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.