Identification of a Polycystin-1 Cleavage Product, P100, That Regulates Store Operated Ca2+ Entry through Interactions with STIM1

Owen M Woodward,Yun Li,Shengqiang Yu,Feng Qian,Claas Wodarczyk,Alessandra Boletta,William B Guggino,Patrick Greenwell

doi:10.1371/journal.pone.0012305

Abstract

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder resulting in large kidney cysts and eventual kidney failure. Mutations in either the PKD1 or PKD2/TRPP2 genes and their respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2) result in ADPKD. PC2 is known to function as a non-selective cation channel, but PC1's function and the function of PC1 cleavage products are not well understood. Here we identify an endogenous PC1 cleavage product, P100, a 100 kDa fragment found in both wild type and epitope tagged PKD1 knock-in mice. Expression of full length human PC1 (FL PC1) and the resulting P100 and C-Terminal Fragment (CTF) cleavage products in both MDCK and CHO cells significantly reduces the store operated Ca2+ entry (SOCE) resulting from thapsigargin induced store depletion. Exploration into the roles of P100 and CTF in SOCE inhibition reveal that P100, when expressed in Xenopus laevis oocytes, directly inhibits the SOCE currents but CTF does not, nor does P100 when containing the disease causing R4227X mutation. Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca2+ sensor protein STIM1 to the cell periphery was significantly altered. In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not. The expression of P100 in CHO cells recapitulates the STIM1 translocation inhibition seen with FL PC1. These data describe a novel polycystin-1 cleavage product, P100, which functions to reduce SOCE via direct inhibition of STIM1 translocation; a function with consequences for ADPKD.

Highlights

Ca2+ homeostasis plays a vital role in the normal development of tubules in the mammalian nephron [1,2]
We show that Polycystin 1 (PC1), through its P100 product, inhibits Ca2+ influx at the plasma membrane, and we provide evidence that PC1 interacts with STIM1, preventing its re-localization after store depletion
Antibody, [16]) (Figure 1A), we detected by Western blot a novel PC1 product of approximately 100 kDa, here termed P100, in the embryo and postnatal mouse, in addition to the previously reported uncleaved full-length and C-terminal fragment (CTF) (Figure 1Bi)

Summary

Introduction

Ca2+ homeostasis plays a vital role in the normal development of tubules in the mammalian nephron [1,2]. Dysregulation of Ca2+ homeostasis is characteristic in the cyst formation associated with Autosomal Dominant Polycystic Kidney Disease (ADPKD)[3], but how dysregulation leads to cyst production is not well understood. ADPKD results from mutations in the polycystin genes PKD1 and PKD2/TRPP2 and mutations in their respective polycystin proteins, Polycystin 1 (PC1) and Polycystin 2 (PC2), both of which have been implicated as significant regulators of intracellular Ca2+ in renal tubules [4]. PC2 is known to regulate ER calcium permeability [7] and modulate IP3R [8] to lower ER Ca2+ stores. PC1’s function in Ca2+ homeostasis is far less clear

Methods

Results

Conclusion