The human polycystin-2 protein represents an integral membrane protein with 6 membrane-spanning domains and intracellular NH2- and COOH-termini
Résumé
PKD2 is one of the two genes mutated in autosomal-dominant polycystic kidney disease (ADPKD). 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 family of cation channels suggest that polycystin-2 contains 6 transmembrane domains and that both the NH2- and COOH-termini extend into the cytoplasm. However, no experimental evidence for this model has been provided so far. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1 to 5 and tagged the constructs at the COOH-terminus with a HA-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, while loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the NH2- and COOH-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 while no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. Our data therefore provide the first experimental evidence for the topological orientation of polycystin-2.
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