Article

Stromal interaction molecule 1 (STIM1), a transmembrane protein with growth suppressor activity, contains an extracellular SAM domain modified by N-linked glycosylation

May 2002
Biochimica et Biophysica Acta 1596(1):131-7

May 2002
1596(1):131-7

DOI:10.1016/S0167-4838(02)00211-X

Source
PubMed

Authors:

Paul V Senior

University of Melbourne

Show all 6 authorsHide

Stromal interaction molecule 1 (STIM1) is a cell surface transmembrane glycoprotein implicated in tumour growth control and stromal-haematopoietic cell interactions. A single sterile alpha motif (SAM) protein-protein interaction domain is modelled within its extracellular region, a subcellular localisation not previously described for other SAM domain-containing proteins. We have defined the transmembrane topology of STIM1 by determining the sites of N-linked glycosylation. We have confirmed that STIM1 is modified by N-linked glycosylation at two sites within the SAM domain itself, deduced as asparagine residues N131 and N171, demonstrating that STIM1 is translocated across the membrane of the endoplasmic reticulum such that the SAM domain resides within the endoplasmic reticulum (ER) lumen. Both N-linked oligosaccharides remain endoglycosidase H-sensitive, indicating absence of full processing within the ER and Golgi. This immature modification is nevertheless sufficient and critical for cell surface expression of STIM1. We show that STIM1-STIM1 homotypic interactions are mediated via the cytoplasmic rather than the extracellular region of STIM1, excluding an essential role for the SAM domain in these protein interactions. These studies provide the first evidence for an extracellular localisation of a SAM domain within any protein, and the first example of a SAM domain modified by N-linked glycosylation.

COVID19 Spike protein enhances cytosol Ca2+ release from ER for its infection through stimulating the association between ACE2 and STIM1

Preprint

Full-text available

Apr 2024

The property and signaling mechanisms of the angiotensin produced by ACE2 (angiotensin converting enzyme 2) have been well studied in the renin–angiotensin system. However, less attention has been attracted to the intracellular regulation of ACE2 protein. Using the information which [⁶⁷¹RKKFPLKIFKKPLKK⁶⁸⁵] motif of STIM1(stromal interaction molecule 1 precursor) is able to bind its partner proteins via two acidic amino acids (DD), we recognized that ACE2 contains the short acidic motif binding motif [⁷⁹⁹DD⁸⁰⁰] on its C-terminus. Moreover, we demonstrated that the protein interaction between STIM1 and ACE2 contributes to Ca²⁺ ion release from ER. We recognized that a novel mechanism of ACE2 regulation is modulated through interaction with STIM1, which binds directly to ACE2 as a COVID19 receptor. Further, our findings provide clues why ACE2 helps to release cytoplasmic Ca²⁺ from store-operated Ca²⁺ entry to stimulate viral coat membrane fusion with host plasma membrane, after COVID19 Spike protein binding to ACE2. Our finding also implicates that COVID19 infection enhances cytoplasmic Ca²⁺ ion concentration by releasing Ca²⁺ ion from ER, through its S protein association with its host ACE2 and subsequent STIM1 activation. Thus, our findings may provide the novel target point to prevent COVID19 pandemic recurring and cure it.

Identification of Glucosidase II that regulates STIM1 activation dynamics

Preprint

Full-text available

Apr 2024

The ubiquitous presence of stromal interaction molecule (STIM), endoplasmic reticulum (ER) Ca2+ sensors, plays a crucial role for maintaining Ca2+ homeostasis and signaling in mammalian cells by linking ER Ca2+ depletion with extracellular Ca2+ influx. Although recent advancements have shed light on glycosylation in STIM proteins, further exploration of deglycosylation enzyme modification and its role remains limited by methodological constraints. In this study, leveraging the miniTurbo-driven proximal biotinylation labeling method, we were able to screen for weak and transiently binding protein molecules within specific environments like ER lumina. Herein, we unveil glucosidase II, comprising GANAB and PRKCSH, as a novel partner of the STIM1 complex. Through investigations into Glucosidase II's (Glu II) regulation of STIM1's Ca2+ affinities both in cellulo and in situ, alongside its impact on store-operated Ca2+ entry (SOCE), we propose a novel mechanism wherein Glu II governs STIM1 activation by modulating Ca2+ binding affinity, thereby adjusting the level of activated STIM1 in response to physiological stimulation.

