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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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Ca2+/CaM competes with STIM2 K-rich domain's binding to PI(4,5)P2(A) Binding of 2 μM reduced GFP, oxidized GFP-STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1–2m, CaM3–4m or CaM1–4m in presence of 100 μM CaCl2 to 2 mol% PI(4,5)P2-containing PM-like liposomes in HK buffer. Binding of dimeric GFP–STIM2K pre-incubated with either 500 μM EGTA or 500 μM EGTA/5 μM CaM to 2 mol% PI(4,5)P2 in HK buffer is also shown. Binding of 2 μM oxidized GFP-STIM2K dimer without Ca2+ to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer was set to 100. (B) Binding of 2 μM reduced GFP, oxidized GFP–STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1-2m, CaM3-4m or CaM1–4m in presence of 100 μM CaCl2 to 5 mol% PI(4,5)P2-containing PC liposomes in HK buffer. Binding of 2 μM oxidized GFP–STIM2K dimer without Ca2+ to PC liposomes with 5 mol% PI(4,5)P2 in HK buffer was set to 100 and bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005 and n.s. according to Student's t test).
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Figure 6: Ca2+/CaM competes with STIM2 K-rich domain's binding to PI(4,5)P2(A) Binding of 2 μM reduced GFP, oxidized GFP-STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1–2m, CaM3–4m or CaM1–4m in presence of 100 μM CaCl2 to 2 mol% PI(4,5)P2-containing PM-like liposomes in HK buffer. Binding of dimeric GFP–STIM2K pre-incubated with either 500 μM EGTA or 500 μM EGTA/5 μM CaM to 2 mol% PI(4,5)P2 in HK buffer is also shown. Binding of 2 μM oxidized GFP-STIM2K dimer without Ca2+ to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer was set to 100. (B) Binding of 2 μM reduced GFP, oxidized GFP–STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1-2m, CaM3-4m or CaM1–4m in presence of 100 μM CaCl2 to 5 mol% PI(4,5)P2-containing PC liposomes in HK buffer. Binding of 2 μM oxidized GFP–STIM2K dimer without Ca2+ to PC liposomes with 5 mol% PI(4,5)P2 in HK buffer was set to 100 and bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005 and n.s. according to Student's t test).

Mentions: In order to test further whether the competition of CaM depends on Ca2+-loading, we pre-incubated GFP-STIM2K dimer with CaM and tested binding to PM-like liposomes with 2 mol% PI(4,5)P2. Compared with binding at 100 μM CaCl2, pre-incubation with 5 μM CaM in presence of CaCl2 reduced binding by 61±1% (Figure 6A). Pre-incubation of GFP–STIM2K dimer with Ca2+-free CaM1–4m (all EF-hand domains mutated), in presence of CaCl2 showed no competition, indicating that Ca2+-loading of CaM is required for competition with lipid binding. Consistently, CaM in presence of EGTA, only had a weak effect on lipid-binding activity of STIM2K dimer and a similar weak effect had the pre-incubation with CaM3–4m (C-terminal lobe Ca2+-binding mutant), suggesting that Ca2+-loading of the C-terminal lobe is required for competition. Mutation of the N-terminal lobe resulted in a CaM variant that showed an intermediate level of competition, i.e. 31±2% reduction in binding to 2 mol% PI(4,5)P2-containing PM-like liposomes. Although this effect is weaker compared with fully Ca2+-loaded CaM, it suggests that semi-Ca2+-loaded CaM at an intermediate cytosolic [Ca2+] may have an inhibitory effect on STIM2-mediated ER–PM contact formation.


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)

Ca2+/CaM competes with STIM2 K-rich domain's binding to PI(4,5)P2(A) Binding of 2 μM reduced GFP, oxidized GFP-STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1–2m, CaM3–4m or CaM1–4m in presence of 100 μM CaCl2 to 2 mol% PI(4,5)P2-containing PM-like liposomes in HK buffer. Binding of dimeric GFP–STIM2K pre-incubated with either 500 μM EGTA or 500 μM EGTA/5 μM CaM to 2 mol% PI(4,5)P2 in HK buffer is also shown. Binding of 2 μM oxidized GFP-STIM2K dimer without Ca2+ to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer was set to 100. (B) Binding of 2 μM reduced GFP, oxidized GFP–STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1-2m, CaM3-4m or CaM1–4m in presence of 100 μM CaCl2 to 5 mol% PI(4,5)P2-containing PC liposomes in HK buffer. Binding of 2 μM oxidized GFP–STIM2K dimer without Ca2+ to PC liposomes with 5 mol% PI(4,5)P2 in HK buffer was set to 100 and bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005 and n.s. according to Student's t test).
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Figure 6: Ca2+/CaM competes with STIM2 K-rich domain's binding to PI(4,5)P2(A) Binding of 2 μM reduced GFP, oxidized GFP-STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1–2m, CaM3–4m or CaM1–4m in presence of 100 μM CaCl2 to 2 mol% PI(4,5)P2-containing PM-like liposomes in HK buffer. Binding of dimeric GFP–STIM2K pre-incubated with either 500 μM EGTA or 500 μM EGTA/5 μM CaM to 2 mol% PI(4,5)P2 in HK buffer is also shown. Binding of 2 μM oxidized GFP-STIM2K dimer without Ca2+ to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer was set to 100. (B) Binding of 2 μM reduced GFP, oxidized GFP–STIM2K dimer without Ca2+ and dimeric GFP–STIM2K pre-incubated with either 100 μM CaCl2, 5 μM CaM, CaM1-2m, CaM3-4m or CaM1–4m in presence of 100 μM CaCl2 to 5 mol% PI(4,5)P2-containing PC liposomes in HK buffer. Binding of 2 μM oxidized GFP–STIM2K dimer without Ca2+ to PC liposomes with 5 mol% PI(4,5)P2 in HK buffer was set to 100 and bars indicate mean±S.D. from at least three experiments (*P<0.05, **P<0.005 and n.s. according to Student's t test).
Mentions: In order to test further whether the competition of CaM depends on Ca2+-loading, we pre-incubated GFP-STIM2K dimer with CaM and tested binding to PM-like liposomes with 2 mol% PI(4,5)P2. Compared with binding at 100 μM CaCl2, pre-incubation with 5 μM CaM in presence of CaCl2 reduced binding by 61±1% (Figure 6A). Pre-incubation of GFP–STIM2K dimer with Ca2+-free CaM1–4m (all EF-hand domains mutated), in presence of CaCl2 showed no competition, indicating that Ca2+-loading of CaM is required for competition with lipid binding. Consistently, CaM in presence of EGTA, only had a weak effect on lipid-binding activity of STIM2K dimer and a similar weak effect had the pre-incubation with CaM3–4m (C-terminal lobe Ca2+-binding mutant), suggesting that Ca2+-loading of the C-terminal lobe is required for competition. Mutation of the N-terminal lobe resulted in a CaM variant that showed an intermediate level of competition, i.e. 31±2% reduction in binding to 2 mol% PI(4,5)P2-containing PM-like liposomes. Although this effect is weaker compared with fully Ca2+-loaded CaM, it suggests that semi-Ca2+-loaded CaM at an intermediate cytosolic [Ca2+] may have an inhibitory effect on STIM2-mediated ER–PM contact formation.

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