Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

Abstract

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ∼2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01±0.10 ns) or Rab7 (2.11±0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78±0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09±0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

Figure 1

Subcellular distributions of (a) AT₁R, (b) Rab5, and (c) Rab7 in AT₁R-HEK293 cells. The AT₁R, 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 AT₁R with DAPI (blue) nuclear stain, (e) overlay of (a) (AT₁R-EGFP, green) and (b) (Rab5-Alexa 546, red), and (f) overlay of (a) and (c) (Rab7-Alexa 633, blue). Bar, 10 μm.

Figure 2

Internalization of AT₁R induced by Ang II (100 nM) treatment for (a–f) 5 min and (g–l) 30 min in AT₁R-HEK293 cells. Ang II induced the cellular internalization of (a,g) AT₁R, but not (b,h) Rab5 or (c,i) Rab7. (d,j) overlays of AT₁R-EGFP (green) and Rab5-Alexa 546 (red); (e,k) overlays of AT₁R-EGFP (green) and Rab7-Alexa 633 (blue), and (f,l) overlays of AT₁R-EGFP (green), Rab5-Alexa 546 (red), and Rab7-Alexa 633 (blue). Bar, 10 μm.

Figure 3

Quantitative analysis of the colocalizations of AT₁R-EGFP with Rab5-Alexa 546, Rab7-Alexa 633, and LAMP1-Alexa 633 in AT₁R-HEK293 cells. AT₁R-HEK293 cells were treated with vehicle (basal) or with Ang II (100 nM) 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 mean±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.

Figure 4

Coimmunoprecipitation of AT₁R-EGFP with (a) Rab5 and (b) Rab7 in AT₁R-HEK293 cells. Cells were treated with Ang II (100 nM), at the indicated periods. One mg of cell lysates were incubated with anti-GFP IgG at 4 °C. The precipitated protein complexes were immunoblotted with (a) Rab5 mAb or (b) anti-Rab7 IgG. Both Rab5 and Rab7 coimmunoprecipitated with AT₁R-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.

Figure 5

Analysis of the AT₁R-EGFP lifetime in the absence of acceptor in AT₁R-HEK293 cells. To separate the AT₁R 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 AT₁R-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 μm. (Color online only.)

Figure 6

Cytoskeletal disruption with cytoskeleton inhibitors in AT₁R-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 μm.

Figure 7

Subcellular distribution of AT₁R-EGFP and LAMP1-Alexa 633 in AT₁R-HEK293 cells. Cells were treated with Ang II (100 nM), at the indicated time periods, as described in Section 2. Green, AT₁R-EGFP; Red, LAMP1-Alexa 633; yellow, colocalization. Bar, 10 μm. (Color online only.)

Figure 8

FLIM-FRET analysis of AT₁R-EGFP interaction with LAMP1-Alexa 555 in AT₁R-HEK293 cells. Cells were treated with (a) vehicle (n=15 cells), Ang II (100 nM) for (b) 5 min (n=8 cells) or (c) 30 min (n=15 cells), as described in Section 2. The AT₁R-EGFP lifetime was analyzed in the presence of LAMP1-Alexa 555. Scale bar, 10 μm.

Figure 9

Comparison of the AT₁R-EGFP lifetimes obtained before and after photobleaching the acceptor, LAMP1-Alexa 555. AT₁R-HEK293 cells were treated with Ang II (100 nM) for 30 min, in the presence of LAMP1-Alexa 555 (acceptor). The DPSS 561 laser was used to photobleach the acceptor. Data are mean±S.E.; ∗ P<0.05, Student’s t-test.