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 $∼2.33ns$. The basal state lifetime was shortened slightly in the presence of Rab5 $(2.01±0.10ns)$ or Rab7 $(2.11±0.11ns)$ 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 $(1.78±0.31ns)$ but was affected minimally in the presence of Rab7-Alexa 555 $(2.09±0.37ns)$. 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.