Characterization of the transmembrane orientation of aquaporin-1 using antibodies to recombinant fusion proteins

Biochemistry
W Daniel StamerJ W Regan

Abstract

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...Continue Reading

References

Mar 1, 1995·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·T C PappasC S Watson
Nov 20, 1957·The Journal of General Physiology·V W SIDEL, A K SOLOMON

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Citations

Mar 10, 2009·Experimental Eye Research·N W BaetzW D Stamer
Jul 29, 2008·Experimental Eye Research·W Daniel StamerC Ross Ethier
Sep 16, 2006·Experimental Eye Research·W D StamerB S McKay
Jun 6, 1998·Journal of Structural Biology·J B HeymannA Engel
Nov 16, 2006·Expert Opinion on Therapeutic Targets·Kandiah JeyaseelanE Marelyn Wintour
Feb 9, 2000·American Journal of Physiology. Renal Physiology·T L PannabeckerW H Dantzler

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