Woodward OM , Li Y , Yu S , Greenwell P , Wodarczyk C , Boletta A , Guggino WB , Qian F .

DK032753-25A1 NIDDK NIH HHS , DK062199 NIDDK NIH HHS , R01 DK062199-09 NIDDK NIH HHS , TCR07005 Telethon, R01 DK062199 NIDDK NIH HHS , P30 DK090868 NIDDK NIH HHS , R01 DK032753 NIDDK NIH HHS , TI_TCR07005 Telethon

	Figure 1. Biochemical characterization of a novel endogenous polycystin-1 product, P100.(A) Pkd1 alleles and schematic diagram of their corresponding polycystin-1 proteins. Pkd1HA is a 3xHA-tagged Pkd1 knock-in allele that produces fully functional PC1 protein. The domains in polycystin-1 are shown. Anti-CC is directed to the cytoplasmic C-terminal tail. The position of uncleaved full-length (uFL), the C-terminal (CTF) and the P100 product is schematically shown. (B) Western blots for wild-type embryo (E18.5), lung (Bi), and kidney (Bii and iii) at different postnatal stages as indicated using anti-CC antibodies after immunoprecipitation. Western blot shown in Bii (enclosed by dashed box) originally from Yu et al 2007; © 2007 by The National Academy of Sciences of the USA. The bands corresponding to uFL, CTF and P100 are indicated. * indicates a band of unknown nature in the lung. (C) Western blot for postnatal kidneys from PKD1v/v mice. (D) Western blot from mouse embryonic fibroblasts (MEF) isolated from Pkd1HA/HA and wild-type 11.5 day old embryos using anti-HA antibody after immunoprecipitation or on the whole cell lysate. (E) Western blot for homozygous Pkd1HA/HA tissues at P20 using anti-HA antibody after immunoprecipitation. The lung of the wild-type littermate (+/+) serves as a negative control.
	Figure 2. In vitro biochemical and functional characterization of polycystin-1 cleavage products.(A) Western blot of PC1-flag protein using anti-CT antibody after immunoprecipitation with flag conjugated beads from MDCK cells with stably transfected, tetracycline inducible PC1 expression. PC1 expression reveals three distinct bands: FL PC1, CTF, and P100. Right two lanes: the effect of 24 hours exposure of PC1 expressing cells to either 10 µM ionomycin or 40 nM thapsigargin (overnight). Blots representative of at least 3 experiments. (B) Fura 2-AM measurements of intracellular Ca2+ in either MDCK cells with (red; n = 5 coverslip, 99 cells) or without (black; n = 5 coverslips, 120 cells) tetracycline induced PC1-flag expression. Recordings began after cells reached steady state in a zero Ca2+, 4 µM thapsigargin ringers. The bath was then replaced with a 5 mM Ca2+ bath solution to measure the store operated calcium entry. Maximum SOC amplitudes (Δf/f) were compared (***p<0.001). (C) Western blot of PC1 and recombinant PC1 cleavage product constructs CTF and P100, in CHO cells. Protein was immunoprecipitated with flag conjugated beads then probed with anti-CT antibodies. Blot representative of at least three experiments. (D) Fura 2-AM measurements in CHO cells transiently transfected with GFP-PC1 (red; n = 12 coverslips, 65 cells) or with empty plasmid (black; n = 7 coverslips, 58 cells). Maximum SOC amplitudes (Δf/f) were compared (**p<0.01). (E) Cartoons of different PC1 constructs used in current study: Full length PC1 with either an N-terminus GFP or C-terminus flag tag; CTF construct with C-terminus flag tag; and P100 construct with both N-terminus myc tags and a C-terminus flag tag.
	Figure 3. Polycystin-1 product CTF does not inhibit SOCE.(A) Western blot of PC1 cleavage product constructs P100 and CTF expressed in Xenopus oocytes, immunoprecipitated with flag conjugated beads and probed with anti-CT antibody. (B) Averaged currents elicited by a −120 mV voltage step in H2O injected control oocytes (black trace, n = 20) or from oocytes expressing CTF (red trace, n = 14). (C) Mean current amplitudes at the transient peak and steady state. ±SEM.
	Figure 4. Polycystin-1 product P100 inhibits SOCE.(A) Averaged currents elicited by a −120 mV voltage step in H2O injected control oocytes (black trace, n = 38) or from oocytes expressing P100 (red trace, n = 18). (B) Mean current amplitudes at the transient peak and steady state. ±SEM; (**p<0.01). (C) Averaged currents form P100 injected oocytes (red trace, n = 11), and the same oocytes after three, 2 second, +60 mV pre-pulses (“induced” black trace, n = 11). For comparison the calculated NFA sensitive Cl- current from supplemental Figure S2E (black dotted trace) and the control currents from 4A (red dotted trace) are included. (D) Mean current amplitudes at the transient peak and steady state, including H2O control data from 4A and calculated ICl- from Figure S2E. ±SEM; (***p<0.001)(*p<0.05). (E) Averaged currents in H2O injected control oocytes (black trace, n = 38), oocytes expressing the AESW construct (red trace, n = 18), or oocytes expressing the P100 R4227X construct (blue trace, n = 13). (F) Mean current amplitudes at the transient peak and steady state; P100X is short for P100 R4227X. ±SEM; (**p<0.01).
	Figure 5. 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).
	Figure 6. 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.
	Figure 7. Model of P100 STIM1 interaction in the ER.(A) When ER Ca2+ stores are full STIM1 has Ca2+ bound and is evenly distributed throughout the endoplasmic reticulum (ER) membrane. Upon ER store depletion, STIM1 may oligomerize but does not translocate to areas of the ER near the plasma membrane (PM) when the polycystin-1 cleavage product P100 is present, inhibiting the activation of store operated Ca2+ (SOC) currents.

