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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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STIM2 K-rich domain binds fully- and semi-Ca2+-loaded CaM in solution(A) Cartoon with illustration of a competition assay with GFP-STIM2K bound to Ca2+/CaM beads and elution of GFP–STIM2K with purified CaM from the beads. (B) Quantification of eluted GFP–STIM2K after incubation with 4 mM EGTA, 1 mM CaCl2, and 75 μM CaM, CaM1-2m, CaM3–4m or CaM1-4m in 1 mM CaCl2. Ca2+-binding EF-hand domains in the N-terminal lobe (CaM1-2m), the C-terminal lobe (CaM3-4m) or in both lobes (CaM1–4m) were mutated. Bars indicate mean±S.D. from at least three experiments (*P<0.05 and n.s. according to Student's t test).
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Figure 4: STIM2 K-rich domain binds fully- and semi-Ca2+-loaded CaM in solution(A) Cartoon with illustration of a competition assay with GFP-STIM2K bound to Ca2+/CaM beads and elution of GFP–STIM2K with purified CaM from the beads. (B) Quantification of eluted GFP–STIM2K after incubation with 4 mM EGTA, 1 mM CaCl2, and 75 μM CaM, CaM1-2m, CaM3–4m or CaM1-4m in 1 mM CaCl2. Ca2+-binding EF-hand domains in the N-terminal lobe (CaM1-2m), the C-terminal lobe (CaM3-4m) or in both lobes (CaM1–4m) were mutated. Bars indicate mean±S.D. from at least three experiments (*P<0.05 and n.s. according to Student's t test).

Mentions: In order to test whether Ca2+/CaM and GFP–STIM2K can form a complex under conditions where GFP–STIM2K bind PI(4,5)P2, we performed in solution-binding experiments with different purified CaM variants and oxidized GFP–STIM2K dimer. CaM is a 17 kDa acidic cytosolic Ca2+-binding protein containing N- and C-terminal lobes connected by a flexible linker. Each lobe has two Ca2+-binding EF-hand domains [31] and since the Ca2+-binding affinity for the C-terminal lobe is several fold higher than for the N-terminal lobe [32,33], CaM can exist in three states: as Ca2+-free, semi Ca2+-loaded or as fully Ca2+-loaded molecule. In order to test under which conditions the K-rich domain of STIM2 interacts with CaM, we mutated EF-hand domains I and II in the N-terminal lobe (CaM1–2m), EF-hand domains III and IV in the C-terminal lobe (CaM3–4m), and all four EF-hand domains (CaM1–4m). Correct folding of the purified proteins was confirmed by trypsin digestion [34], where fully Ca2+-loaded CaM and C-terminal lobe Ca2+-loaded CaM (CaM1–2m) showed increased protease protection in the presence of CaCl2 (Supplementary Figures S3A and S3C; available at http://www.bioscirep.org/bsr/033/bsr033e077add.htm) compared with the unloaded CaM1–4m and the N-terminal lobe Ca2+-loaded CaM (CaM3–4m) (Figures S3B and S3D). To compare the binding of GFP–STIM2K dimer to semi and fully Ca2+-loaded CaM, we performed a competition experiment with GFP–STIM2K bound to Ca2+/CaM beads (cartoon Figure 4A). Incubation of immobilized GFP–STIM2K dimer with purified CaM in CaCl2 buffer eluted 45±7% of GFP–STIM2K from the Ca2+/CaM beads compared with only 7±1 and 8±2% elution after incubation with CaM1–4m and CaM3–4m, respectively (Figure 4B). This showed that Ca2+ loading of CaM is required for binding to STIM2 K-rich domain and that mutation of the EF-hand domains in the C-terminal lobe abolished binding. We confirmed these data by in solution-binding experiments under conditions that allow binding of GFP–STIM2K dimer to PI(4,5)P2. Incubation of 10 μM CaM with 10 μM GFP–STIM2K dimer in a buffer with 1 mM CaCl2 led to complex formation, which after separation by gel filtration mainly eluted in fractions 6–8 compared with elution of GFP–STIM2K dimer in fractions 7–14 and CaM in 10–14 (Supplementary Figure S4A; available at http://www.bioscirep.org/bsr/033/bsr033e077add.htm). Mutation of all four Ca2+-binding sites of CaM (CaM1–4m) or mutation of the Ca2+-binding sites in the C-terminal lobe (CaM3–4m) abolished interaction with GFP–STIM2K dimer (Figures S4B and S4C), while mutation of Ca2+-binding sites in the N-terminal lobe (CaM1–2m) had a milder effect (Figure S4D). This weak binding of the C-terminal lobe Ca2+-loaded CaM to GFP-STIM2K dimer was confirmed by elution of GFP-STIM2K with CaM1-2m from CaM beads (Figure 4B). However, compared with elution with fully Ca2+-loaded CaM, competition with semi Ca2+-loaded CaM (CaM1–2m) eluted only 21±5% of the bound GFP–STIM2K dimer compared with 45±7% with fully Ca2+-loaded CaM (Figure 4B). This suggests that semi- and fully Ca2+-load CaM bind with different affinities to STIM2 K-rich domain dimer.


