Molecular anatomy of the early events in STIM1 activation - oligomerization or conformational change?

Decreased luminal endoplasmic reticulum (ER) Ca2+ concentration triggers oligomerization and clustering of the ER Ca2+ sensor STIM1 to promote its association with plasma membrane Orai1 Ca2+ channels leading to increased Ca2+ influx. A key step in STIM1 activation is the release of its SOAR domain from an intramolecular clamp formed with the STIM1 first coiled-coil (CC1) region. Using a truncated STIM1(1-343) molecule that captures or releases the isolated SOAR domain depending on luminal ER Ca2+ concentrations, we analyzed the early molecular events that control the intramolecular clamp formed between the CC1 and SOAR domains. We found that STIM1 forms constitutive dimers, and its CC1 domain can bind the SOAR domain of another STIM1 molecule in trans. Artificial oligomerization failed to liberate the SOAR domain or activate STIM1 unless the luminal Ca2+-sensing domains were removed. We propose that the release of SOAR from its CC1 interaction is controlled by changes in the orientation of the two CC1 domains in STIM1 dimers. Ca2+ unbinding in the STIM1 luminal domains initiates the conformational change allowing SOAR domain liberation and clustering, leading to Orai1 channel activation.

Polyunsaturated fatty acids inhibit stimulated coupling between the ER Ca(2+) sensor STIM1 and the Ca(2+) channel protein Orai1 in a process that correlates with inhibition of stimulated STIM1 oligomerization.

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts, and we find that acute addition of micromolar PUFAs such as linoleic acid effectively inhibit of Ca(2+) responses in mast cells stimulated by antigen-mediated crosslinking of FcεRI or by the SERCA pump inhibitor, thapsigargin. In contrast, the saturated fatty acid, stearic acid, with the same carbon chain length as linoleic acid does not inhibit these responses. Consistent with this inhibition of store-operated Ca(2+) entry (SOCE), linoleic acid inhibits antigen-stimulated granule exocytosis to a similar extent. Using the fluorescently labeled plasma membrane Ca(2+) channel protein, AcGFP-Orai1, together with the labeled ER Ca(2+) sensor protein, STIM1-mRFP, we monitor stimulated coupling of these proteins that is essential for SOCE with a novel spectrofluorimetric resonance energy transfer method. We find effective inhibition of this stimulated coupling by linoleic acid that accounts for the inhibition of SOCE. Moreover, we find that linoleic acid induces some STIM1-STIM1 association, while inhibiting stimulated STIM1 oligomerization that precedes STIM1-Orai1 coupling. We hypothesize that linoleic acid and related PUFAs inhibit STIM1-Orai1 coupling by a mechanism that involves perturbation of ER membrane structure, possibly by disrupting electrostatic interactions important in STIM1 oligomerization. Thisarticle is part of a Special Issue entitled Tools to study lipid functions.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

LIPID transfer proteins regulate store-operated calcium entry via control of plasma membrane phosphoinositides.

The ER-resident proteins STIM1 together with the plasma membrane (PM)-localized Orai1 channels constitute the molecular components of the store-operated Ca2+ entry (SOCE) pathway. Prepositioning of STIM1 to the peripheral ER close to the PM ensures its efficient interaction with Orai1 upon a decrease in the ER luminal Ca2+ concentration. The C-terminal polybasic domain of STIM1 has been identified as mediating the interaction with PM phosphoinositides and hence positions the molecule to ER-PM contact sites. Here we show that STIM1 requires PM phosphatidylinositol 4-phosphate (PI4P) for efficient PM interaction. Accordingly, oxysterol binding protein related proteins (ORPs) that work at ER-PM junctions and consume PI4P gradients exert important control over the Ca2+ entry process. These studies reveal an important connection between non-vesicular lipid transport at ER-PM contact sites and regulation of ER Ca2+store refilling.

Dependence of STIM1/Orai1-mediated calcium entry on plasma membrane phosphoinositides.

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain.