Control of STIM and Orai function by post-translational modifications

Article

Jan 2022
CELL CALCIUM

Store-operated calcium entry (SOCE) is mediated by the endoplasmic reticulum (ER) Ca²⁺ sensors stromal interaction molecules (STIM1 and STIM2) and the plasma membrane Orai (Orai1, Orai2, Orai3) Ca²⁺ channels. Although primarily regulated by ER Ca²⁺ content, there have been numerous studies over the last 15 years demonstrating that all 5 proteins are also regulated through post-translational modification (PTM). Focusing primarily on phosphorylation, glycosylation and redox modification, this review focuses on how PTMs modulate the key events in SOCE; Ca²⁺ sensing, STIM translocation, Orai interaction and/or Orai1 activation.

Endoplasmic Reticulum‐Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling

Article

Full-text available

Apr 2021
INT J MOL SCI

In eukaryotic cells, ultimate specificity in activation and action—for example, by means of second messengers—of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple—sometimes opposite—cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER–PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

Physiopathologie de la myopathie à agrégats tubulaires

Thesis

Jan 2017

Georges Arielle Peche

La myopathie à agrégats tubulaires (TAM) est une maladie génétique qui se caractérise par la présence d’agrégats tubulaires dans les biopsies musculaires de patients. Notre équipe a identifié pour la première fois des mutations dans STIM1 comme étant à l’origine de cette maladie. STIM1 (stromal interaction molecule 1) est le senseur calcique du réticulum sarco/endoplasmique (RE/RS). En effet, en cas de diminution du calcium (Ca2+) dans le RE/RS, STIM1 se déplie, oligomérise et migre à proximité de la membrane plasmique (MP) pour activer le canal calcique ORAI1 et permettre le remplissage des stocks. Ce mécanisme est le «store-operated Ca2+ entry» (SOCE). D’autres équipes ont rapporté une mutation dans STIM1 (p.R304W) conduisant à une TAM associée à d’autres symptômes, ou encore syndrome de Stormorken. Ainsi, ce travail a eu pour but d’étudier et de comparer l’impact des mutations TAM et Stormorken à différents niveaux du SOCE. Nous avons ainsi montré que les mutations TAM et Stormorken conduisent à une augmentation de l’expression de STIM1, à la formation de clusters constitutifs de STIM1 à proximité de la MP, ainsi qu’au recrutement du canal ORAI1 et à l’activation de la voie du NFAT, dépendante du Ca2+.

The regulation of STIM1 translocation to the plasma membrane

Article

STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia

Article

Full-text available

Apr 2019

Background Dysregulation in calcium (Ca²⁺) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca²⁺ pathway has been associated with disease progression, the importance of the newly described constitutive Ca²⁺ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca²⁺ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. Methods An extensive analysis of the Ca²⁺ entry (CE) pathway in CLL B cells was performed including constitutive Ca²⁺ entry, basal Ca²⁺ levels, and store operated Ca²⁺ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. Results Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca²⁺ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca²⁺ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup. Conclusions These data establish the critical role of a newly discovered BCR independent Ca²⁺ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane. Electronic supplementary material The online version of this article (10.1186/s40425-019-0591-3) contains supplementary material, which is available to authorized users.

Mechanisms of STIM1-mediated endoplasmic reticulum-plasma membrane (ER-PM) contact formation

