Distinct subcellular expression of endogenous polycystin-2 in the plasma membrane and Golgi apparatus of MDCK cells

Abstract

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.

Gpnmb Is Induced in Macrophages by IFN-γ and Lipopolysaccharide and Acts as a Feedback Regulator of Proinflammatory Responses

“…This hypothesis was supported by several observations, as follows: first, the substantial colocalization of GPNMB with ␤-COP, a protein that associates with membranes of the Golgi complex (29); second, GPNMB staining was dispersed into the ER and endosomal compartments when the Golgi network was disrupted by BFA treatment. GPNMB localization to the Golgi apparatus is also consistent with evidence that GPNMB is a highly glycosylated protein (28,37) and that it contains a polycystic kidney diseases domain that is shared with the polycystin family of vesicle-trafficking mediators (44). Differences in the distribution of GPNMB-and transGolgi-derived vesicles containing the soluble N-ethylmaleimidesensitive factor attachment protein receptor proteins Stx6 and Vti1b (data not shown) suggested that GPNMB may be predominantly associated with cis-Golgi membranes.…”

Genetic mechanisms and signaling pathways in autosomal dominant polycystic kidney disease

“…A number of different localizations of the PCs have been proposed, including localization to the ER (likely a major site of PC2) or to the apical and basolateral membranes, or secretion on microvesicles (exosomes) (56,58,59). Although these localizations are likely, there are several lines of evidence that primary cilia are central to pathogenesis in PKD, making it a ciliopathy (3).…”

The left-right axis in the mouse: from origin to morphology

“…Given that Pkd2 is expressed ubiquitously, Pkd2 may potentially function during LR patterning at any site between, and including, the node and LPM. The subcellular localization of Pkd2 has also been controversial, as it has been detected in association with a variety of organelles, including the plasma membrane, Golgi apparatus (Scheffers et al, 2002), mitotic spindle (Rundle et al, 2004) and cilia (Yoder et al, 2002). If it is localized to the surface of node cilia, Pkd2 may serve as a mechanosensory, as proposed by the two-cilia model (McGrath et al, 2003;Tabin and Vogan, 2003), which proposes that two kinds of node cilia exist in the node: motile cilia that are positive for a dynein called Lrd (Supp et al, 1997), which generate the flow; and immotile Lrd-negative cilia, which presumably act as mechanosensors.…”