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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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P100 physically interacts with STIM1.(A) CHO cells transiently transfected with STIM1 and either P100 or CTF construct; immunoprecipitated with anti-STIM1 antibody, then probed with anti-CT;or immunoprecipitated with flag conjugated beads then probed with the anti-CT antibody to verify transfection of PC1 products. * denotes endogenous STIM1 only. (B) STIM1 expression was verified by probing the same lysate and immunoprecipitate used in A and B with anti-STIM1 antibody. Blots representative of at least 3 experiments. (C) To confirm the pull down of P100 by STIM1, the reverse was attempted, pulling down STIM1 with P100. CHO cells were again transiently transfected with STIM1 and P100 or STIM1 alone. The Flag tagged P100 was immunoprecipitated with flag conjugated beads then probed with anti-STIM1 antibody. The expression of STIM1 was verified by probing the lysate directly with the anti-STIM1 antibody. (D) The expression of P100 was confirmed by probing the lysate and the immunoprecipitate with anti-Flag. Blots representative of at least 3 experiments. (E) CHO cells transfected with YFP-STIM1 and P100 (right) or the empty plasmid (left) and imaged first in a 5 mM Ca2+ bath solution (top) then again after a 10 min incubation in a zero Ca2+ bath with 8 µM thapsigargin (bottom). P100 retards the peripheral YFP puncta formation.
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pone-0012305-g006: P100 physically interacts with STIM1.(A) CHO cells transiently transfected with STIM1 and either P100 or CTF construct; immunoprecipitated with anti-STIM1 antibody, then probed with anti-CT;or immunoprecipitated with flag conjugated beads then probed with the anti-CT antibody to verify transfection of PC1 products. * denotes endogenous STIM1 only. (B) STIM1 expression was verified by probing the same lysate and immunoprecipitate used in A and B with anti-STIM1 antibody. Blots representative of at least 3 experiments. (C) To confirm the pull down of P100 by STIM1, the reverse was attempted, pulling down STIM1 with P100. CHO cells were again transiently transfected with STIM1 and P100 or STIM1 alone. The Flag tagged P100 was immunoprecipitated with flag conjugated beads then probed with anti-STIM1 antibody. The expression of STIM1 was verified by probing the lysate directly with the anti-STIM1 antibody. (D) The expression of P100 was confirmed by probing the lysate and the immunoprecipitate with anti-Flag. Blots representative of at least 3 experiments. (E) CHO cells transfected with YFP-STIM1 and P100 (right) or the empty plasmid (left) and imaged first in a 5 mM Ca2+ bath solution (top) then again after a 10 min incubation in a zero Ca2+ bath with 8 µM thapsigargin (bottom). P100 retards the peripheral YFP puncta formation.

Mentions: We over expressed STIM1 and P100 in CHO cells and found that P100 and STIM1 could be co-precipitated using an anti-STIM1 antibody, and visualized with the anti-PC1 CT antibody (Figure 6A). Consistent with the lack of SOCE inhibition, CTF was not pulled down with the anti-STIM1 antibody (Figure 6A). CTF and P100 expression was confirmed by immunoprecipitating the same lysate with Flag congregated beads then probing with the anti-PC1 CT antibody (Figure 6A). STIM1 expression was also verified under each condition (Figure 6B). The STIM1/P100 interaction was confirmed by reciprocal co-immunoprecipitation using flag-conjugated beads and visualized with the anti-STIM1 antibody (Figure 6C). The possible functional interaction of P100 and STIM1 was assessed like full length PC1 above. CHO cells were transfected with YFP-STIM1 and either an empty plasmid or P100 and photographed in 5mM Ca2+ ringers and again 10 minutes after exposure to 8 µM thapsigargin (Figure 6E). In CHO cells expressing YFP-Stim1 alone, ten minutes of thapsigargin treatment altered the STIM1 localization from a diffuse ER pattern to dramatic puncta around the periphery of the cell. However, the co-expression of YFP-Stim1 and P100 presents a different localization pattern, with very little of the puncta signal or YFP signal just beneath the plasma membrane. The inhibition of STIM1 re-localization in the presence of the P100 is not total, but does suggest that PC1 regulates Ca2+ entry through the generation of P100.