Article

Jan 2014

Shan-Hua Chung

The coupling of endoplasmic reticulum (ER) and plasma membrane (PM) is crucial for calcium (Ca2+) homeostasis. STIM1 and STIM2 are type I membrane proteins of the ER and function as Ca2+ sensors in a process known as store-operated calcium entry (SOCE). They sense a drop in luminal Ca2+ concentration and undergo conformational changes and oligomerization. The active oligomerized STIM proteins translocate to ER-PM contact sites, where they bind to phosphoinositides (PIPs) at the inner leaflet of the PM via their lysine (K)- rich domains and activate Orai1, a pore-forming Ca2+ release-activated Ca2+ (CRAC) channel subunit in the PM. I found that STIM2, but not STIM1, contains a di-lysine ER-retention signal. This signal restricts the function of STIM2 as Ca2+ sensor to the ER while STIM1 can reach the PM via the classical secretary pathway. The intracellular distribution of STIM1 is regulated in a cell-cycle-dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its K-rich domain and a di-arginine ER retention signal. SOCE enhances ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1’s role as multifunctional component in Ca2+-signaling. In contrast to mitotic cells, interphase cells retain most of their STIM1 intracellularly. Under resting condition, the ER-resident STIMs are preferentially located in PI(4,5)P2 containing preexisting ER-PM contact sites, which are expanded upon ER Ca2+ depletion. The lipid-binding, K-rich domains are required to localize STIM proteins in preexisting ERPM contact sites. Moreover, STIM2 recruits ER more efficiently to the PM. This is consistent with the fact that STIM2 has higher lipid-binding affinity and lower activation threshold than STIM1 and that STIM2 functions as a regulator of basal Ca2+ homeostasis. Finally, I studied the role of microtubules in ER-PM contact site formation. I observed that STIM1 aligns along microtubules. Alignment of STIM proteins with microtubules is a conserved process. In addition to accumulation of STIM1 at microtubule plus ends, STIM1 moves along microtubules in an EB-1-independent manner. I identified two EB-1- independent microtubule-binding sites located within the C-terminus of STIM1 and found that oligomerization increases the EB-1-independent microtubule-binding affinity of STIM1. However, the physiological function of this EB1-independent microtubule binding activity remains elusive.

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

Article

Full-text available

Feb 2014
J GEN PHYSIOL

Orai proteins contribute to Ca(2+) entry into cells through both store-dependent, Ca(2+) release-activated Ca(2+) (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca(2+) (ARC) and leukotriene C4 (LTC4)-regulated Ca(2+) (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum-resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.

Regulation of cellular Mn homeostasis : unexpected functions of TMEM165, SERCA and SPCA1

Thesis

Dec 2020

Marine Houdou

Glycosylation is a universal cellular process in all living organisms where monosaccharides are added one by one onto an acceptor molecule, most of the time a protein, a lipid or another monosaccharide. In eukaryotes, many glycosylation pathways occur simultaneously, resulting in the biosynthesis of a broad variety of glycan structures with different functions. In humans, if one -or more- glycosylation reactions are genetically impaired, Congenital Disorders of Glycosylation (CDG) appear. One of them, TMEM165-CDG, was identified in 2012 by our group and is at the heart of this work. Pathogenic mutations in TMEM165 gene cause severe glycosylation defects mainly characterized by hypo-galactosylated N-glycan structures. While characterizing these glycosylation abnormalities, a link has rapidly been established by the team between TMEM165 deficiency and Golgi manganese (Mn2+) homeostasis disruption. Therefore, and based on previous work, TMEM165 was assumed to act as a Ca2+/Mn2+ antiporter, allowing the import of Mn2+ into the Golgi lumen in order to sustain an adequate ionic environment, required for all glycosylation reactions. Interestingly, we also found that exogenous addition of Mn2+ in the culture medium of TMEM165 deficient cells completely rescues the N-glycosylation defects observed in these cells. Moreover, TMEM165, like Gdt1p its yeast ortholog, is a protein highly sensitive to Mn2+, being rapidly degraded via the lysosomal pathway in the presence of high Mn2+ concentrations. All in all, a close link exists between TMEM165/Gdt1p, Golgi Mn2+ homeostasis and Golgi glycosylation; the three major aspects focused in my PhD research. More precisely, my thesis focuses on (i) understanding the mechanisms of Mn2+-induced glycosylation rescue in TMEM165 deficient cells and (ii) the potential links between different key players acting in the regulation of the secretory pathway ionic homeostasis which are the Sarco/Endoplasmic Reticulum calcium (Ca2+)-ATPase SERCA2, TMEM165 and SPCA1 (Secretory Pathway Ca2+/Mn2+-ATPase), the only pump of the Golgi apparatus known to import both Ca2+ and Mn2+ in the Golgi lumen. Through the use of isogenic human cell lines knockout for either TMEM165 or ATP2C1 and yeasts lacking Gdt1p and/or Pmr1p, we highlighted three main concepts that closely link these proteins: TMEM165 (Gdt1p), SPCA1 (Pmr1p) and SERCA2. On the one hand, we demonstrated that the activity of SERCA pumps is crucial to sustain Golgi glycosylation reactions in absence of TMEM165 by their contribution in Mn2+ pumping and redistribution into the Golgi lumen. On the other hand, TMEM165 was found essential for maintaining Golgi glycosylation reactions in absence of both SPCA1 and when SERCA2 are inhibited by pharmacological agents. Moreover, we also shed light on the fact that expression and stability of TMEM165 (in humans) and Gdt1p (in yeast) were directly linked to the capacities of SPCA1 and Pmr1p to import Mn2+ into the Golgi lumen. Although differences exist between humans and yeast Saccharomyces cerevisiae, all of our work illustrates the crucial importance of the ionic homeostasis of the Golgi apparatus to sustain Golgi glycosylation reactions.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation

