Inhibition of Endocytosis Causes Phosphorylation (S256)-Independent Plasma Membrane Accumulation of AQP-2

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Title: Inhibition of Endocytosis Causes Phosphorylation (S256)-Independent Plasma Membrane Accumulation of AQP-2
Authors: Lu, MD, PhD, Hua A.J.; Sun, Tian-Xiao; Bouley, Ph.D., Richard; Blackburn, Karen; McLaughlin, M.D., Margaret Elise; Brown, Dennis
Publisher: American Journal of Physiology: Renal Physiology
Date Published: February 01, 2004
Reference Number: 628
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Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane AQP2 in epithelial cells stably transfected with wild type AQP2. We now show a similar effect of K44A dynamin in LLC-PK1 cells transfected with an S256-phosphorylation deficient AQP2 mutant, AQP2(S256A), and in AQP2-transfected IMCD cells. More acute blockade of endocytosis in these cells with the cholesterol-depleting agent, methyl-beta- cyclodextrin (mbetaCD - 10 mM) resulted in a rapid and extensive cell surface accumulation of both wild-type AQP2 and AQP2(S256A) within 15 min after treatment. This effect was similar to that induced by treatment of the cells with vasopressin. Blockade of endocytosis by mbetaCD was confirmed using quantitative analysis of FITC-dextran uptake and AQP2 membrane insertion was verified by cellsurface biotinylation. These data indicate that AQP2 recycles constitutively and rapidly between intracellular stores and the cell surface in LLC-PK1 and IMCD cells. The constitutive trafficking process is not dependent on phosphorylation of the serine 256 residue of AQP2 which is, however, an essential step for regulated, vasopressin/cAMPmediated translocation of AQP2. Our data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin-receptor (V2R)- and phosphorylation- independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQP2.

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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

The classic model for urine concentration describes the anti-diuretic hormone, arginine vasopressin (AVP) binding with the vasopressin-2 receptor (V2R) in the membranes of the principal cells in the kidney collecting duct. The binding of AVP with its receptor, V2R, raises the level of adenylyl cyclase in the cell. This elevates cyclic adenosine monophosphate (cAMP). cAMP is activated by protein kinase A (PKA), and this leads to a process called phosphorylation (the binding of a phosphate group to a molecule) of the water channel protein, aquaporin-2 (AQP2). Upon phosphorylation, AQP2 moves from within the cell to the cell membrane, where it is inserted. Once in the membrane, AQP2 acts as a channel through which water can pass. Water passing through AQP2 is an essential aspect of the kidney’s ability to concentrate urine.

The movement of AQP2 from within the cell interior to the cell membrane is called exocytosis. Its movement back to the cell interior after serving its function is called endocytosis. The precise mechanics of both AQP2 exocytosis and endocytosis are not fully understood, but until recently the movement of AQP2 to the cell membrane was thought to be dependent on the molecular process initiated when AVP binds with V2R.

However, recent research using laboratory cell cultures indicates that AQP2 can travel to the cell membrane independently of the AVP/V2R initiated process. For example, AQP2 can reach cell culture membrane when cyclic guanosine monophosphate (cGMP) is stimulated, and it has also been shown that AQP2 moves continuously between the cell membrane and cell interior independent of any sort of hormonal stimulation.

Lu, et al., have investigated ways in which AQP2 can gather in the cell membrane independent of AVP. They have shown AQP2 could be prevented from traveling back to the cell interior by inhibiting the process that normally takes it back to the cell interior. In their present study, Lu, et al., used a method of inhibiting AQP2 endocytosismembrane cholesterol depletion with methyl-b-cyclodextrin (mbCD) – that took only a matter of minutes to produce its effects. This resulted in an accumulation of AQP2 in the cell membrane that was as similar in number and almost as fast as the accumulation of AQP2 caused by AVP.

The researchers were also able to determine whether phosphorylation was necessary for cell membrane accumulation of AQP2. Though phosphorylation is definitely for AQP2 membrane accumulation when it uses the pathway stimulated by AVP/V2R, the research indicating that AQP2 also traveled to and from the cell membrane independently of any hormone suggested that phosphorylation might not always be necessary.

Using two different types of laboratory cell cultures, Lu, et al., determined that they could inhibit AQP2 from returning to the cell interior in a manner that results in rapid accumulation of AQP2 in the cell membrane. Further, this accumulation was independent of AQP2 phosphorylation at Serine 256. (In the classic model that begins with AVP/V2R binding, AQP2 must be phosphorylated at a specific place on it: the serine amino acid residue that is the 256th residue in the chain of amino acid residues that comprise the AQP2 protein.)

The picture the researchers’ study indicates is that AQP2 travels to the cell membrane by at least two pathways: one that is regulated by the hormone AVP, and another that needs no hormone to signal the AQP2 to move to the membrane. In this latter, constitutive, pathway, AQP2s are constantly traveling to and from the cell membrane. The fact that the AQP2s that take this pathway can rapidly accumulate in the membrane when their return trip is inhibited may have important implications for people with X-linked NDI. This is because people with X-linked NDI do not respond to AVP, and thus their AQP2 cannot accumulate in the cell membrane as it should. Should a treatment protocol arise from this research that allows rapid accumulation of AQP2 in the cell membrane by working with another AQP2 pathway, it could prove very beneficial for people with X-linked NDI.