The trafficking of proteins such as aquaporin-2 (AQP2) in the exocytotic pathway requires an active actin cytoskeleton network, but the mechanism is incompletely understood. Here, we show that the actin-related protein (Arp) 2/3 complex, a key factor in actin filament branching and polymerization, is involved in the shuttling of aquaporin-2 (AQP2) between the trans Golgi network (TGN) and the plasma membrane. Arp2/3 inhibition (using CK-666) or siRNA knockdown blocks vasopressin induced AQP2 membrane accumulation, and induces the formation of distinct AQP2 perinuclear patches positive for markers of TGN-derived clathrin coated vesicles. After a 20oC cold block, AQP2 formed perinuclear patches due continuous endocytosis coupled with inhibition of exit from the TGN-associated vesicles. Upon rewarming, AQP2 normally leaves the TGN, and redistributes into the cytoplasm, entering the exocytotic pathway. Inhibition of Arp2/3 blocked this process, and trapped AQP2 in clathrin positive vesicles. Taken together, these results suggest that Arp2/3 is essential for AQP2 trafficking, specifically for its delivery into the post-TGN exocytotic pathway to the plasma membrane.

Vasopressin (VP) regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the C-terminus, and among them, serine-256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of the serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells, and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and western blotting with phospho-specific antibodies. Using methyl-b-cyclodextrin (MBCD), cold block or bafilomycin, and Taxol, we blocked AQP2 at the plasma membrane, in the peri-nuclear trans-Golgi network (TGN), and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared to baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the TGN, or in cytoplasmic vesicles, and that this event is dependent on the expression of PKA in these cells.

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