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.
Upon activation, the angiotensin (Ang) II type 1 receptor (AT1Rs) rapidly undergoes endocytosis. After a series of
intracellular processes, the internalized AT1Rs recycle back to the plasma membrane or are trafficked to proteasomes or
lysosomes for degradation. We recently reported that AT1Rs degrades in proteasomes upon stimulation of the D5
dopamine receptor (D5R) in human renal proximal tubule and HEK-293 cells. This is in contrast to the degradation of
AT1R in lysosomes upon binding Ang II. However, the dynamic regulation of the AT1Rs in lysosomes is not well
understood. Here we investigated the AT1Rs lysosomal degradation using FRET-FLIM in HEK 293 cells heterologously
expressing the human AT1R tagged with EGFP as the donor fluorophore. Compared to its basal state, the lifetime of
AT1Rs decreased after a 5-minute treatment with Ang II treatment and colocalized with Rab5 but not Rab7 and LAMP1.
With longer Ang II treatment (30 min), the AT1Rs lifetime decreased and co-localized with Rab5, as well as Rab7 and
LAMP1. The FLIM data are corroborated with morphological and biochemical co-immunoprecipitation studies. These
data demonstrate that Ang II induces the internalization of AT1Rs into early sorting endosomes prior to trafficking to late
endosomes and subsequent degradation in lysosomes.
The recycling of G-protein-coupled receptors (GPCR) to the cell surface after internalization plays an important role in the regulation of overall GPCR activity. The angiotensin II type I receptor (AT1R) belongs to class B GPCRs that recycle slowly back to the cell surface. Previous studies have proposed that Rab11 controls the recycling of AT1R; however, recent reports show that Rab4, a rapid recycling regulator, co-localizes also with internalized AT1R. Different from the subcellular co-localization provided by fluorescence microscopy, fluorescence resonance energy transfer (FRET) microscopy provided the spatial relationship of AT1R with Rab4 and Rab11 in the nanometer-range proximity during the entire course of AT1R recycling. During the early recycling stage, internalized AT1Rs were mainly associated with Rab4 in the cytoplasm. During the mid-recycling stage, AT1Rs were associated with both Rab4 and Rab11 in the perinuclear compartments. However, during the late-recycling stage, AT1Rs were mainly associated with Rab11, both in the perinuclear compartments and the plasma membrane. Co-immunoprecipitation data confirmed these dynamic associations, which were disrupted by silencing of either the Rab4 or Rab11 gene. Based on these observations, we propose a Rab4 and Rab11 coordinated model for AT1R recycling.
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