Polarized Expression of the Vasopressin V2 Receptor in Madin-Darby Canine Kidney Cells
| Title: | Polarized Expression of the Vasopressin V2 Receptor in Madin-Darby Canine Kidney Cells |
|---|---|
| Authors: | Andersen-Beckh, Bettina; Dehe, Marcel; Schulein, Ralf; Wiesner, Burkhard; Rutz, Claudia; Liebenhoff, Ursula; Rosenthal, Walter; Oksche, Alexander |
| Publisher: | Kidney International |
| Date Published: | August 01, 1999 |
| Reference Number: | 493 |
BACKGROUND: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented.
METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody.
RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell.
CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.
METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody.
RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell.
CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.
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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 Madin-Darby canine kidney (MDCK) II cells are a polarized cell line derived from the canine kidney collecting duct. The authors of this paper report on their successful expression of human V2Rs in MDCK II cell lines.
The authors were able to track the V2Rs in the MDCK II cells. They found that normal V2Rs were located in the sides of the cell membranes, whereas a mutant V2R was unable to reach the side membrane and was instead retained within the cell. This transportation defect of the mutant V2R has previously been found numerous times in research using non-polarized cell systems. The authors conclude that the MDCK II cells are suitable for analyzing transport-defective mutants of V2Rs.