The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini.
PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.
Institute for Molecular and Cellular Anatomy, University of Regensburg, Germany.
Helen
H
Hoffmeister
Anna-Rachel
AR
Gallagher
Anne
A
Rascle
Ralph
R
Witzgall
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D050396:TRPP Cation Channels / Q000737:chemistry* / Q000378:metabolism
D050396:TRPP Cation Channels