Oligomerization of the Ca2+ sensor, STIM1, in the endoplasmic reticulum (ER) membrane, caused by depletion of ER Ca2+ stores, results in STIM1 coupling to the plasma membrane Ca2+ channel protein, Orai1, to activate Ca2+ influx in a process known as store-operated Ca2+ entry. We use fluorimetry-based fluorescence resonance energy transfer (FRET) to monitor changes in STIM1 oligomerization in COS7 cells transfected with STIM1 constructs containing selected truncations, deletions, and point mutations, and labeled with donor and acceptor fluorescent proteins at either the luminal (N-terminal) or the cytoplasmic (C-terminal) ends. Our results with sequential truncations of STIM1 from the C-terminus support previous evidence that the CRAC activation domain (CAD/SOAR, human sequence 342-448) is an oligomer-promoting segment of STIM1, and they show that truncation just after CAD/SOAR (1-448) causes significantly elevated basal cytoplasmic Ca2+ and spontaneous STIM1 clustering. We find that a 14 amino acid sequence just C-terminal of CAD/SOAR (449-462) prevents spontaneous clustering and activation of STIM1 in COS7 cells. In response to store depletion, C-terminally labeled STIM1 without CAD/SOAR clusters together with CAD/SOAR-containing STIM1 constructs. However, these donor-acceptor pairs do not undergo a stimulated increase in FRET, exhibiting instead a decrease in FRET consistent with a stimulated conformational extension in full length STIM1. We find that the 14 amino acid sequence plays a regulatory role in this process. Overall, our FRET results provide evidence in live cells that Ca2+ store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.

Activation of STIM1-Orai1 involves an intramolecular switching mechanism.

Stromal interaction molecule 1 (STIM1) stimulates calcium ion (Ca(2+)) entry through plasma membrane Orai1 channels in response to decreased Ca(2+) concentrations in the endoplasmic reticulum lumen. We identified an acidic motif within the STIM1 coiled-coil region that keeps its Ca(2+) activation domain [Ca(2+) release-activated Ca(2+) (CRAC) activation domain/STIM1-Orai activating region (CAD/SOAR)]-a cytoplasmic region required for its activation of Orai1-inactive. The sequence of the STIM1 acidic motif shows substantial similarity to that of the carboxyl-terminal coiled-coil segment of Orai1, which is the postulated site of interaction with STIM1. Mutations within this acidic region rendered STIM1 constitutively active, whereas mutations within a short basic segment of CAD/SOAR prevented Orai1 activation. We propose that the CAD/SOAR domain is released from an intramolecular clamp during STIM1 activation, allowing the basic segment to activate Orai1 channels. This evolutionarily conserved mechanism of STIM1 activation resembles the regulation of protein kinases by intramolecular silencing through pseudosubstrate binding.

Store-operated Ca2+ influx and subplasmalemmal mitochondria.

Calcium depletion of the endoplasmic reticulum (ER) induces oligomerisation, puncta formation and translocation of the ER Ca(2+) sensor proteins, STIM1 and -2 into plasma membrane (PM)-adjacent regions of the ER, where they activate the Orai1, -2 or -3 proteins present in the opposing PM. These proteins form ion channels through which store-operated Ca(2+) influx (SOC) occurs. Calcium ions exert negative feed-back on SOC. Here we examined whether subplasmalemmal mitochondria, which reduce this feed-back by Ca(2+) uptake, are located within or out of the high-Ca(2+) microdomains (HCMDs) formed between the ER and plasmalemmal Orai1 channels. For this purpose, COS-7 cells were cotransfected with Orai1, STIM1 labelled with YFP or mRFP and the mitochondrially targeted Ca(2+) sensitive fluorescent protein inverse-Pericam. Depletion of ER Ca(2+) with ATP+thapsigargin (in Ca(2+)-free medium) induced the appearance of STIM1 puncta in the < or =100 nm wide subplasmalemmal space, as examined with TIRF. Mitochondria were located either in the gaps between STIM1-tagged puncta or in remote, STIM1-free regions. After addition of Ca(2+) mitochondrial Ca(2+) concentration increased irrespective of the mitochondrion-STIM1 distance. These observations indicate that mitochondria are exposed to Ca(2+) diffused laterally from the HCMDs formed between the PM and the subplasmalemmal ER.