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

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.

Novel Functional Complexity of Polycystin-1 by GPS Cleavage In Vivo: Role in Polycystic Kidney Disease

“…One possible explanation for the observed Pc1 deN excess (about 10-fold) is that Pc1 cFL at the plasma membrane continuously undergoes subunit dissociation followed by internalization and degradation of the resulting dissociated CTF via its cytoplasmic PEST domain (58)(59)(60). An alternative explanation for the finding is the previously described cleavage events in the C-terminal tail of the CTF (59,61,62), which may result in C-terminal fragments that are translocated to the nucleus for signaling (59,61). Since Pc1 deN is predicted to contain no TM domain, it may be associated to the membrane via another cell surface receptor(s) and/or by lipid modifications, as previously proposed for CIRL/ latrophilin and Sonic hedgehog (63,64).…”

Stanniocalcin 2 Is a Negative Modulator of Store-Operated Calcium Entry

“…STIM1 serves as both the sensor of ER Ca 2ϩ levels and the activator of plasma membrane SOCs (6). Recent work has identified several SOCE regulatory proteins that appear to function by binding STIM1 (19,48,59,60). However, to our knowledge, STC2 represents the first ER luminal protein with the ability to both bind STIM1 and regulate the overall magnitude of SOCE.…”

Pkd1l1 establishes left-right asymmetry and physically interacts with Pkd2

“…6A, blue arrow). Cleavage at an equivalent position in Pkd1 has recently been described (Woodward et al, 2010), suggesting that this cleavage site is conserved between polycystin-1 family members. During our IP experiments, we found that the Pkd1l1-GFP 80 kDa band, which we named Pkd1l1_53, co-purified with Myc-PKD2 upon GFP or Myc IP (Fig.…”