Polycystin-1 interacts with inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling with implications for polycystic kidney disease - PubMed (original) (raw)

Polycystin-1 interacts with inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling with implications for polycystic kidney disease

Yun Li et al. J Biol Chem. 2009.

Abstract

The PKD1 or PKD2 genes encode polycystins (PC) 1 and 2, which are associated with polycystic kidney disease. Previously we demonstrated that PC2 interacts with the inositol 1,4,5-trisphosphate receptor (IP(3)R) to modulate Ca(2+) signaling. Here, we investigate whether PC1 also regulates IP(3)R. We generated a fragment encoding the last six transmembrane (TM) domains of PC1 and the C-terminal tail (QIF38), a section with the highest homology to PC2. Using a Xenopus oocyte Ca(2+) imaging system, we observed that expression of QIF38 significantly reduced the initial amplitude of IP(3)-induced Ca(2+) transients, whereas a mutation lacking the C-terminal tail did not. Thus, the C terminus is essential to QIF38 function. Co-immunoprecipitation assays demonstrated that through its C terminus, QIF38 associates with the IP(3)-binding domain of IP(3)R. A shorter PC1 fragment spanning only the last TM and the C-terminal tail also reduced IP(3)-induced Ca(2+) release, whereas another C-terminal fragment lacking any TM domain did not. Thus, only endoplasmic reticulum-localized PC1 can modulate IP(3)R. Finally, we show that in the polarized Madin-Darby canine kidney cells, heterologous expression of full-length PC1 resulted in a smaller IP(3)-induced Ca(2+) response. Overexpression of the IP(3)-binding domain of IP(3)R reversed the inhibitory effect of PC1, suggesting interaction of full-length PC1 (or its cleavage forms) with endogenous IP(3)R in Madin-Darby canine kidney cells. These results indicate that the behavior of full-length PC1 in mammalian cells is congruent with that of PC1 C-terminal fragments in the oocyte system. These data demonstrate that PC1 inhibits Ca(2+) release, perhaps opposing the effect of PC2, which facilitates Ca(2+) release through the IP(3)R.

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Figures

FIGURE 1.

FIGURE 1.

Overexpression of QIF38 significantly decreases the amplitude of Ca2+ transients induced by IP3. A, these panels graphically show full-length PC1, QIF38, and R4227X. The dark portion of PC1 is the region of homology with PC2. B, the traces represent change in fluorescence (Δ_F_/F) showing IP3-induced Ca2+ transients in control Xenopus oocytes injected with H2O (n = 44) and those overexpressing QIF38 (n = 39) or the C-terminal truncation mutant R4227X (n = 46). C, left panel, the histograms show the average of the amplitude of Ca2+ transients; the asterisk indicates statistical significance compared with the control (p < 0.00001, Student's t test). Middle panel, the histogram shows the averages of half decay times (_T_½) of the Ca2+ transients. Right panel, immunoblot (IB) shows the similar expression levels of QIF38 and R4227X in the oocytes.

FIGURE 2.

FIGURE 2.

QIF38 physically associates with exogenously expressed type-1 IP3R, as well as the endogenously expressed type-3 IP3R. A, QIF38 physically associates with the exogenously expressed type-1 IP3R. HEK-293 cells were co-transfected with QIF38 and IP3R-1 or transfected with IP3R-1 alone as the negative control. Immunocomplexes were pulled down by FLAG antibody-conjugated beads. IP3R type-1 polyclonal antibody was used to probe the IP3R-1, and monoclonal Myc antibody was used to detect QIF38. Top panels, the immunoblotting (IB) was used to detect the IP3R-1 from the total cell lysates (left panel) and from the co-IP immunocomplex (right panel). Bottom panels, the immunoblot was used to detect QIF38 from the total cell lysates (left panel) and from the co-IP immunocomplex (right panel). B, QIF38 physically associates with endogenous type-3 IP3R. HEK-293 cells were transfected with QIF38 alone. Endogenous type-3 IP3R were pulled down by type-3 IP3R antibody. An unrelated HA antibody served as the negative control. Monoclonal Myc antibody was used to detect QIF38. Top panel, the immunoblot reveals QIF38 from the co-IP immunocomplex. Note that a nonspecific band (∼105 kDa indicated as “NS band”) above the QIF38 band was also pulled down in the co-IP immunocomplex, because it can be recognized by both monoclonal HA and IP3R-3 antibody. Bottom panel, the immunoblot reveals the endogenously expressed IP3R-3.

FIGURE 3.

FIGURE 3.

ER-localized PC1 fragments. A, an immunostaining of QIF38 and R4227X as detected with monoclonal Myc antibody (1:500). B, co-localization of QIF38 with IP3R. HEK-293 cells were co-transfected with QIF38 and IP3R-1. QIF38 was detected by monoclonal Myc antibody (1:250) and Alexa488 conjugated goat anti-mouse IgG (1:200). IP3R-1 was revealed by polyclonal anti-IP3R type-1 antibody (1:100) and Cy3-conjugated donkey anti-rabbit IgG (1:500). The scale bar represents 10 μm. C, Endo-H and PNGase F sensitivity assay with protein samples obtained from wild type murine embryonic fibroblasts. Endogenous PC1 was detected by a combination of immunoprecipitation and Western blot using PC1-CC antibody. The first lane of the blot represents control, untreated with neither Endo-H nor PNGase F. The asterisk on the blot shows the presence of full-length PC1 (marked FL) in the untreated control sample (first lane). Two black arrows point to the Endo-H resistant (upper) and -sensitive (lower) bands (second lane) of the C-terminal fragment of PC1 (CTF). IB, immunoblot.