Baba, Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum. 2006, Pubmed

Baba, Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum. 2006, Pubmed
Barish, A transient calcium-dependent chloride current in the immature Xenopus oocyte. 1983, Pubmed , Xenbase
Basavanna, The isolated polycystin-1 COOH-terminal can activate or block polycystin-1 signaling. 2007, Pubmed
Bertuccio, Polycystin-1 C-terminal cleavage is modulated by polycystin-2 expression. 2009, Pubmed
Chauvet, Mechanical stimuli induce cleavage and nuclear translocation of the polycystin-1 C terminus. 2004, Pubmed
Chiu, Soft substrate up-regulates the interaction of STIM1 with store-operated Ca2+ channels that lead to normal epithelial cell apoptosis. 2008, Pubmed
Grimm, Polycystin-1 distribution is modulated by polycystin-2 expression in mammalian cells. 2003, Pubmed
Hanaoka, Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. , Pubmed
Hartzell, Activation of different Cl currents in Xenopus oocytes by Ca liberated from stores and by capacitative Ca influx. 1996, Pubmed , Xenbase
Hogan, Dissecting ICRAC, a store-operated calcium current. 2007, Pubmed
Hooper, Expression of polycystin-1 enhances endoplasmic reticulum calcium uptake and decreases capacitative calcium entry in ATP-stimulated MDCK cells. 2005, Pubmed
Köttgen, TRPP2 and autosomal dominant polycystic kidney disease. 2007, Pubmed
Lal, Polycystin-1 C-terminal tail associates with beta-catenin and inhibits canonical Wnt signaling. 2008, Pubmed
Li, Overexpression of Bax induces down-regulation of store-operated calcium entry in prostate cancer cells. 2008, Pubmed
Li, Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation. 2007, Pubmed
Li, Polycystin-1 interacts with inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling with implications for polycystic kidney disease. 2009, Pubmed , Xenbase
Li, Polycystin 2 interacts with type I inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling. 2005, Pubmed , Xenbase
Liou, Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion. 2007, Pubmed
Luik, Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. 2008, Pubmed
Manzati, The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca(2+)-dependent pathway(s). 2005, Pubmed
Mochizuki, PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. 1996, Pubmed
Nauli, Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. 2003, Pubmed
Orrenius, Regulation of cell death: the calcium-apoptosis link. 2003, Pubmed
Park, STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. 2009, Pubmed
Prakriya, Orai1 is an essential pore subunit of the CRAC channel. 2006, Pubmed
Qian, Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1-associated mutations. 2002, Pubmed
Simons, Polycystic kidney disease: cell division without a c(l)ue? 2006, Pubmed
Smyth, Phosphorylation of STIM1 underlies suppression of store-operated calcium entry during mitosis. 2009, Pubmed
Somlo, Human disease: calcium signaling in polycystic kidney disease. 2001, Pubmed
Stathopulos, Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry. 2008, Pubmed
Sutters, Autosomal dominant polycystic kidney disease: molecular genetics and pathophysiology. 2003, Pubmed
Vanden Abeele, Bcl-2-dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells. 2002, Pubmed
Vanoverberghe, Ca2+ homeostasis and apoptotic resistance of neuroendocrine-differentiated prostate cancer cells. 2004, Pubmed
Wegierski, TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum. 2009, Pubmed , Xenbase
Wei, Characterization of cis-autoproteolysis of polycystin-1, the product of human polycystic kidney disease 1 gene. 2007, Pubmed
Wildman, The isolated polycystin-1 cytoplasmic COOH terminus prolongs ATP-stimulated Cl- conductance through increased Ca2+ entry. 2003, Pubmed
Wilson, Polycystic kidney disease. 2004, Pubmed
Wodarczyk, A novel mouse model reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional cilia and hydrocephalus. 2009, Pubmed
Yao, Calcium current activated by depletion of calcium stores in Xenopus oocytes. 1997, Pubmed , Xenbase
Yu, Essential role of cleavage of Polycystin-1 at G protein-coupled receptor proteolytic site for kidney tubular structure. 2007, Pubmed