Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking.
Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)-dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference-mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.
Noda
Yumi
Y
Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan. ynodmed2@tmd.ac.jp
Horikawa
Saburo
S
Kanda
Eiichiro
E
Yamashita
Maho
M
Meng
Hu
H
Eto
Kayoko
K
Li
Yuhua
Y
Kuwahara
Michio
M
Hirai
Keiji
K
Pack
Changi
C
Kinjo
Masataka
M
Okabe
Shigeo
S
Sasaki
Sei
S
D000199:Actins / Q000378:metabolism*; D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D002460:Cell Line; D016764:Cell Polarity; D002470:Cell Survival; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D051399:Aquaporin 2; D008081:Liposomes; D011994:Recombinant Proteins; D014335:Tropomyosin; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases