Aquaporin 2 (AQP2) and vasopressin type 2 receptor (V2R) endocytosis in kidney epithelial cells: AQP2 is located in 'endocytosis-resistant' membrane domains after vasopressin treatment.
BACKGROUND
Aquaporin 2 (AQP2) plays an important, VP (vasopressin)-regulated role in water reabsorption by the kidney. The amount of AQP2 expressed at the surface of principal cells results from an equilibrium between the AQP2 in intracellular vesicles and the AQP2 on the plasma membrane. VP shifts the equilibrium in favour of the plasma membrane and this allows osmotic equilibration to occur between the collecting duct lumen and the interstitial space. Membrane accumulation of AQP2 could result from a VP-induced increase in exocytosis, a decrease in endocytosis, or both. In the present study, we further investigated AQP2 accumulation at the cell surface, and compared it with V2R (VP type 2 receptor) trafficking using cells that express epitope-tagged AQP2 and V2R.


RESULTS
Endocytosis of V2R and of AQP2 are independent events that can be separated temporally and spatially. The burst of endocytosis seen after VP addition to target cells, when AQP2 accumulates at the cell surface, is primarily due to internalization of the V2R. Increased endocytosis is not induced by forskolin, which also induces membrane accumulation of AQP2 by direct stimulation of adenylate cyclase. This indicates that cAMP elevation is not the primary cause of the initial, VP-induced endocytic process. After VP exposure, AQP2 is not located in endosomes with internalized V2R. Instead, it remains at the cell surface in 'endocytosis-resistant' membrane domains, visualized by confocal imaging. After VP washout, AQP2 is progressively internalized with the fluid-phase marker FITC-dextran, indicating that VP washout releases an endocytotic block that maintains AQP2 at the cell surface. Finally, polarized application of VP to filter-grown cells shows that apical VP can induce basolateral endocytosis and V2R down-regulation, and vice versa.


CONCLUSIONS
After VP stimulation of renal epithelial cells, AQP2 accumulates at the cell surface, while the V2R is actively internalized. This endocytotic block may involve a reduced capacity of phosphorylated AQP2 to interact with components of the endocytotic machinery. In addition, a complex cross-talk exists between the apical and basolateral plasma-membrane domains with respect to endocytosis and V2R down-regulation. This may be of physiological significance in down-regulating the VP response in the kidney in vivo.
Bouley
Richard
R
Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA.
Hawthorn
Gayle
G
Russo
Leileata M
LM
Lin
Herbert Y
HY
Ausiello
Dennis A
DA
Brown
Dennis
D
D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003911:Dextrans / Q000737:chemistry; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D007668:Kidney / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008297:Male; D018613:Microscopy, Confocal; D051381:Rats; D017207:Rats, Sprague-Dawley; D020239:Receptor Cross-Talk / Q000502:physiology; D017483:Receptors, Vasopressin / Q000378:metabolism*; D013552:Swine; D013997:Time Factors; D014162:Transfection; D014667:Vasopressins / Q000494:pharmacology*
D051399:Aquaporin 2; D003911:Dextrans; D017483:Receptors, Vasopressin; C015219:fluorescein isothiocyanate dextran; D014667:Vasopressins; D006735:Horseradish Peroxidase; D016650:Fluorescein-5-isothiocyanate