Localization of AQP5/AQP8 chimeras in MDCK-II cells: exchange of the N- and C-termini.
AQP5 and AQP8 possess targeting/retention motifs which mediate their localization to the apical and basolateral membranes, respectively, of polarized MDCK-II cells. As targeting/retention motifs have been localized to the N- or C-termini of other AQPs, we sought the location of such motifs in AQPs 5 and 8 by exchanging their corresponding N- or C-termini and examining the expression, localization, and function of the resultant chimeras. We did not detect the expression of constructs in which the C-terminus of AQP5 was replaced by the C-terminus of AQP8. Substitution of the N-terminus of AQP8 for the N-terminus of AQP5 generated a construct which was trapped intracellularly and did not significantly facilitate transepithelial fluid movement. In contrast, modifications of the N- and C-termini of AQP8 were better tolerated. Substitution of either AQP8 terminus by the corresponding AQP5 terminus generated constructs which localized to basolateral membranes and facilitated transepithelial fluid movement. Our results suggest that, unlike the other AQP targeting/retention signals reported thus far, an AQP8 basolateral targeting/retention motif might reside between the two cytosolic termini.
Wellner
Robert B
RB
Gene Transfer Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Cranciofacial Research, National Institutes of Health, DHHS, Bethesda, MD 20892, USA.
Cotrim
Ana P
AP
Hong
Sohee
S
Swaim
William D
WD
Baum
Bruce J
BJ
D000818:Animals; D051402:Aquaporin 5; D020346:Aquaporins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
D051402:Aquaporin 5; D020346:Aquaporins; D007473:Ion Channels; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C108330:aquaporin 8