ABSTRACT
The cloning of the PKD1 and PKD2 genes has led to promising new insight into the mechanisms that are responsible for cyst development in patients with autosomal dominant polycystic kidney disease. Although the dominant pattern of inheritance would argue for haploinsufficiency, a gain of function, or a dominant negative mechanism, there is good evidence that autosomal dominant polycystic kidney disease behaves like a recessive disease on a cellular level (two-hit mechanism of cystogenesis). For testing of whether other pathomechanisms in addition to the two-hit hypothesis can explain cyst formation, two transgenic rat lines that contain a truncated human polycystin-2 cDNA were generated. The protein product lacks almost the entire COOH-terminus and mimics mutations that frequently are found in patients. The transgene-encoded mRNA could be detected in multiple tissues of both transgenic lines, with the highest expression in the kidney. Both lines present with renal cysts that originate predominantly from the proximal tubule; in the tubular epithelial cells, the epitope-tagged mutant protein was detected in the brush border and in primary cilia. Further evidence of the involvement of primary cilia stems from the finding of retinal degeneration in the transgenic rats and from the fact that stably transfected LLC-PK(1) cells that inducibly produced the truncated polycystin-2 protein elaborated shorter cilia. Other experimental approaches, such as a knock-in strategy, will be necessary to validate these results, but this is the first preliminary evidence that cyst formation is due not only to somatic mutations.
Subject(s)
Mutation , Polycystic Kidney, Autosomal Dominant/genetics , Retinal Degeneration/genetics , TRPP Cation Channels/chemistry , Animals , Animals, Genetically Modified , Blotting, Western , Cells, Cultured , Cilia/metabolism , Cilia/pathology , Electrophoresis, Polyacrylamide Gel , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Humans , Immunoprecipitation , Male , Polycystic Kidney, Autosomal Dominant/pathology , Rats , Rats, Sprague-Dawley , Retinal Degeneration/pathology , Ribonuclease, Pancreatic/metabolism , Sequence Deletion , TRPP Cation Channels/metabolism , TransfectionABSTRACT
Employing a yeast two-hybrid screen with the COOH terminus of polycystin-2, one of the proteins mutated in patients with polycystic kidney disease, we were able to isolate a novel protein that we call PIGEA-14 (polycystin-2 interactor, Golgi- and endoplasmic reticulum-associated protein with a molecular mass of 14 kDa). Molecular modeling only predicts a coiled-coil motif, but no other functional domains, in PIGEA-14. In a subsequent two-hybrid screen using PIGEA-14 as a bait, we found GM130, a component of the cis-compartment of the Golgi apparatus. Co-expression of the PIGEA-14 and PKD2 cDNAs in LLC-PK(1) and HeLa cells resulted in a redistribution of PIGEA-14 and polycystin-2 to the trans-Golgi network, which suggests that PIGEA-14 plays an important role in regulating the intracellular location of polycystin-2 and possibly other intracellular proteins. Our results also indicate that the intracellular trafficking of polycystin-2 is regulated both at the level of the endo-plasmic reticulum and that of the trans-Golgi network.
