Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells

Background. The vasopressin V2 receptor is expressed in the polarized princ ipal cell of the renal collecting duct. Inactivating mutations of the vasop ressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI) . Most of the mutant V2 receptors show transport defects, as analyzed in no n-polarized cells, but data pertaining to polarized cells have not previous ly been presented. Methods. Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [H-3]-arginine vasop ressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 re ceptors were immunocytochemically localized using the tyramide signal ampli fication 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 I I clones. However, the use of standard immunofluorescence methodology for t hese MDCK II clones yielded only weak signals. With the tyramide signal amp lification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical m embrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor N DI mutant L44P, fluorescent signals were found exclusively within the cell. Conclusion. The wild-type V2 receptor is expressed mainly in the lateral me mbrane, 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 re ceptor.