Characterization of the transmembrane orientation of aquaporin-1 using antibodies to recombinant fusion proteins.
Aquaporin-1 (AQP1) is a member of a family of integral membrane proteins, the aquaporins, which function as molecular channels for the movement of water across the plasma membrane. While the primary structure of AQP1 has been obtained from the cloning of its cDNA, its secondary structure is less certain. In this study, antibodies have been generated to defined regions of AQP1 in order to characterize its secondary structure. The antibodies were produced in chickens against glutathione S-transferase fusion proteins which represented loops C and E, and the carboxyl terminus of AQP1 as defined in the six-transmembrane model of Preston and Agre [(1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11110]. Characterization of the antibodies showed that they recognized their corresponding fusion proteins as well as native AQP1 in erythrocytes and recombinant AQP1 expressed in COS7 cells. They differed, however, with respect to the specific conditions required for recognition. Thus, the anti-C-terminal antibodies recognized COS7 cells transfected with AQP1 that were fixed and permeabilized but did not recognize live cells (unpermeabilized). Conversely, antibodies to loop C labeled both live and fixed cells, while antibodies to loop E labeled live cells but not fixed. The data indicate that the carboxyl terminus of aquaporin-1 is intracellular and that loops C and E are extracellular. Furthermore, antibody recognition of loop E is very sensitive to the labeling conditions which may reflect an active role in the functional protein.
Stamer
W D
WD
Department of Pharmacology & Toxicology, University of Arizona, Tucson 85714, USA. stamer@tonic.pharm.arizona.edu
Snyder
R W
RW
Regan
J W
JW
D000818:Animals; D000906:Antibodies; D051398:Aquaporin 1; D020346:Aquaporins; D019556:COS Cells; D002645:Chickens; D017931:DNA Primers; D005982:Glutathione Transferase; D015151:Immunoblotting; D007150:Immunohistochemistry; D007473:Ion Channels / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008961:Models, Structural; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D020346:Aquaporins; D017931:DNA Primers; D007473:Ion Channels; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D051398:Aquaporin 1; D005982:Glutathione Transferase