Syntaxin 5 regulates the endoplasmic reticulum channel-release properties of polycystin-2

Abstract

Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca(2+)) release channel. We performed a yeast two-hybrid screen by using the NH(2) terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca(2+) levels, decreased endoplasmic reticulum (ER) Ca(2+) stores, and reduced Ca(2+) release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca(2+) levels. Our data suggest that PC2 in the ER plays a role in cellular Ca(2+) homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca(2+) from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca(2+) signaling associated with polycystic kidney disease.

Fig. 1.

Interaction of PC2 with both long and short forms of Stx5 in cells and tissue. (A) LLC-PK₁ cells with stable expression of full-length PC2 were transiently transfected with either Stx5S or Stx5L and immunoprecipitation was performed with anti-PC2 monoclonal antibody YCE2 (PC2) or culture medium (C) as control. The top half was immunoblotted with anti-PC2 polyclonal antisera (YCC2) and the bottom with anti-Stx5 polyclonal antisera. Stx5S and Stx5L coimmunoprecipitate with PC2. (B) Native Stx5 coimmunoprecipitation with PC2 from conditionally immortalized mouse kidney cell lines (M and C) made from Pkd2-BAC transgenic mice. Cell lysates were immunoprecipitated by using anti-Stx5 antisera or nonimmune rabbit sera (NIS). The top half was immunoblotted with polyclonal anti-PC2 and the bottom with anti-Stx5. (C) Native Stx-5 coimmunoprecipitation with PC2 from M1 cells by using the same approach as panel (B). (D) Native Stx-5 coimmunoprecipitation with PC2 from Pkd2-BAC transgenic mouse kidney tissue lysates by using the approach described in (B) (*, nonspecific band).

Fig. 2.

Stx5 interacts directly with the NH₂-terminal 72 aa region of PC2. (A) Far Western in vitro binding assays mapping the PC2 interaction domain of Stx5. MBP-fusion constructs containing the F1, F2, F7, and F8 portions of Stx5 (see Fig. S1A) were expressed, purified, separated by PAGE, and transferred onto PVDF membranes. Purified GST fusion protein containing the cytoplasmic NH₂-terminal region of PC2 was incubated with the immobilized Stx5 peptides and specific binding was detected by using an NH₂-terminal anti-PC-2 polyclonal antiserum (YCB9, Top). F1, F2, and F8 bound PC2; F7 did not. A parallel PVDF membrane was stained with Coomassie blue as an MBP-fusion protein loading control (Bottom). (B) Coimmunoprecipitation of nested NH₂-terminal PC2 constructs with Stx5. PC2-L703X with an intact NH₂ terminus and an HA epitope tag and three in-frame deletions of this truncated PC2 (Δ5-72, Δ72-130, and Δ130-220) were stably expressed in LLC-PK₁ cells at comparable levels (Top) (22). Endogenous Stx5 was immunoprecipitated from lysates of each cell line (Bottom). Intact PC2-L703X and deletion forms Δ72-130 and Δ130-220 were successfully coimmunoprecipitated with Stx5, but PC2-L703X deleted for amino acids 5–72 (Δ5-72) did not interact with Stx5 (Middle). *, expected migration of PC2-L703X intact and deletion constructs (Middle). (C) Far Western mapping of the direct interaction between PC2 and Stx5. MBP-fusion protein F1 was expressed, purified, separated by PAGE, and transferred onto PVDF membranes. Parallel blots were incubated with cell lysates from each of three stable cell lines expressing the HA-tagged deletion constructs of PC2-L703X and binding of the latter were detected by anti-HA antisera. The Δ72-130 and Δ130-220 but not the Δ5-72 forms of PC2 showed direct interaction with Stx5 (Top). Subsequent far Western blotting with the COOH-terminal anti-PC2 antiserum (YCC2) that does not recognize the COOH-terminal truncated PC2-L703X forms detected directed interaction of endogenous full-length PC2 with Stx5 in all three samples (Bottom).

Fig. 3.

Binding of Stx5 inhibits the channel activity of PC2. (A and B) ER-derived vesicles from LLC-PK₁ cells overexpressing full length PC2 were fused to lipid bilayers. Single channel currents generated by PC2 were recorded with 53-mM barium on the ER luminal side as the sole current carrier at a holding potential of 0 mV in the presence of 300-nM free Ca²⁺ on the cytoplasmic side. Channel openings are shown as downward deflections and horizontal lines indicate zero current (C) and successive channel openings (O₁, O₂). Representative channel traces in the presence of increasing concentrations (ng/ml) on the cytoplasmic side of the fusion protein Stx5-F7 containing only the NH₂-terminal coiled coil domain (A) and fusion protein Stx5-F1 containing the entire cytosolic domain of Stx5S (B) are shown. F1 had no effect on channel activity when added to the ER luminal side (data not shown). (C) Representative channel traces when Stx5-F1 is applied to bilayers fused with ER-derived vesicles from LLC-PK₁ cells expressing the Δ(5-72)PC2 construct that cannot bind Stx5, showing that this form of PC2 retains channel activity but Stx5-F1 can no longer block this activity after deletion of the binding domain. (D) Summary data of open probabilities for PC2 and Δ(5-72)PC2 over an extended range of Stx5-F7 or Stx5-F1 peptide concentrations in single channel studies under conditions described in (A–C). The solid line is a theoretical fit for the Stx5-F7 with PC2 (open squares) and Stx5-F1 with Δ(5-72)PC2 (open circles); the dashed curve corresponds to a sigmoid fit for the Stx5-F1 dependence of PC2 open probability with an IC₅₀ of 80 ng/ml. Individual points with error bars are the mean ± SEM.

Fig. 4.

ER Ca²⁺ channel activity of PC-2 is regulated by Stx5. LLC-PK₁ cells stably expressing empty vector, full length PC2 or Δ(5-72)PC-2 were stimulated with 200nM AVP in the absence of extracellular Ca²⁺. (A) Representative traces during AVP stimulation (gray bar) show the baseline response of vector-expressing cells (solid line) and the increased amplitude and duration of ER Ca²⁺ release in cells over-expressing PC2 (dotted line). The Ca²⁺ response in cells expressing Δ(5-72)PC2 (dashed line) is reduced below the vector-only response. (B) Mean amplitude (± SEM) of the peak increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) above baseline in response to AVP stimulation of LLC-PK₁ cells expressing empty vector, full length PC2 or Δ(5-72)PC2. (C) Baseline [Ca²⁺]_i before AVP stimulation in each of the cell lines expressed as a percentage of Ca²⁺ levels in LLC-PK₁ cells expressing only empty vector. (D) ER Ca²⁺ content expressed as a percent of ER content in cells expressing only empty vector. (E) Basal [Ca²⁺]_i in Pkd2^+/− (n = 104), Pkd2^−/− (n = 109) and Pkd2^−/− PC2 cells (n = 83) re-expressing PC2 on a null background. ***, P < 0.001; **, P < 0.01.