Functional Role of the NPxxY Motif in Internalization of the Type 2 Vasopressin Receptor in LLC-PK1 Cells

Title: Functional Role of the NPxxY Motif in Internalization of the Type 2 Vasopressin Receptor in LLC-PK1 Cells
Authors: Bouley, Ph.D., Richard; Sun, Tian-Xiao; Chenard, Melissa; McLaughlin, M.D., Margaret Elise; McKee, Mary; Lin, M.D., Ph.D., Herbert Y.; Brown, Dennis; Ausiello, M.D., Dennis A.
Publisher: American Journal of Physiology: Cell Physiology
Date Published: October 01, 2003
Reference Number: 619
Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, FLAG-tagged wild-type V2R and a Y325F mutant V2R were expressed in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [(3)H]-AVP binding and immunofluorescence, but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor down-regulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells, nor for adenylyl cyclase-mediated signal transduction.

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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 vasopressin 2 receptor protein (V2R) must move from the cell interior to the cell membrane to perform its function of linking with the hormone, vasopressin (AVP). The binding of AVP with V2R signals a molecular sequence which allows the kidney to concentrate urine. After a sustained interaction between AVP and V2R, a process called downregulation occurs: the V2R stops responding to AVP and this stops signaling the molecular process responsible for urine concentration. This is a normal cycle that occurs in the kidney collecting duct cells.

The V2R’s time dependent loss of sensitivity to AVP (downregulation) is believed to depend on changes in the shape of the V2R stimulated AVP. This allows the V2R to be phosphorylated (i.e., have a phosphate group attached to it), desensitized so it signals less when AVP binds with it, internalized (i.e., moved back into the cell interior), and once internalized, sequestered.

Though the exact details involved in V2R’s internalization (i.e., its movement from the cell membrane to the cell interior) are not known, Bouley, et al., had reason to think that the amino acid sequence NPxxY, consisting of the amino acid residue, Asparagine (N) linked to proline (P) followed by any two amino acids (represented by xx) and ending with the amino acid residue tyrosine (Y), plays an essential role in the internalization of V2R. NPxxY is located in the tail end of the V2R protein.

To determine the role of the NPxxY motif in the AVP induced internalization, cell surface expression, and molecular signaling of V2R, these researchers traced the movements in laboratory cell cultures of both V2R with the NPxxY motif intact and the movements of a mutant V2R where the motif was disrupted due to a phenylalanine amino acid residue (F) being in the spot occupied by tyrosine in a normal V2R. This mutant is called (V2R-Y325F). Bouley, et al., found that both the V2R and the V2R-Y325F were distributed through the cell membranes in a similar manner. They both were able to link and bind with AVP and initiate the molecular sequence that leads to urine concentration, but V2R-Y325F did not return to the cell interior.

The researchers conclude that the NPxxY motif in the V2R plays a major role in the V2R’s ability to return to the cell interior, but that it is not essential for the distribution of V2R in the cell membrane or for the molecular sequence that occurs when AVP and V2R bind. They also discovered that the protein, clathrin, plays a major role in the internalization of V2R.