section 2. Green, ${\mathrm{AT}}_1\mathrm{R}$ -EGFP; Red, LAMP1-Alexa 633; yellow, colocalization. Bar, $10\mu \mathrm{m}$ . (Color online only.)

3.2.

Coimmunoprecipitation of ${\mathrm{AT}}_1\mathrm{R}$ with Rab Proteins

In agreement with the morphological observations, both Rab5 [Fig. 4 ] and Rab7 [Fig. 4] coimmunoprecipitated with GFP antibody after $5\min$ of Ang II treatment, that became more obvious after $30\min$ of treatment (Fig. 4). ${\mathrm{AT}}_1\mathrm{R}$ (-EGFP) also coimmunoprecipitated with Rab5 and Rab7 antibodies with Ang II treatment (data not shown). There was no coimmunoprecipitation in the basal state, which is consistent with the absence of obvious colocalization between ${\mathrm{AT}}_1\mathrm{R}$ and Rab5 or Rab7 observed with confocal microscopy (Fig. 1), but not with a previous report using GST-tagged Rab5 in transiently transfected COS7 cells.⁶ Whether or not the discrepancy is caused by the tagging of Rab5²⁸ or cell-specific trafficking remains to be determined.

Fig. 4

Coimmunoprecipitation of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP with (a) Rab5 and (b) Rab7 in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. Cells were treated with Ang II $\left(100\mathrm{nM}\right)$ , at the indicated periods. One mg of cell lysates were incubated with anti-GFP IgG at $4°\mathrm{C}$ . The precipitated protein complexes were immunoblotted with (a) Rab5 mAb or (b) anti-Rab7 IgG. Both Rab5 and Rab7 coimmunoprecipitated with ${\mathrm{AT}}_1\mathrm{R}$ -GFP in the samples treated with Ang II for $5\min$ and $30\min$ . NR, normal rabbit IgG; LC, IgG light chain; NS, nonspecific band; Lys, whole cell lysate.

3.3.

Dynamic Interaction of ${\mathrm{AT}}_1\mathrm{R}$ with Rab Proteins

To investigate further the interaction between ${\mathrm{AT}}_1\mathrm{R}$ and Rab proteins, FLIM-FRET analyses were performed in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells with ${\mathrm{AT}}_1\mathrm{R}$ -EGFP, as the donor fluorophore, and Rab5 or Rab7 Fab conjugated Alexa Fluor 555, as the acceptor fluorophores. First, we measured the fluorescence lifetime of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP, in absence of any acceptor. Without Ang II treatment, the average lifetime was $\sim 2.33\mathrm{ns}$ [Fig. 5, 5, 5 ]. In the absence of any acceptor, Ang II treatment, for $5\min$ or $30\min$ [Fig. 5, 5, 5], did not change that lifetime (Table 1 ). We also measured the lifetime of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP in the presence of Fab of Alexa Fluor 555 that was not conjugated to Rab proteins. Additional controls included EGFP, as the donor, in the presence or absence of Alexa Fluor 555, as the acceptor. The EGFP lifetimes obtained in all these controls were similar (Table 1).

Fig. 5

Analysis of the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime in the absence of acceptor in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. To separate the ${\mathrm{AT}}_1\mathrm{R}$ images from the background, the threshold was set at 50 (the minimum number of photon counts in the peak). The histograms (b,e) show the distribution of lifetime images of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP (a,d). The decay graphs (c,f) show the trace of the fit (red) to the photon decay data (blue) at a particular pixel in the lifetime image. Scale bar, $10\mu \mathrm{m}$ . (Color online only.)

Table 1

Lifetimes of AT1R -EGFP in AT1R -HEK293 cells. Cells were treated with Ang II (100nM) at the indicated time periods and fixed, as described in Section 2. N , number of cells analyzed; data are mean±S.E. ; ANOVA, Student–Newman–Keuls test. No significant differences among the control groups were detected.

Next, we studied the lifetime of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP in the presence of Rab proteins conjugated with Fab of Alexa Fluor 555. In the basal state, ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime was not changed by the presence of Rab7-Alexa 555, but slightly decreased in the presence of Rab5-Alexa 555 (Table 2 ), compared to that measured from the donor-alone control samples (Table 1). Treatment with Ang II for $5\min$ shortened the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime in the presence of Rab5-Alexa 555, but not in the presence of Rab7-Alexa 555, relative to their corresponding values at basal state (Table 2), indicating FRET occurrence between ${\mathrm{AT}}_1\mathrm{R}$ -EGFP with Rab5-Alexa 555, but not with Rab7-Alexa 555. When the Ang II treatment was increased to $30\min$ , the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime was shortened in the presence of both Rab5 and Rab7 labeled with Alexa 555 (Table 2), indicating FRET occurrence between ${\mathrm{AT}}_1\mathrm{R}$ and these two Alexa 555-labeled Rab proteins.

Table 2

Effect of Ang II on the lifetime of AT1R -EGFP in AT1R -HEK293 cells. Cells were treated with Ang II (100nM) at the indicated time periods and fixed, as described in Section 2. Data are mean±S.E. ; N , number of cells analyzed; ANOVA, Student-Newman-Keuls test.

3.4.

