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


CONCLUSIONS
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.
Andersen-Beckh
B
B
Rudolf-Buchheim-Institut für Pharmakologie, Giessen, Germany. Bettina.Andersen@pharma.medm.uni-giessen.de
Dehe
M
M
Schülein
R
R
Wiesner
B
B
Rutz
C
C
Liebenhoff
U
U
Rosenthal
W
W
Oksche
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D015342:DNA Probes; D003928:Diabetic Nephropathies / Q000235:genetics / Q000503:physiopathology; D004285:Dogs; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007686:Kidney Tubules, Distal / Q000737:chemistry* / Q000166:cytology* / Q000201:enzymology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D017483:Receptors, Vasopressin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D014162:Transfection; D014316:Tritium; D014960:X Chromosome
D015342:DNA Probes; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D014316:Tritium; D000262:Adenylyl Cyclases