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)

P100 physically interacts with STIM1.(A) CHO cells transiently transfected with STIM1 and either P100 or CTF construct; immunoprecipitated with anti-STIM1 antibody, then probed with anti-CT;or immunoprecipitated with flag conjugated beads then probed with the anti-CT antibody to verify transfection of PC1 products. * denotes endogenous STIM1 only. (B) STIM1 expression was verified by probing the same lysate and immunoprecipitate used in A and B with anti-STIM1 antibody. Blots representative of at least 3 experiments. (C) To confirm the pull down of P100 by STIM1, the reverse was attempted, pulling down STIM1 with P100. CHO cells were again transiently transfected with STIM1 and P100 or STIM1 alone. The Flag tagged P100 was immunoprecipitated with flag conjugated beads then probed with anti-STIM1 antibody. The expression of STIM1 was verified by probing the lysate directly with the anti-STIM1 antibody. (D) The expression of P100 was confirmed by probing the lysate and the immunoprecipitate with anti-Flag. Blots representative of at least 3 experiments. (E) CHO cells transfected with YFP-STIM1 and P100 (right) or the empty plasmid (left) and imaged first in a 5 mM Ca2+ bath solution (top) then again after a 10 min incubation in a zero Ca2+ bath with 8 µM thapsigargin (bottom). P100 retards the peripheral YFP puncta formation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925899&req=5

pone-0012305-g006: P100 physically interacts with STIM1.(A) CHO cells transiently transfected with STIM1 and either P100 or CTF construct; immunoprecipitated with anti-STIM1 antibody, then probed with anti-CT;or immunoprecipitated with flag conjugated beads then probed with the anti-CT antibody to verify transfection of PC1 products. * denotes endogenous STIM1 only. (B) STIM1 expression was verified by probing the same lysate and immunoprecipitate used in A and B with anti-STIM1 antibody. Blots representative of at least 3 experiments. (C) To confirm the pull down of P100 by STIM1, the reverse was attempted, pulling down STIM1 with P100. CHO cells were again transiently transfected with STIM1 and P100 or STIM1 alone. The Flag tagged P100 was immunoprecipitated with flag conjugated beads then probed with anti-STIM1 antibody. The expression of STIM1 was verified by probing the lysate directly with the anti-STIM1 antibody. (D) The expression of P100 was confirmed by probing the lysate and the immunoprecipitate with anti-Flag. Blots representative of at least 3 experiments. (E) CHO cells transfected with YFP-STIM1 and P100 (right) or the empty plasmid (left) and imaged first in a 5 mM Ca2+ bath solution (top) then again after a 10 min incubation in a zero Ca2+ bath with 8 µM thapsigargin (bottom). P100 retards the peripheral YFP puncta formation.
Mentions: We over expressed STIM1 and P100 in CHO cells and found that P100 and STIM1 could be co-precipitated using an anti-STIM1 antibody, and visualized with the anti-PC1 CT antibody (Figure 6A). Consistent with the lack of SOCE inhibition, CTF was not pulled down with the anti-STIM1 antibody (Figure 6A). CTF and P100 expression was confirmed by immunoprecipitating the same lysate with Flag congregated beads then probing with the anti-PC1 CT antibody (Figure 6A). STIM1 expression was also verified under each condition (Figure 6B). The STIM1/P100 interaction was confirmed by reciprocal co-immunoprecipitation using flag-conjugated beads and visualized with the anti-STIM1 antibody (Figure 6C). The possible functional interaction of P100 and STIM1 was assessed like full length PC1 above. CHO cells were transfected with YFP-STIM1 and either an empty plasmid or P100 and photographed in 5mM Ca2+ ringers and again 10 minutes after exposure to 8 µM thapsigargin (Figure 6E). In CHO cells expressing YFP-Stim1 alone, ten minutes of thapsigargin treatment altered the STIM1 localization from a diffuse ER pattern to dramatic puncta around the periphery of the cell. However, the co-expression of YFP-Stim1 and P100 presents a different localization pattern, with very little of the puncta signal or YFP signal just beneath the plasma membrane. The inhibition of STIM1 re-localization in the presence of the P100 is not total, but does suggest that PC1 regulates Ca2+ entry through the generation of P100.

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