Cytoskeleton-Dependent ${\mathrm{AT}}_1\mathrm{R}$ Interactions with Rab Proteins

Next, we studied the role of the cytoskeleton in the interaction of ${\mathrm{AT}}_1\mathrm{R}$ with Rab5 or Rab7 by cytoskeleton polymerization inhibitors. Latrunculin A, a blocker of the polymerization of actin filaments [Fig. 6 ] but not microtubules [Fig. 6], did not affect ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime in the presence of Rab5- or Rab7-Alexa 555 without Ang II treatment (Table 2). However, latrunculin A pretreatment blocked the ability of Ang II to shorten the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime in the presence of Rab5- or Rab7-Alexa 555 (Table 2), indicating the requirement of intact actin for Rab5 and Rab7 to regulate ${\mathrm{AT}}_1\mathrm{R}$ intracellular trafficking. Nocodazole, which disrupts the polymerization of microtubules [Fig. 6] but not actin [Fig. 6], also had no significant effect on ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime on samples not treated with Ang II, in the presence of Rab5- or Rab7-Alexa 555 (Table 2). However, in contrast to latrunculin A pretreatment, nocodazole pretreatment did not block the ability of Ang II to shorten the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime, in the presence of Rab5- or Rab7-Alexa 555. The ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetimes measured after Ang II treatment with and without nocodazole pretreatment were similar (Table 2), indicating that intact microtubules are not required in the Rab5/7-mediated intracellular trafficking of ${\mathrm{AT}}_1\mathrm{R}$ .

Fig. 6

Cytoskeletal disruption with cytoskeleton inhibitors in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. Cells were treated with (a,b) vehicle (DMSO), (c,d) latrunculin A, or (e,f) nocodozale, and stained with fluorescein phalloidin to visualize F-actin (a,c,e) and anti-tubulin to visualize microtubules (b,d,f), as described in Section 2. Scale bar, $10\mu \mathrm{m}$ .

3.5.

FLIM-FRET Analysis of ${\mathrm{AT}}_1\mathrm{R}$ Association with Lysosomes

Following stimulation with Ang II, ${\mathrm{AT}}_1\mathrm{R}$ has been proposed to traffic to Rab7-positive late endosomes for eventual degradation in lysosomes.^{1, 3, 4} Therefore, we next investigated the relationship between ${\mathrm{AT}}_1\mathrm{R}$ and LAMP1, a marker protein for lysosomes. Confocal microscopy showed the colocalization of ${\mathrm{AT}}_1\mathrm{R}$ with LAMP1 following a $30\text{-}\min$ Ang II treatment, but only modest colocalization with a $5\text{-}\min$ Ang II treatment, close to the basal state (Figs. 3 and 7 ).

To corroborate the morphological studies, FLIM-FRET experiments were performed to observe the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime, in the presence of LAMP1-Alexa 555. Similar to the basal state with Rab5-Alexa 555, there was a modest decrease in ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime in the presence of LAMP1-Alexa 555 [Fig. 8 ], compared to that obtained with the donor-alone samples. The decrease in ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime in the basal state could have been caused by the acidic environment of lysosomes (see Section 4). With $5\text{-}\min$ Ang II treatment, no obvious lifetime change was observed [Fig. 8], compared to its basal state, indicating no FRET occurrence. The $30\text{-}\min$ Ang II treatment resulted in a dramatic decrease in the lifetime of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP, in some ${\mathrm{AT}}_1\mathrm{R}$ locations, indicating FRET occurrence between ${\mathrm{AT}}_1\mathrm{R}$ -EGFP and LAMP1-Alexa 555 in those locations [Fig. 8], suggesting this portion of ${\mathrm{AT}}_1\mathrm{R}$ degraded in lysosomes. Chloroquine, a lysosome inhibitor, abrogated the lifetime change that occurred with the $30\text{-}\min$ Ang II treatment (data not shown).

Fig. 8

FLIM-FRET analysis of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP interaction with LAMP1-Alexa 555 in ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells. Cells were treated with (a) vehicle ( $n=15$ cells), Ang II $\left(100\mathrm{nM}\right)$ for (b) $5\min$ ( $n=8$ cells) or (c) $30\min$ ( $n=15$ cells), as described in Section 2. The ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetime was analyzed in the presence of LAMP1-Alexa 555. Scale bar, $10\mu \mathrm{m}$ .

To confirm the observation of FRET occurrence between ${\mathrm{AT}}_1\mathrm{R}$ -EGFP and LAMP1-Alexa 555 with the $30\text{-}\min$ Ang II treatment detected by FLIM-FRET analysis, acceptor photobleaching experiments were performed. The average lifetime of ${\mathrm{AT}}_1\mathrm{R}$ -EGFP was $1.82\pm 0.04\mathrm{ns}$ ( $n=89$ cells) after photobleaching the acceptor, which was significantly longer than that obtained prior to bleaching ( $1.53\pm 0.07\mathrm{ns}$ , $n=74$ cells). In contrast, photobleaching the EGFP alone-Alexa 555 alone samples by the DPSS 561 laser (using the same power and duration of photobleaching) did not change the donor lifetime (Fig. 9 ). These studies support the conclusion of an association of ${\mathrm{AT}}_1\mathrm{R}$ with LAMP1 and that following Ang II stimulation, the ${\mathrm{AT}}_1\mathrm{R}$ is trafficked to lysosomes for eventual degradation.

Fig. 9

Comparison of the ${\mathrm{AT}}_1\mathrm{R}$ -EGFP lifetimes obtained before and after photobleaching the acceptor, LAMP1-Alexa 555. ${\mathrm{AT}}_1\mathrm{R}$ -HEK293 cells were treated with Ang II $\left(100\mathrm{nM}\right)$ for $30\min$ , in the presence of LAMP1-Alexa 555 (acceptor). The DPSS 561 laser was used to photobleach the acceptor. Data are $\text{mean}\pm \mathrm{S.E.}$ ; ∗ $P<0.05$ , Student’s t-test.