MABS2145-100UG Sigma-AldrichAnti-Polycystin-2 Antibody, clone YCE2

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited nephropathy responsible for 4%-10% of end-stage renal disease cases. Mutations in the genes encoding polycystin-1 (PC1, PKD1) or polycystin-2 (PC2, PKD2) cause ADPKD, and PKD1 mutations are associated with more severe renal disease. PC1 has been shown to form a complex with PC2, and the severity of PKD1-mediated disease is associated with the level of the mature PC1 glycoform. Here, we demonstrated that PC1 and PC2 first interact in the ER before PC1 cleavage at the GPS/GAIN site and determined that PC2 acts as an essential chaperone for PC1 maturation and surface localization. The chaperone function of PC2 was dependent on the presence of the distal coiled-coil domain and was disrupted by pathogenic missense mutations. In Pkd2-/- mice, complete loss of PC2 prevented PC1 maturation. In Pkd2 heterozygotes, the 50% PC2 reduction resulted in a nonequimolar reduction (20%-25%) of the mature PC1 glycoform. Interbreeding between various Pkd1 and Pkd2 models revealed that animals with reduced levels of functional PC1 and PC2 in the kidney exhibited severe, rapidly progressive disease, illustrating the importance of complexing of these proteins for function. Our results indicate that PC2 regulates PC1 maturation; therefore, mature PC1 levels are a determinant of disease severity in PKD2 as well as PKD1.

Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser(812), a putative casein kinase II (CK2) substrate domain. Ser(812) can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca(2+) but there is a shift in this Ca(2+) dependence such that S812A is 10-fold less sensitive to Ca(2+) activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca(2+) transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser(812) modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.