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)

STIM2 K-rich domain binds fully- and semi-Ca2+-loaded CaM in solution(A) Cartoon with illustration of a competition assay with GFP-STIM2K bound to Ca2+/CaM beads and elution of GFP–STIM2K with purified CaM from the beads. (B) Quantification of eluted GFP–STIM2K after incubation with 4 mM EGTA, 1 mM CaCl2, and 75 μM CaM, CaM1-2m, CaM3–4m or CaM1-4m in 1 mM CaCl2. Ca2+-binding EF-hand domains in the N-terminal lobe (CaM1-2m), the C-terminal lobe (CaM3-4m) or in both lobes (CaM1–4m) were mutated. Bars indicate mean±S.D. from at least three experiments (*P<0.05 and n.s. according to Student's t test).
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Figure 4: STIM2 K-rich domain binds fully- and semi-Ca2+-loaded CaM in solution(A) Cartoon with illustration of a competition assay with GFP-STIM2K bound to Ca2+/CaM beads and elution of GFP–STIM2K with purified CaM from the beads. (B) Quantification of eluted GFP–STIM2K after incubation with 4 mM EGTA, 1 mM CaCl2, and 75 μM CaM, CaM1-2m, CaM3–4m or CaM1-4m in 1 mM CaCl2. Ca2+-binding EF-hand domains in the N-terminal lobe (CaM1-2m), the C-terminal lobe (CaM3-4m) or in both lobes (CaM1–4m) were mutated. Bars indicate mean±S.D. from at least three experiments (*P<0.05 and n.s. according to Student's t test).
Mentions: In order to test whether Ca2+/CaM and GFP–STIM2K can form a complex under conditions where GFP–STIM2K bind PI(4,5)P2, we performed in solution-binding experiments with different purified CaM variants and oxidized GFP–STIM2K dimer. CaM is a 17 kDa acidic cytosolic Ca2+-binding protein containing N- and C-terminal lobes connected by a flexible linker. Each lobe has two Ca2+-binding EF-hand domains [31] and since the Ca2+-binding affinity for the C-terminal lobe is several fold higher than for the N-terminal lobe [32,33], CaM can exist in three states: as Ca2+-free, semi Ca2+-loaded or as fully Ca2+-loaded molecule. In order to test under which conditions the K-rich domain of STIM2 interacts with CaM, we mutated EF-hand domains I and II in the N-terminal lobe (CaM1–2m), EF-hand domains III and IV in the C-terminal lobe (CaM3–4m), and all four EF-hand domains (CaM1–4m). Correct folding of the purified proteins was confirmed by trypsin digestion [34], where fully Ca2+-loaded CaM and C-terminal lobe Ca2+-loaded CaM (CaM1–2m) showed increased protease protection in the presence of CaCl2 (Supplementary Figures S3A and S3C; available at http://www.bioscirep.org/bsr/033/bsr033e077add.htm) compared with the unloaded CaM1–4m and the N-terminal lobe Ca2+-loaded CaM (CaM3–4m) (Figures S3B and S3D). To compare the binding of GFP–STIM2K dimer to semi and fully Ca2+-loaded CaM, we performed a competition experiment with GFP–STIM2K bound to Ca2+/CaM beads (cartoon Figure 4A). Incubation of immobilized GFP–STIM2K dimer with purified CaM in CaCl2 buffer eluted 45±7% of GFP–STIM2K from the Ca2+/CaM beads compared with only 7±1 and 8±2% elution after incubation with CaM1–4m and CaM3–4m, respectively (Figure 4B). This showed that Ca2+ loading of CaM is required for binding to STIM2 K-rich domain and that mutation of the EF-hand domains in the C-terminal lobe abolished binding. We confirmed these data by in solution-binding experiments under conditions that allow binding of GFP–STIM2K dimer to PI(4,5)P2. Incubation of 10 μM CaM with 10 μM GFP–STIM2K dimer in a buffer with 1 mM CaCl2 led to complex formation, which after separation by gel filtration mainly eluted in fractions 6–8 compared with elution of GFP–STIM2K dimer in fractions 7–14 and CaM in 10–14 (Supplementary Figure S4A; available at http://www.bioscirep.org/bsr/033/bsr033e077add.htm). Mutation of all four Ca2+-binding sites of CaM (CaM1–4m) or mutation of the Ca2+-binding sites in the C-terminal lobe (CaM3–4m) abolished interaction with GFP–STIM2K dimer (Figures S4B and S4C), while mutation of Ca2+-binding sites in the N-terminal lobe (CaM1–2m) had a milder effect (Figure S4D). This weak binding of the C-terminal lobe Ca2+-loaded CaM to GFP-STIM2K dimer was confirmed by elution of GFP-STIM2K with CaM1-2m from CaM beads (Figure 4B). However, compared with elution with fully Ca2+-loaded CaM, competition with semi Ca2+-loaded CaM (CaM1–2m) eluted only 21±5% of the bound GFP–STIM2K dimer compared with 45±7% with fully Ca2+-loaded CaM (Figure 4B). This suggests that semi- and fully Ca2+-load CaM bind with different affinities to STIM2 K-rich domain dimer.

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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