Mutations in sixth transmembrane domain of AQP2 inhibit its translocation induced by vasopression.
Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK(1) cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.
Yamashita
Y
Y
2nd Department of Internal Medicine, Bunkyo-ku, Tokyo 113-8519, Japan. ynodmed2@med.tmd.ac.jp
Hirai
K
K
Katayama
Y
Y
Fushimi
K
K
Sasaki
S
S
Marumo
F
F
D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000235:genetics*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D018374:LLC-PK1 Cells; D016253:Microscopy, Immunoelectron; D009154:Mutation / Q000502:physiology*; D051381:Rats; D013552:Swine; D013997:Time Factors; D014018:Tissue Distribution; D014667:Vasopressins / Q000494:pharmacology*
C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D014667:Vasopressins