Modifying the NH2 and COOH termini of aquaporin-5: effects on localization in polarized epithelial cells.
To reengineer polarized epithelial cell functions directly in situ, or ex vivo in the fabrication of an artificial organ, it is necessary to understand mechanisms that account for polarized membrane sorting. We have used the aquaporins (AQPs), a family of homotetrameric water channel proteins, as model membrane proteins for this purpose. AQP monomers contain six transmembrane-spanning domains linked by five interconnecting loops, with the NH2 and COOH termini residing in the cytosol. AQP5 is localized in the apical membranes of several different epithelia in vivo, and in stably transfected MDCK-II cells grown as a polarized monolayer. We wished to identify a structural region(s) within rat AQP5 (rAQP5) important for apical localization, and to study the MDCK-II cell localization of rAQP5s modified in either their NH2 or COOH terminus. We show that the NH2- terminal region does not play a major role in apical localization as deletion of the NH2 terminus produced a modified rAQP5 construct (AQP5-NT(del)) that was stably expressed and localized primarily to the apical membranes of MDCK-II cells. Attachment of a FLAG epitope to the NH2 terminus of AQP5 (AQP5(flag) construct) also did not perturb apical localization. In addition, we found that the exchange of NH2-terminal regions between rAQP5 and human AQP1 (hAQP1; a nonpolarized AQP isoform) produced a modified rAQP5 construct (AQP5-1NT) and a modified hAQP1 construct (AQP1-5NT), each of which localized as the parental AQP (apically, and to both apical and basolateral membranes, respectively). In contrast, we found that deletion of the COOH terminus resulted in a modified rAQP5 construct (AQP5-CT(del)) that was unstably expressed and localized to intracellular site(s) in MDCK-II cells. Substitution of the COOH terminus of AQP1 with the COOH terminus of AQP5 also produced a construct (AQP1-5CT) transiently expressed in intracellular compartment(s). However, substitution of the COOH terminus of rAQP5 with the COOH terminus of hAQP1 produced a modified rAQP5 construct (AQP5-1CT) that was stably expressed and localized to basolateral membranes, suggesting the loss of an apical targeting/retention signal from rAQP5, the gain of a basolateral targeting/retention signal from hAQP1, or a combination of these two possibilities.
Wellner
Robert B
RB
Gene Transfer Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-1190, USA.
Hong
Sohee
S
Cotrim
Ana P
AP
Swaim
William D
WD
Baum
Bruce J
BJ
D000818:Animals; D051402:Aquaporin 5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012235:Rhodamines; D014162:Transfection
D051402:Aquaporin 5; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012235:Rhodamines; C009434:tetramethylrhodamine isothiocyanate; D016650:Fluorescein-5-isothiocyanate