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Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids.

Bhardwaj R, Müller HM, Nickel W, Seedorf M - Biosci. Rep. (2013)

Bottom Line: We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation.Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner.Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

View Article: PubMed Central - PubMed

Affiliation: *Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

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α-helicity of STIM2 K-rich domain contributes in binding to Ca2+/CaM(A) Binding of 1 μM reduced GFP, GFP–STIM1C, GFP–STIM1C∆K, GFP–STIM2C and GFP-STIM2C∆K to Ca2+/CaM beads in 1 mM CaCl2 and GFP–STIM1C and GFP-STIM2C in 1 mM EGTA. (B) Binding of 1 μM reduced GFP, GFP–STIM1K, GFP–STIM1K–P675A, GFP–STIM1K–P682A, GFP–STIM1K–P675A–P682A, GFP–STIM2K and GFP–STIM2K–K743P to Ca2+/CaM beads in 1 mM CaCl2. (C) Binding of 1 μM GFP–STIM1K, GFP–STIM1K–P675A–P682A, GFP–STIM1K+LC, GFP–STIM1K+LC–P675A–P682A, GFP–STIM2K, and GFP–STIM2K–C725A (all dialysed against DTT-free buffer) to Ca2+/CaM beads in 1 mM CaCl2. Bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005, n.s. according to Student's t test).
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Figure 3: α-helicity of STIM2 K-rich domain contributes in binding to Ca2+/CaM(A) Binding of 1 μM reduced GFP, GFP–STIM1C, GFP–STIM1C∆K, GFP–STIM2C and GFP-STIM2C∆K to Ca2+/CaM beads in 1 mM CaCl2 and GFP–STIM1C and GFP-STIM2C in 1 mM EGTA. (B) Binding of 1 μM reduced GFP, GFP–STIM1K, GFP–STIM1K–P675A, GFP–STIM1K–P682A, GFP–STIM1K–P675A–P682A, GFP–STIM2K and GFP–STIM2K–K743P to Ca2+/CaM beads in 1 mM CaCl2. (C) Binding of 1 μM GFP–STIM1K, GFP–STIM1K–P675A–P682A, GFP–STIM1K+LC, GFP–STIM1K+LC–P675A–P682A, GFP–STIM2K, and GFP–STIM2K–C725A (all dialysed against DTT-free buffer) to Ca2+/CaM beads in 1 mM CaCl2. Bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005, n.s. according to Student's t test).

Mentions: In order to characterize the interaction between CaM and STIM1 and STIM2 in more detail, we developed a fluorescence-based in vitro CaM-binding assay. Since many complexes of CaM with its targets form via hydrophobic interactions with an α-helical structure in the target [29], we established the CaM-binding assay in detergent-free buffer system. The resulting unspecific interactions were minimized by the use of reaction tubes with modified surfaces and bound proteins were eluted with high salt and the metal chelator EGTA. First, we analysed binding of the entire CTDs to CaM-beads. Under reducing conditions at 1 μM protein concentration in 1 mM CaCl2, 64±8% of GFP–STIM1C and 61±9% of GFP–STIM2C were bound to CaM-beads, while addition of EGTA completely abolished the binding (Figure 3A). Free GFP showed no binding, demonstrating that this assay is specific. Deletion of the K-rich domains of STIM1C and STIM2C (GFP–STIM1C∆K and GFP–STIM2C∆K) reduced the binding to Ca2+/CaM to 41±6 and 30±4%, respectively, suggesting these domains contribute to Ca2+/CaM binding (Figure 3A). Binding of GFP–STIM1C∆K and GFP–STIM2C∆K is consistent with the presence of additional Ca2+/CaM-binding sites, which are predicted in the CAD of STIM1 and STIM2 [20,30]. We confirmed the results from the fluorescence-based assay by analysis of GFP–STIM2C and GFP–STIM2C∆K binding to Ca2+/CaM by Western blotting using GFP-specific antibody and found 67±5 and 27±4% bound GFP–STIM2C and GFP–STIM2C∆K (Supplementary Figure S2; available at http://www.bioscirep.org/bsr/033/bsr033e077add.htm). This is similar to the values observed by our fluorescence-based assay. Thereafter, we employed this quantitative fluorescence-based assay for all following experiments.


Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids.

Bhardwaj R, Müller HM, Nickel W, Seedorf M - Biosci. Rep. (2013)

α-helicity of STIM2 K-rich domain contributes in binding to Ca2+/CaM(A) Binding of 1 μM reduced GFP, GFP–STIM1C, GFP–STIM1C∆K, GFP–STIM2C and GFP-STIM2C∆K to Ca2+/CaM beads in 1 mM CaCl2 and GFP–STIM1C and GFP-STIM2C in 1 mM EGTA. (B) Binding of 1 μM reduced GFP, GFP–STIM1K, GFP–STIM1K–P675A, GFP–STIM1K–P682A, GFP–STIM1K–P675A–P682A, GFP–STIM2K and GFP–STIM2K–K743P to Ca2+/CaM beads in 1 mM CaCl2. (C) Binding of 1 μM GFP–STIM1K, GFP–STIM1K–P675A–P682A, GFP–STIM1K+LC, GFP–STIM1K+LC–P675A–P682A, GFP–STIM2K, and GFP–STIM2K–C725A (all dialysed against DTT-free buffer) to Ca2+/CaM beads in 1 mM CaCl2. Bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005, n.s. according to Student's t test).
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Figure 3: α-helicity of STIM2 K-rich domain contributes in binding to Ca2+/CaM(A) Binding of 1 μM reduced GFP, GFP–STIM1C, GFP–STIM1C∆K, GFP–STIM2C and GFP-STIM2C∆K to Ca2+/CaM beads in 1 mM CaCl2 and GFP–STIM1C and GFP-STIM2C in 1 mM EGTA. (B) Binding of 1 μM reduced GFP, GFP–STIM1K, GFP–STIM1K–P675A, GFP–STIM1K–P682A, GFP–STIM1K–P675A–P682A, GFP–STIM2K and GFP–STIM2K–K743P to Ca2+/CaM beads in 1 mM CaCl2. (C) Binding of 1 μM GFP–STIM1K, GFP–STIM1K–P675A–P682A, GFP–STIM1K+LC, GFP–STIM1K+LC–P675A–P682A, GFP–STIM2K, and GFP–STIM2K–C725A (all dialysed against DTT-free buffer) to Ca2+/CaM beads in 1 mM CaCl2. Bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005, n.s. according to Student's t test).
Mentions: In order to characterize the interaction between CaM and STIM1 and STIM2 in more detail, we developed a fluorescence-based in vitro CaM-binding assay. Since many complexes of CaM with its targets form via hydrophobic interactions with an α-helical structure in the target [29], we established the CaM-binding assay in detergent-free buffer system. The resulting unspecific interactions were minimized by the use of reaction tubes with modified surfaces and bound proteins were eluted with high salt and the metal chelator EGTA. First, we analysed binding of the entire CTDs to CaM-beads. Under reducing conditions at 1 μM protein concentration in 1 mM CaCl2, 64±8% of GFP–STIM1C and 61±9% of GFP–STIM2C were bound to CaM-beads, while addition of EGTA completely abolished the binding (Figure 3A). Free GFP showed no binding, demonstrating that this assay is specific. Deletion of the K-rich domains of STIM1C and STIM2C (GFP–STIM1C∆K and GFP–STIM2C∆K) reduced the binding to Ca2+/CaM to 41±6 and 30±4%, respectively, suggesting these domains contribute to Ca2+/CaM binding (Figure 3A). Binding of GFP–STIM1C∆K and GFP–STIM2C∆K is consistent with the presence of additional Ca2+/CaM-binding sites, which are predicted in the CAD of STIM1 and STIM2 [20,30]. We confirmed the results from the fluorescence-based assay by analysis of GFP–STIM2C and GFP–STIM2C∆K binding to Ca2+/CaM by Western blotting using GFP-specific antibody and found 67±5 and 27±4% bound GFP–STIM2C and GFP–STIM2C∆K (Supplementary Figure S2; available at http://www.bioscirep.org/bsr/033/bsr033e077add.htm). This is similar to the values observed by our fluorescence-based assay. Thereafter, we employed this quantitative fluorescence-based assay for all following experiments.

Bottom Line: We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation.Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner.Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

View Article: PubMed Central - PubMed

Affiliation: *Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

Show MeSH