Limits...
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.

Show MeSH
Tetramerization of STIM1 K-rich domain leads to PI(4,5)P2 binding(A) Schematic outline of human STIM1 and STIM2 domain structure (drawn to scale). SS (signal sequence), EF-hand domain (EF), SAM, TMD (transmembrane domain), CC domains 1–3, CAD, the K-rich domains with their amino acid sequences and the orientations of N- and C-termini are shown. Cysteine residues are depicted as grey circles. (B) Purified GFP-tagged CTDs of STIM1 and STIM2 were run on S200 gel-filtration column and the elution profiles of GFP–STIM1C (grey), GFP–STIM2C (black) and of the indicated marker proteins are shown. (C) Binding of 1 μM of each GFP, GFP–STIM1C, GFP–STIM1K, GFP-tagged GCN4 leucine zipper (GFP–Zipper) and GFP–Zipper with STIM1 K-rich domain (GFP–Zipper–STIM1K) to PM-like liposomes with 5 mol% PI(4,5)P2 in presence of 1 mM DTT. Binding of GFP–STIM1C was set to 100 and bars indicate mean±S.D. from at least three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814058&req=5

Figure 1: Tetramerization of STIM1 K-rich domain leads to PI(4,5)P2 binding(A) Schematic outline of human STIM1 and STIM2 domain structure (drawn to scale). SS (signal sequence), EF-hand domain (EF), SAM, TMD (transmembrane domain), CC domains 1–3, CAD, the K-rich domains with their amino acid sequences and the orientations of N- and C-termini are shown. Cysteine residues are depicted as grey circles. (B) Purified GFP-tagged CTDs of STIM1 and STIM2 were run on S200 gel-filtration column and the elution profiles of GFP–STIM1C (grey), GFP–STIM2C (black) and of the indicated marker proteins are shown. (C) Binding of 1 μM of each GFP, GFP–STIM1C, GFP–STIM1K, GFP-tagged GCN4 leucine zipper (GFP–Zipper) and GFP–Zipper with STIM1 K-rich domain (GFP–Zipper–STIM1K) to PM-like liposomes with 5 mol% PI(4,5)P2 in presence of 1 mM DTT. Binding of GFP–STIM1C was set to 100 and bars indicate mean±S.D. from at least three experiments.

Mentions: The CTDs of STIM1 and STIM2 are exposed to the cytosol and comprise three CC domains with an overlapping CAD, variable regions and K-rich domains (Figure 1A). Binding to PI(4,5)P2-containing liposomes having a PM-like lipid composition with cholesterol and sphingolipids depends on electrostatic interaction of the K-rich domains with PI(4,5)P2 [6].


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)

Tetramerization of STIM1 K-rich domain leads to PI(4,5)P2 binding(A) Schematic outline of human STIM1 and STIM2 domain structure (drawn to scale). SS (signal sequence), EF-hand domain (EF), SAM, TMD (transmembrane domain), CC domains 1–3, CAD, the K-rich domains with their amino acid sequences and the orientations of N- and C-termini are shown. Cysteine residues are depicted as grey circles. (B) Purified GFP-tagged CTDs of STIM1 and STIM2 were run on S200 gel-filtration column and the elution profiles of GFP–STIM1C (grey), GFP–STIM2C (black) and of the indicated marker proteins are shown. (C) Binding of 1 μM of each GFP, GFP–STIM1C, GFP–STIM1K, GFP-tagged GCN4 leucine zipper (GFP–Zipper) and GFP–Zipper with STIM1 K-rich domain (GFP–Zipper–STIM1K) to PM-like liposomes with 5 mol% PI(4,5)P2 in presence of 1 mM DTT. Binding of GFP–STIM1C was set to 100 and bars indicate mean±S.D. from at least three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814058&req=5

Figure 1: Tetramerization of STIM1 K-rich domain leads to PI(4,5)P2 binding(A) Schematic outline of human STIM1 and STIM2 domain structure (drawn to scale). SS (signal sequence), EF-hand domain (EF), SAM, TMD (transmembrane domain), CC domains 1–3, CAD, the K-rich domains with their amino acid sequences and the orientations of N- and C-termini are shown. Cysteine residues are depicted as grey circles. (B) Purified GFP-tagged CTDs of STIM1 and STIM2 were run on S200 gel-filtration column and the elution profiles of GFP–STIM1C (grey), GFP–STIM2C (black) and of the indicated marker proteins are shown. (C) Binding of 1 μM of each GFP, GFP–STIM1C, GFP–STIM1K, GFP-tagged GCN4 leucine zipper (GFP–Zipper) and GFP–Zipper with STIM1 K-rich domain (GFP–Zipper–STIM1K) to PM-like liposomes with 5 mol% PI(4,5)P2 in presence of 1 mM DTT. Binding of GFP–STIM1C was set to 100 and bars indicate mean±S.D. from at least three experiments.
Mentions: The CTDs of STIM1 and STIM2 are exposed to the cytosol and comprise three CC domains with an overlapping CAD, variable regions and K-rich domains (Figure 1A). Binding to PI(4,5)P2-containing liposomes having a PM-like lipid composition with cholesterol and sphingolipids depends on electrostatic interaction of the K-rich domains with PI(4,5)P2 [6].

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