FIGURE 4.

FIGURE 4.

Overexpression of NNY38 or QIF38F-A mutation significantly decreases the amplitude of the Ca2+ transients induced by IP3. A, left panels graphically show portions of QIF38F-A and NNY38. The right panel is the immunoblot (IB) showing the expression of NNY38, QIF38, R4227X, and QIF38F-A in Xenopus oocytes as detected by PC1-CT antibody (1:1000). B, histograms show the averages of the amplitude of IP3-induced Ca2+ transients from oocytes injected with H2O (n = 35), oocytes overexpressing QIF38 (n = 20), NNY38 (n = 17), or QIF38F-A (n = 19). The bars represent the standard error, and the asterisk indicates statistical significance compared with the control (p < 0.001, Student's t test). C, NNY38 and F-A mutation physically associated with exogenous expressed type-1 IP3R. HEK-293 cells were transiently transfected with IP3R-1 alone or co-transfected with QIF38, NNY38, or QIF38F-A. FLAG antibody-conjugated beads were used to pull down the immunocomplexes. IP3R type-1 polyclonal antibody was used to probe the IP3R-1, and monoclonal Myc antibody was used to detect QIF38 or its various mutants. Top panel, the immunoblot was used to detect the IP3R-1 from the co-IP immunocomplexes. Middle panel, the immunoblot was used to detect the IP3R-1 from total cell lysates. Bottom panel, the immunoblot was used to detect QIF38, NNY38, and the F-A mutation from the co-IP immunocomplex.

FIGURE 5.

FIGURE 5.

QIF38, but not C-terminal truncated R4227X, associates with the IP3-binding domain of IP3R. A, figure shows the putative structure of IP3R, its HA-tagged constructs of its suppressor domain (1–225), and its IP3-binding domain (amino acids 226–604). B, HEK-293 cells were transfected with HA-tagged IP3R domains alone or co-transfected together with QIF38. Immunocomplexes were pulled down by FLAG antibody (Ab)-conjugated beads. Monoclonal HA antibody was used to detect HA-tagged IP3R domains. Left panel, the immunoblot (IB) was used to probe HA-tagged IP3R domains from the co-IP immunocomplex. The black arrowhead indicates the ∼40-kDa IP3-binding domain (amino acids 226–604) from the co-IP. The white arrowhead indicates the absence of the band at ∼30 kDa, showing that QIF38 failed to pull down the suppressor domain (1–225). Middle panel, immunoblot of the total cell lysate detects the HA-tagged N-terminal domains of IP3R. Right panel, immunoblot of pulled down QIF38. C, HEK-293 cells co-transfected with HA-tagged IP3-binding domain (amino acids 226–604) with QIF38 or R4227X. Immunocomplexes were pulled down by FLAG antibody-conjugated beads. Monoclonal HA antibody was used to detect the HA-tagged IP3-binding domain. Left panel, immunoblot shows that the IP3-binding domain was co-immunoprecipitated with QIF38, but not with R4227X. Middle panel, immunoblot of total cell lysate detects the IP3-binding domain. Right panel, immunoblot of pulled down QIF38 and R4227X.

FIGURE 6.

FIGURE 6.

Expression of PC1 C-terminal tail significantly decreases the amplitude of Ca2+ transients induced by IP3. A, on the left are the C-terminal FLAG-tagged constructs AESW and RWRYH. On the right are subcellular localizations of AESW and RWRYH as revealed by immunostaining and detected with polyclonal FLAG antibody or PC1-CT and Cy3-conjugated donkey anti-rabbit IgG. The bar indicates 10 μm. B, the Western blot shows oocyte expression of AESW and RWRYH as detected by PC1-CT antibody. C, expression of PC1 C-terminal tail AESW associates with the reduced amplitude of IP3-induced Ca2+ transients, whereas its cytosolic portion, RWRYH, does not. Histograms show the average of the amplitude of Ca2+ transients, and an asterisk indicates statistical significance (p < 0.0001, Student's t test).

FIGURE 7.

FIGURE 7.

Full-length PC1 expression in polarized MDCK cells is associated with a reduced Ca2+ transient in response to 100 μm ATP stimulation, which can be rescued by overexpression of its binding partner, IP3R 226–604 fragment. A, full-length PC1 stably transfected MDCK cells displayed a reduced ATP-induced Ca2+ transient. The traces represent Ca2+ transients in response to ATP stimulation in the control MDCKzeo (F6, n = 9) and PC1-stable MDCKPKD1Zeo (C8/68, n = 13). B, histograms show the averages of the initial amplitude of Ca2+ transients. An asterisk indicates statistical significance (p < 0.0001, Student's t test). C, overexpression of the IP3R 226–604 fragment in PC1-expressing MDCK cells significantly increased Ca2+ release in response to 100 μ

m

ATP. The histogram shows the averages of initial amplitude of Ca2+ transient in response to ATP in four groups: control MDCK cells (MDCKzeo, F6) transfected with either GFP (F6+GFP, n = 9) or IP3R 226–604 (F6+226–604, n = 8), and PC1-expressing MDCK cells (MDCKPKD1Zeo, C8/68) transfected with either GFP (C8+GFP, n = 7) or IP3R 226–604 fragment (C8+226–604, n = 6). An asterisk indicates a statistical significance (p < 0.05 between the first two groups and p < 0.005 between the last two groups by Student's t test, respectively).

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