Heat shock protein 70 interacts with aquaporin-2 and regulates its trafficking.
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.
Lu
Hua A J
HAJ
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. Electronic address: halu@partners.org.
Sun
Tian-Xiao
TX
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
Matsuzaki
Toshiyuki
T
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
Yi
Xian-Hua
XH
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
Eswara
Jairam
J
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
Bouley
Richard
R
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
McKee
Mary
M
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
Brown
Dennis
D
Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. Electronic address: brown@receptor.mgh.harvard.edu.
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D051399:Aquaporin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014168:Transferrin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D014667:Vasopressins / Q000494:pharmacology
D051399:Aquaporin 2; D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494373:Hspa8 protein, rat; D014168:Transferrin; D014667:Vasopressins; D000255:Adenosine Triphosphate