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Inhibition of actin‐related protein (Arp) 2/3 complex blocks vasopressin‐induced AQP2 membrane accumulation
Author(s) -
Liu Chen-Chung Steven,
Cheung Pui Wen,
Bouley Richard,
Brown Dennis
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09019
Subject(s) - microbiology and biotechnology , aquaporin 2 , endosome , biology , cytoplasm , actin , actin cytoskeleton , cytoskeleton , chemistry , water channel , cell , biochemistry , intracellular , mechanical engineering , engineering , inlet
Aquaporin 2 (AQP2) is a water channel protein located primarily on principal cells of kidney collecting ducts, and is crucial for regulating body water homeostasis. Regulation of AQP2 trafficking is subject to hormonal control, mainly via the canonical vasopressin (VP) signaling pathway which stimulates AQP2 membrane accumulation. Active actin cytoskeleton remodeling also plays an important role in AQP2 trafficking, but the mechanism is incompletely understood. Using immunohistochemistry and confocal microscopy, we discovered that actin‐related protein (Arp) 2/3 complex, an actin nucleator, was highly expressed in inner medullary principal cells where it colocalized with AQP2. Using an Arp2/3 complex inhibitor, CK‐666, we found that VP‐induced AQP2 membrane accumulation was inhibited both in rat kidneys and LLC‐AQP2 cells in vitro. Instead of distributing throughout the cytoplasm, AQP2 in cells treated with CK‐666 was concentrated in vesicles forming a perinuclear patch, which was also positive for Rab‐11 (a recycling endosome marker) and clathrin (a trans Golgi Network (TGN) marker). Similar perinuclear AQP2 patches appear in cells incubated at 20°C (cold block), which allows endocytosis to continue, but prevents protein exit from the TGN. By rewarming the cells to 37°C, these perinuclear patches dissipate, and AQP2 quickly redistributes throughout the cytoplasm (cold block release). However, we found that in cells exposed to a 20°C cold block and treated with CK‐666, AQP2 patches failed to dissipate upon rewarming, suggesting that CK‐666 blocked release of AQP2 from the TGN in the exocytotic pathway. This effect of CK‐666 was independent of VP signaling, and did not alter the VP‐induced phosphorylation state of AQP2 at residues serine‐256, S269 and S261. In conclusion, inhibition of the Arp2/3 complex blocks VP‐induced AQP2 plasma membrane accumulation by blocking AQP2 exocytosis at the level of the TGN and the recycling endosome, but did not affect VP signaling pathway. This result suggests that actin filament nucleation and growth via Arp2/3 activity is essential for AQP2 recycling and trafficking. Support or Funding Information CCL is supported by NIH training grant 5T32DK007540. PWC is supported by NIH grant DK115901. DB is supported by NIH grant DK096586