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Identification of a polycystin-1 cleavage product, P100, that regulates store operated Ca entry through interactions with STIM1.

Woodward OM, Li Y, Yu S, Greenwell P, Wodarczyk C, Boletta A, Guggino WB, Qian F - PLoS ONE (2010)

Bottom Line: 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 Ca(2+) entry (SOCE) resulting from thapsigargin induced store depletion.Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca(2+) sensor protein STIM1 to the cell periphery was significantly altered.In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

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 Ca(2+) 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 Ca(2+) 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.

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PC1 inhibits STIM1 translocation after ER Ca2+ depletion.(A) MDCK cells stably transfected with either mouse PC1 (+mPC1) or an empty vector (-mPC1) and transiently transfected with YFP-STIM1 imaged in 5 mM Ca2+ ringers and again after 15 min in zero Ca2+ with 4 µM thapsigargin. (B) Translocation of the YFP-STIM1 was monitored as the ratio of peripheral YFP signal (FP) to the total YFP signal per cell (FTot). For mPC1 expressing MDCK cells, n = 4 coverslips, 12 cells; for control cells, n = 4 coverslips, 47 cells. Scale bar in images is 20 µM. ±SEM; (***p<0.001).
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pone-0012305-g005: PC1 inhibits STIM1 translocation after ER Ca2+ depletion.(A) MDCK cells stably transfected with either mouse PC1 (+mPC1) or an empty vector (-mPC1) and transiently transfected with YFP-STIM1 imaged in 5 mM Ca2+ ringers and again after 15 min in zero Ca2+ with 4 µM thapsigargin. (B) Translocation of the YFP-STIM1 was monitored as the ratio of peripheral YFP signal (FP) to the total YFP signal per cell (FTot). For mPC1 expressing MDCK cells, n = 4 coverslips, 12 cells; for control cells, n = 4 coverslips, 47 cells. Scale bar in images is 20 µM. ±SEM; (***p<0.001).

Mentions: We hypothesized that PC1, through the actions of its P100 product, may exert its inhibitory effect on SOCE by interfering with STIM1 in the ER, retarding STIM1's ability to transduce a Ca2+ depletion signal to the SOCE channels of the plasma membrane. Functionally, we sought to address this by measuring the amount of STIM1 translocation to the periphery with or without the expression of PC1. We chose to use a MDCK cell line stably expressing mouse PC1, which we compared to empty vector control cells. The MDCK (with or without mPC1) cells were transfected with a YFP-STIM1 construct and photographed in high Ca2+ ringers (5 mM) after 15 minutes exposure to 4 µM thapsigargin (Figure 5A). Similar to a methodology used by Luik et al (2008)[27] the YFP signal of the periphery was compared to the total signal to create a FP/FTotal ratio for the STIM1 fluorescence for before and after exposure to thapsigargin. In cells expressing mPC1, thapsigargin elicited no significant change in the STIM1 fluorescence ratio (p<0.298), suggesting that STIM1 did not translocate after ER store depletion. In the control cells with no mPC1 expression, thapsigargin induced a significant change in the STIM1 ratio (p<0.001). Interestingly, the expression of PC1 also limited the overall STIM1 fluorescence in the high Ca2+ ringer (p<0.001) in addition to the translocation of STIM1.


Identification of a polycystin-1 cleavage product, P100, that regulates store operated Ca entry through interactions with STIM1.

Woodward OM, Li Y, Yu S, Greenwell P, Wodarczyk C, Boletta A, Guggino WB, Qian F - PLoS ONE (2010)

PC1 inhibits STIM1 translocation after ER Ca2+ depletion.(A) MDCK cells stably transfected with either mouse PC1 (+mPC1) or an empty vector (-mPC1) and transiently transfected with YFP-STIM1 imaged in 5 mM Ca2+ ringers and again after 15 min in zero Ca2+ with 4 µM thapsigargin. (B) Translocation of the YFP-STIM1 was monitored as the ratio of peripheral YFP signal (FP) to the total YFP signal per cell (FTot). For mPC1 expressing MDCK cells, n = 4 coverslips, 12 cells; for control cells, n = 4 coverslips, 47 cells. Scale bar in images is 20 µM. ±SEM; (***p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925899&req=5

pone-0012305-g005: PC1 inhibits STIM1 translocation after ER Ca2+ depletion.(A) MDCK cells stably transfected with either mouse PC1 (+mPC1) or an empty vector (-mPC1) and transiently transfected with YFP-STIM1 imaged in 5 mM Ca2+ ringers and again after 15 min in zero Ca2+ with 4 µM thapsigargin. (B) Translocation of the YFP-STIM1 was monitored as the ratio of peripheral YFP signal (FP) to the total YFP signal per cell (FTot). For mPC1 expressing MDCK cells, n = 4 coverslips, 12 cells; for control cells, n = 4 coverslips, 47 cells. Scale bar in images is 20 µM. ±SEM; (***p<0.001).
Mentions: We hypothesized that PC1, through the actions of its P100 product, may exert its inhibitory effect on SOCE by interfering with STIM1 in the ER, retarding STIM1's ability to transduce a Ca2+ depletion signal to the SOCE channels of the plasma membrane. Functionally, we sought to address this by measuring the amount of STIM1 translocation to the periphery with or without the expression of PC1. We chose to use a MDCK cell line stably expressing mouse PC1, which we compared to empty vector control cells. The MDCK (with or without mPC1) cells were transfected with a YFP-STIM1 construct and photographed in high Ca2+ ringers (5 mM) after 15 minutes exposure to 4 µM thapsigargin (Figure 5A). Similar to a methodology used by Luik et al (2008)[27] the YFP signal of the periphery was compared to the total signal to create a FP/FTotal ratio for the STIM1 fluorescence for before and after exposure to thapsigargin. In cells expressing mPC1, thapsigargin elicited no significant change in the STIM1 fluorescence ratio (p<0.298), suggesting that STIM1 did not translocate after ER store depletion. In the control cells with no mPC1 expression, thapsigargin induced a significant change in the STIM1 ratio (p<0.001). Interestingly, the expression of PC1 also limited the overall STIM1 fluorescence in the high Ca2+ ringer (p<0.001) in addition to the translocation of STIM1.

Bottom Line: 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 Ca(2+) entry (SOCE) resulting from thapsigargin induced store depletion.Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca(2+) sensor protein STIM1 to the cell periphery was significantly altered.In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

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 Ca(2+) 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 Ca(2+) 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.

Show MeSH
Related in: MedlinePlus