STIM1, but not STIM2, is required for proper agonist-induced Ca2+ signaling
Introduction
Inositol 1,4,5-trisphosphate (IP3) is a second messenger involved in setting up intracellular Ca2+ signals [1]. IP3 binds to the IP3 receptor (IP3R), a Ca2+-release channel located in the endoplasmic reticulum (ER) [1] and in the Golgi apparatus [2]. The IP3R becomes less sensitive to IP3 when the Ca2+ concentration ([Ca2+]) in the lumen of the store ([Ca2+]lum) decreases [3], [4], [5], [6], [7]. The stores sometimes have to be extensively depleted before IP3R sensitivity decreases [8], [9], [10]. The effect of luminal Ca2+ on the IP3R might arise from an interaction of released Ca2+ with Ca2+-binding sites on the cytosolic side of the IP3R [11], [12], [13]. Ca2+ may however also act from the lumen of the store via the luminal Ca2+-binding site of the IP3R [14] or via associated proteins. Binding of ERp44 protein-disulfide isomerase to a luminal site of the IP3R1 inhibits channel activity and this inhibition becomes more pronounced at low levels of store loading [15].
The stromal interaction molecules STIM1 and STIM2 are ER Ca2+ sensors involved in activation of Ca2+ entry when stores lose Ca2+ [16], [17]. These single-pass transmembrane proteins have near their N-terminus in the ER lumen two adjacent regions containing negative charges. One is typical for an EF-hand Ca2+-binding motif [18]. It binds Ca2+ with a dissociation constant in the range of [Ca2+]lum occurring within the ER [19]. Release of ER Ca2+ leads to an unfolding of the EF-hand and adjoining 5-helix sterile α motif protein interaction domain and a rapid oligomerization with similar domains of neighboring STIM molecules [20]. Further aggregation involves interaction with cytoplasmic C-terminal coiled–coiled regions. These STIM aggregates move to ER–plasma membrane appositions, recruit ORAI1 by binding its C-terminus, and trigger ORAI1 opening and Ca2+ influx by binding to an N-terminal region of ORAI1. The most recent advances in the understanding of STIM/ORAI signaling have been discussed by Hogan et al. [21]. STIM1 not only translocates during ER Ca2+-store depletion, but also during Ca2+ oscillations, and transiently activates Ca2+-entry channels in the plasma membrane. This finding indicates that STIM1 is also activated by physiological Ca2+ signaling in response to low concentrations of agonist and thus plays a central role in the spatiotemporal profile of intracellular Ca2+ dynamics [22], [23].
It was previously suggested that STIM1 may interact with the IP3R and in this way reduce the store Ca2+ content and agonist-induced Ca2+ release [24], [25]. In addition, in pancreatic acinar cells STIM1 translocates after ER Ca2+ depletion from the apical part of the ER, where IP3Rs reside, to the more lateral and basal regions of the ER, with a segregation of IP3Rs from STIM1 [26]. We therefore now investigated whether STIM1 and STIM2 would interfere with the function of the IP3R. We studied mouse embryonic fibroblasts (MEFs) from Stim1−/− and Stim2−/− mice and compared them with cells from wild-type mice. We first investigated in plasma-membrane permeabilized cells whether STIM1 or STIM2 would also act as luminal Ca2+ sensor for the IP3R. Decreasing the store Ca2+ content increased the EC50 and decreased the Hill coefficient for IP3-induced Ca2+ release. This effect occurred both in the presence and absence of STIM proteins, indicating that these proteins were not the luminal Ca2+ sensor that controls the loading dependence of the release. We then examined the intracellular Ca2+ release induced by extracellular ATP in intact cells. We found reduced ATP-induced rises in free cytosolic [Ca2+] ([Ca2+]cyt) in STIM1-deficient cells, while the function of the IP3R was not affected. We conclude that (i) the STIM proteins are not the luminal Ca2+ sensor for the IP3R, and (ii) that a direct effect on the IP3R is not involved in the reduced ATP-induced intracellular Ca2+ release in STIM1-deficient cells.
Section snippets
Cell culture
Wild-type, Stim1−/− and Stim2−/− MEFs were established from embryos at day 14.5 obtained by intercrossing of Stim1+/− or Stim2+/− mice [27]. The cells were cultured at 37 °C in a 9% CO2 incubator in DMEM/Ham's F12 medium supplemented with 10% fetal calf serum, 3.8 mM l-glutamine, 85 IU ml−1 penicillin and 85 μg ml−1 streptomycin. All media were obtained from Invitrogen (Paisley, UK).
For the 45Ca2+ fluxes, the cells were seeded in 12-well clusters (Costar, MA, 4 cm2) at a density of approximately 104
STIM1 and STIM2 expression in wild-type, Stim1−/− and Stim2−/− MEFs
The mRNA levels of STIM1 and STIM2 were quantified using qRT-PCR in wild-type MEFs. STIM2-mRNA levels were 1.47 (± 0.18)-fold higher than those of STIM1 (n = 5, with each measurement performed in triplicate). We also assessed whether knocking out STIM1 affected the expression of STIM2, and vice versa. Expression of STIM2 was not affected by knocking out STIM1 (STIM2 expression in Stim1−/− and Stim2−/− MEFs compared to wild-type MEFs, which was set at 1: 0.918 ± 0.067 and 0.037 ± 0.007, respectively).
Conclusions
We have directly tested the hypothesis raised by Varga-Szabo et al. [24] and Braun et al. [25] that STIM1 may interact with the IP3R and in this way reduce the store Ca2+ content and agonist-induced Ca2+ release. We found that STIM1 and STIM2 were not the luminal Ca2+ sensor that controls the loading dependence of the IP3-induced Ca2+ release. This finding suggests that another luminal Ca2+ sensor like ERp44 protein-disulfide isomerase [15] might be involved, or that the effect of luminal Ca2+
Acknowledgements
We thank Marina Crabbé, Anja Florizoone and Irene Willems for their technical assistance. This work was supported by Grant GOA/09/12 from the Research Council of the K.U.Leuven, by FWO-grant G.0724.09N from the Fonds Wetenschappelijk Onderzoek–Vlaanderen, and by the Interuniversity Poles of Attraction Programme-Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs, IUAP P6/28-C. Jean-Paul Decuypere was funded by a Ph.D. grant of the Institute for
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These authors contributed equally to this work.