Heat Shock Protein 70 Interacts with Aquaporin-2 (AQP2) and Regulates Its Trafficking

Title: Heat Shock Protein 70 Interacts with Aquaporin-2 (AQP2) and Regulates Its Trafficking
Authors: Lu, MD, PhD, Hua A.J.; Sun, Tian-Xiao; Matsuzaki, Toshiyuki; Yi, Xian-Hua; Eswara, Jairam; Bouley, Ph.D., Richard; McKee, Mary; Brown, Dennis
Publisher: Journal of Biological Chemistry
Date Published: September 28, 2007
Reference Number: 725
The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

The aquaporin 2 protein (AQP2) plays a major role in the kidney’s ability to reabsorb body water and therefore concentrate urine. It resides in the principal cells of the kidney collecting duct. In order to perform its function as a channel through which water can enter the cell, it travels from the cell interior to a section of the cell membrane called the apical membrane. This movement from the cell interior to membrane is called exocytosis. After AQP2 has performed its function as a water channel, it travels from the apical membrane back to the cell interior. This movement is called endocytosis. The term trafficking is used to describe the overall movement of AQP2 to and from the membrane.

AQP2 traffics to and from the apical membrane in a regular cycle. But it also responds to a molecular sequence initiated when the antidiuretic hormone, arginine vasopressin (AVP) binds with the vasopressin 2 receptor (V2R). When this binding occurs, AQP2 rapidly traffics to the cell membrane in greater numbers than normal. The trafficking is a complex process involving other cellular organisms. The AQP2 travels to and from the membrane in tiny sacs called vesicles. These vesicles are coated with the protein, clathrin. Other cytosolic proteins are involved in the trafficking as are such cytoskeletal components as actin filament, myosins and microtubules.

Lu, et al., conducted a series of experiments designed to see if the 70 kD heat shock proteins, hsc70 and hsp70 directly interacted with AQP2, and, if so, at what stage of its trafficking process. They were familiar with research revealing that heat shock protein 70 plays a role in endocytosis in general. And more recent research showed that hsc70 participates in the ATPase-dependent uncoating of clathrin-coated vesicles during endocytosis.

Using several different experimental techniques such as mass spectrometry, co-immunoprecipitation assays and direct binding analyses, Lu, et al., confirmed that hsp70 and hsc70 directly interacts with AQP2. Further experimentation suggested further details such as the importance of Ser256 in this interaction. When amino acids combine to form proteins, the amino acids so formed are called amino acid residues. The 256th amino acid residue in the chain of residues that form AQP2 is a serine amino acid. It is called Ser256, and it occurs in the tail end of the AQP2. Lu, et al.’s, experiments suggest that Ser256 plays an important role in the hsc70/AQP2 interaction. Ser256 is a place that accepts phosphate molecules for AQP2 in a process called phosphorylation. AQP2 phophorylation increases when AVP is administered to cell cultures containing AQP2. The research team’s data suggests that AVP enhances the interaction of AQP2 and heat shock protein 70 in cells.

Hypothesizing that hsc70 is crucial in AQP2’s return to the cell interior from the cell membrane, the researchers reduced the number of hsc70 in their experimental cell cultures and found that this induced AQP2 accumulation in cell membranes. Hu, et al., suggest that phosphorylated AQP2 accumulates in the cell membrane after being treated with AVP, and then afterwards AQP2 associates with hsc70 during its return to the cell interior via clathrin coated vesicles. The researchers’ finding that AQP2s with mutations at Ser256 have greatly reduced interaction with hsc70 supports their idea that Ser256 plays a pivotal role in the interaction of AQP2 and hsc70.

Nephrogenic Diabetes Insipidus (NDI) is a disorder characterized by the kidney’s inability to respond to AVP, with the result that AQP2 can not travel to the apical membrane in great enough numbers to allow water through the cell membrane. Some members of the researcher team have previously speculated on the possibility of inhibiting AQP2s return from the cell membrane as a way to keep enough AQP2s at the membrane to allow the kidney to absorb sufficient amounts of body water to alleviate the symptoms of NDI. Lu, et al.’s, research contributes to the growing body of knowledge that may lead to such a therapeutic intervention in NDI patients.