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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+ inhibits binding of STIM2 K-rich domain dimer to PI(4,5)P2 liposomes(A) Binding of 1 μM GFP and oxidized GFP-STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of GFP–STIM2K dimer in HK buffer with indicated Ca2+ concentrations. Binding of dimeric GFP–STIM2K in HK buffer without Ca2+ was set to 100. (B) Binding of 2 μM GFP and oxidized GFP–STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM oxidized GFP–STIM2K and GFP-STIM2K pre-incubated for 1 h with 100 μM CaCl2/5 μM CaM to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer with 100 μM CaCl2. Binding of GFP–STIM2K without CaCl2 and CaM was set to 100. (C) Binding of 2 μM GFP–STIM2K dimer to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM dimeric GFP–STIM2K pre-incubated for 1 h with 5 μM CaM to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer with 50, 100 and 200 μM CaCl2. Binding of GFP–STIM2K dimer in HK buffer without Ca2+ 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 5: Ca2+ inhibits binding of STIM2 K-rich domain dimer to PI(4,5)P2 liposomes(A) Binding of 1 μM GFP and oxidized GFP-STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of GFP–STIM2K dimer in HK buffer with indicated Ca2+ concentrations. Binding of dimeric GFP–STIM2K in HK buffer without Ca2+ was set to 100. (B) Binding of 2 μM GFP and oxidized GFP–STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM oxidized GFP–STIM2K and GFP-STIM2K pre-incubated for 1 h with 100 μM CaCl2/5 μM CaM to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer with 100 μM CaCl2. Binding of GFP–STIM2K without CaCl2 and CaM was set to 100. (C) Binding of 2 μM GFP–STIM2K dimer to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM dimeric GFP–STIM2K pre-incubated for 1 h with 5 μM CaM to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer with 50, 100 and 200 μM CaCl2. Binding of GFP–STIM2K dimer in HK buffer without Ca2+ 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: Based on the binding of semi- and fully Ca2+-loaded CaM to the K-rich domain of STIM2, we hypothesized that high cytosolic [Ca2+] interferes with binding of STIM2 to PM lipids in vivo. This may lead to a negative-feedback regulation of STIM2-mediated ER–PM contact formation and Ca2+ entry. In order to investigate the effect of Ca2+/CaM on binding of GFP–STIM2K dimer to PM-like liposomes, we first tested whether Ca2+ alone has any effect on the interaction between the K-rich domain dimer and PI(4,5)P2. Therefore we measured the binding of 1 μM oxidized GFP–STIM2K to PM-like liposomes with 5 mol% PI(4,5)P2 in Ca2+-free buffer and set this value to 100 (Figure 5A). An increase of [Ca2+] up to 100 μM had no severe effect and allowed 88% binding, whereas higher [Ca2+] between 250 μM and 1 mM abolished the interaction completely (Figure 5A). However, when we used higher STIM2K dimer concentration the binding to 2 mol% PI(4,5)P2-containing liposomes was resistant to 200 μM CaCl2 (Figure 5C) as compared with binding of 1 μM protein in presence of 250 μM CaCl2 (Figure 5A). Thus, the binding of dimeric STIM2 K-rich domain to PM lipids is sensitive to high [Ca2+], suggesting that sustained local increase of cytosolic Ca2+ may interfere with 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+ inhibits binding of STIM2 K-rich domain dimer to PI(4,5)P2 liposomes(A) Binding of 1 μM GFP and oxidized GFP-STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of GFP–STIM2K dimer in HK buffer with indicated Ca2+ concentrations. Binding of dimeric GFP–STIM2K in HK buffer without Ca2+ was set to 100. (B) Binding of 2 μM GFP and oxidized GFP–STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM oxidized GFP–STIM2K and GFP-STIM2K pre-incubated for 1 h with 100 μM CaCl2/5 μM CaM to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer with 100 μM CaCl2. Binding of GFP–STIM2K without CaCl2 and CaM was set to 100. (C) Binding of 2 μM GFP–STIM2K dimer to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM dimeric GFP–STIM2K pre-incubated for 1 h with 5 μM CaM to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer with 50, 100 and 200 μM CaCl2. Binding of GFP–STIM2K dimer in HK buffer without Ca2+ 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 5: Ca2+ inhibits binding of STIM2 K-rich domain dimer to PI(4,5)P2 liposomes(A) Binding of 1 μM GFP and oxidized GFP-STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of GFP–STIM2K dimer in HK buffer with indicated Ca2+ concentrations. Binding of dimeric GFP–STIM2K in HK buffer without Ca2+ was set to 100. (B) Binding of 2 μM GFP and oxidized GFP–STIM2K dimer to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM oxidized GFP–STIM2K and GFP-STIM2K pre-incubated for 1 h with 100 μM CaCl2/5 μM CaM to PM-like liposomes with 5 mol% PI(4,5)P2 in HK buffer with 100 μM CaCl2. Binding of GFP–STIM2K without CaCl2 and CaM was set to 100. (C) Binding of 2 μM GFP–STIM2K dimer to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer without Ca2+ and binding of 2 μM dimeric GFP–STIM2K pre-incubated for 1 h with 5 μM CaM to PM-like liposomes with 2 mol% PI(4,5)P2 in HK buffer with 50, 100 and 200 μM CaCl2. Binding of GFP–STIM2K dimer in HK buffer without Ca2+ 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: Based on the binding of semi- and fully Ca2+-loaded CaM to the K-rich domain of STIM2, we hypothesized that high cytosolic [Ca2+] interferes with binding of STIM2 to PM lipids in vivo. This may lead to a negative-feedback regulation of STIM2-mediated ER–PM contact formation and Ca2+ entry. In order to investigate the effect of Ca2+/CaM on binding of GFP–STIM2K dimer to PM-like liposomes, we first tested whether Ca2+ alone has any effect on the interaction between the K-rich domain dimer and PI(4,5)P2. Therefore we measured the binding of 1 μM oxidized GFP–STIM2K to PM-like liposomes with 5 mol% PI(4,5)P2 in Ca2+-free buffer and set this value to 100 (Figure 5A). An increase of [Ca2+] up to 100 μM had no severe effect and allowed 88% binding, whereas higher [Ca2+] between 250 μM and 1 mM abolished the interaction completely (Figure 5A). However, when we used higher STIM2K dimer concentration the binding to 2 mol% PI(4,5)P2-containing liposomes was resistant to 200 μM CaCl2 (Figure 5C) as compared with binding of 1 μM protein in presence of 250 μM CaCl2 (Figure 5A). Thus, the binding of dimeric STIM2 K-rich domain to PM lipids is sensitive to high [Ca2+], suggesting that sustained local increase of cytosolic Ca2+ may interfere with 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