Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy.
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.
Children's Research Institute, Children's National Medical Center, Center for Molecular Physiology Research, Washington, DC 20010, USA.
Hewang
H
Li
Peiying
P
Yu
Yuansheng
Y
Sun
Robin A
RA
Felder
Ammasi
A
Periasamy
Pedro A
PA
Jose
D000199:Actins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1
D000199:Actins; D049452:Green Fluorescent Proteins; D051907:Lysosomal Membrane Proteins; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C485184:enhanced green fluorescent protein