Store-operated Ca2+ Entry-associated Regulatory factor (SARAF) Plays an Important Role in the Regulation of Arachidonate-regulated Ca2+ (ARC) Channels

Abstract

The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.

Keywords: ARC channels; SARAF; arachidonic acid (AA) (ARA); calcium channel; calcium release-activated calcium channel protein 1 (ORAI1); protein-protein interaction; stromal interaction molecule 1 (STIM1).

FIGURE 1.

SARAF modulates arachidonic acid-evoked Ca²⁺ entry in neuroblastoma SH-SY5Y cells. SH-SY5Y cells were loaded with fura-2 and resuspended in a medium containing 1.2 mm Ca²⁺ or in a Ca²⁺-free medium (1.5 mm EGTA added) as described under “Experimental Procedures.” Elevations in [Ca²⁺]_i were monitored by using the 340/380 nm fluorescence ratio and presented as ΔF₃₄₀/F₃₈₀. A, SH-SY5Y cells overexpressing SARAF (SARAF) and mock-treated cells (Control and bottom panel) were treated with AA (8 μm) in the presence of 1.2 mm extracellular Ca²⁺ or in a Ca²⁺-free medium as indicated. B, SH-SY5Y cells transfected with si SARAF, alone or in combination with SARAF overexpression plasmid, or scramble plasmids, as indicated, were stimulated with AA (8 μm) in the presence of 1.2 mm extracellular Ca²⁺. C, bar graph indicates the amount of Ca²⁺ entry after cell stimulation with AA in the presence of extracellular Ca²⁺. Data are expressed as mean ± S.E. * and **, p < 0.05 and p < 0.01, respectively. D, cells overexpressing SARAF or transfected with si SARAF and their respective controls were lysed, and whole cell lysates were subjected to 10% SDS-PAGE and subsequent Western blotting with a specific anti-SARAF antibody. Membranes were reprobed with anti-β-actin antibody for protein loading control. The panels show results from one experiment representative of 4 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. E, SH-SY5Y cells transfected with si Orai3, alone or in combination with SARAF overexpression plasmid or siRNA SARAF, or scramble plasmid, as indicated, were stimulated with AA (8 μm) in the presence of 1.2 mm extracellular Ca²⁺. F, cells transfected with si Orai3 or scramble plasmid were lysed, and whole cell lysates were subjected to 10% SDS-PAGE and subsequent Western blotting with a specific anti-Orai3 antibody. Membranes were reprobed with anti-β-actin antibody for protein loading control. The panels show results from one experiment representative of 4 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.

FIGURE 2.

SOCE and ARC-mediated Ca²⁺ entry require STIM1 and are modulated by SARAF. A, SH-SY5Y cells overexpressing SARAF (SARAF) and mock-treated cells (Control) were treated in a Ca²⁺-free medium (1.5 mm EGTA added) with 1 μm TG followed by addition Ca²⁺ to the medium (final concentration 1.2 mm) to initiate Ca²⁺ entry. Cells were then stimulated with AA (8 μm) to evoke store-independent Ca²⁺ entry via ARC channels. B, NG115-401L cells were treated in a Ca²⁺-free medium (1.5 mm EGTA added) with 1 μm TG followed by addition of Ca²⁺ (final concentration 1.2 mm). Cells were then stimulated with AA (8 μm). The traces shown are representative of ten to twelve independent experiments.

FIGURE 3.

SARAF is constitutively expressed in the plasma membrane and interacts with Orai1. A, SH-SY5Y cells were mixed with ice-cold Söerscen's buffer containing 2.5 mg EZ-Link sulfo-NHS-LC-biotin, and cell surface proteins were labeled by biotinylation, as described under “Experimental Procedures.” Labeled proteins were extracted with streptavidin-coated agarose beads and analyzed by SDS-PAGE and Western blotting using the anti-SARAF antibody. These results are representative of four separate experiments. B, SH-SY5Y cells were pretreated for 30 min with 10 μm jasplakinolide and then were stimulated in the presence of 1.2 mm extracellular Ca²⁺ with AA (8 μm) and lysed. Whole cell lysates were immunoprecipitated with anti-SARAF antibody. Immunoprecipitates were analyzed by Western blotting (WB) using anti-Orai1 antibody (top) and reprobed with anti-SARAF antibody (bottom) for protein loading control. Positions of molecular mass markers are shown on the right. HC, heavy chain of the Ig used for immunoprecipitation. Histograms indicate the quantification of SARAF-Orai1 interaction presented as percentage of control (cells not stimulated with AA). Data are expressed as mean ± S.E. of six independent experiments. C, schematic presentation of the effect of jasplakinolide on actin filament reorganization. Actin polymerization induced by jasplakinolide forms a tight cortical actin barrier, displacing the ER and preventing physical interaction with the plasma membrane.

FIGURE 4.

SARAF modulates the ability of AA to promote cell survival. SH-SY5Y cells overexpressing SARAF (SARAF) and mock-treated cells were treated for 24h with ATRA (5–15 μm) alone or in combination with the PLA2 inhibitor AACOCF3 (10 μm). Apoptosis was determined as described under “Experimental Procedures.” Data are expressed as mean ± S.E. of four independent experiments. *, p < 0.05 as compared with Control.