Distinct subcellular expression of endogenous polycystin-2 in the plasma membrane and Golgi apparatus of MDCK cells.
Polycystin-2 is a predicted integral membrane protein with non-selective cation channel activity. The protein is encoded by the PKD2 gene, which is mutated in approximately 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 can interact with the transmembrane protein polycystin-1, the product of the PKD1 gene. However, endoplasmic reticulum (ER) localization was reported for (heterologously expressed) polycystin-2 in cultured cells and baso-lateral localization has been reported in renal tissues. Using two polyclonal antisera raised against polycystin-2 we demonstrated distinct expression of the endogenous protein in the Golgi apparatus and the plasma membrane of MDCK cells. In contrast, most of the heterologously expressed polycystin-2 (PC2-EGFP) remained in the ER, substantially overlapping with the staining pattern of protein-disulfide isomerase (PDI), a marker for the ER. Only in a small subset of these cells weak plasma membrane signals were observed. Membrane staining was also suggested by immunoelectron microscopy and was confirmed by subcellular fractionation on sucrose density gradients. The plasma membrane staining disappeared following extraction with a buffer containing Triton X-100, whereas signals for polycystin-1 and E-cadherin remained visible, suggesting that polycystin-2 is neither tightly bound to the Triton X-100 insoluble cytoskeleton, nor to these proteins. We conclude that endogenous polycystin-2 is transported via the Golgi apparatus to the plasma membrane and has a broader membrane localization than polycystin-1. These data suggest that polycystin-2 can move freely in certain regions of the membrane where it probably functions as a channel, activated by, or in complex with, polycystin-1.
Scheffers
Martijn S
MS
Department of Human and Clinical Genetics, Sylvius Laboratory, Leiden University Medical Center, 2333AL Leiden, The Netherlands.
Le
Hang
H
van der Bent
Paola
P
Leonhard
Wouter
W
Prins
Frans
F
Spruit
Lia
L
Breuning
Martijn H
MH
de Heer
Emile
E
Peters
Dorien J M
DJ
D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D006056:Golgi Apparatus / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D016891:Polycystic Kidney, Autosomal Dominant / Q000378:metabolism*; D011817:Rabbits; D013552:Swine; D050396:TRPP Cation Channels; D014162:Transfection
D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D050396:TRPP Cation Channels