Article

Full-text available

Dec 2022

Simple Summary Breast cancer cells exhibit several differences in store-operated Ca²⁺ entry (SOCE) as compared to non-tumoral breast epithelial cells due to altered expression and post-translational modification of STIM proteins and Orai channels, as well as their modulators. The aim of this study was to analyze Orai1 and STIM1 N-linked glycosylation in SOCE in breast cancer cells and to ascertain the potential functional relevance of this post-translational modification in the development of cancer hallmarks. Using glycosylation-deficient STIM1 and Orai1 mutants we have found SOCE in breast cancer cells is insensitive to N-linked glycosylation of these proteins, a mechanism that might be relevant to evade apoptosis. Abstract N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca²⁺ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca²⁺ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

STIM and Orai Mediated Regulation of Calcium Signaling in Age-Related Diseases

Article

Full-text available

Apr 2022

Tight spatiotemporal regulation of intracellular Ca2+ plays a critical role in regulating diverse cellular functions including cell survival, metabolism, and transcription. As a result, eukaryotic cells have developed a wide variety of mechanisms for controlling Ca2+ influx and efflux across the plasma membrane as well as Ca2+ release and uptake from intracellular stores. The STIM and Orai protein families comprising of STIM1, STIM2, Orai1, Orai2, and Orai3, are evolutionarily highly conserved proteins that are core components of all mammalian Ca2+ signaling systems. STIM1 and Orai1 are considered key players in the regulation of Store Operated Calcium Entry (SOCE), where release of Ca2+ from intracellular stores such as the Endoplasmic/Sarcoplasmic reticulum (ER/SR) triggers Ca2+ influx across the plasma membrane. SOCE, which has been widely characterized in non-excitable cells, plays a central role in Ca2+-dependent transcriptional regulation. In addition to their role in Ca2+ signaling, STIM1 and Orai1 have been shown to contribute to the regulation of metabolism and mitochondrial function. STIM and Orai proteins are also subject to redox modifications, which influence their activities. Considering their ubiquitous expression, there has been increasing interest in the roles of STIM and Orai proteins in excitable cells such as neurons and myocytes. While controversy remains as to the importance of SOCE in excitable cells, STIM1 and Orai1 are essential for cellular homeostasis and their disruption is linked to various diseases associated with aging such as cardiovascular disease and neurodegeneration. The recent identification of splice variants for most STIM and Orai isoforms while complicating our understanding of their function, may also provide insight into some of the current contradictions on their roles. Therefore, the goal of this review is to describe our current understanding of the molecular regulation of STIM and Orai proteins and their roles in normal physiology and diseases of aging, with a particular focus on heart disease and neurodegeneration.

Cellular and Mitochondrial Calcium Communication in Obstructive Lung Disorders

Article

Full-text available

Mar 2021
MITOCHONDRION

Calcium (Ca²⁺) signalling is well known to dictate cellular functioning and fate. In recent years, the accumulation of Ca²⁺ in the mitochondria has emerged as an important factor in Chronic Respiratory Diseases (CRD) such as Asthma and Chronic Obstructive Pulmonary Disease (COPD). Various reports underline an aberrant increase in the intracellular Ca²⁺, leading to mitochondrial ROS generation, and further activation of the apoptotic pathway in these diseases. Mitochondria contribute to Ca²⁺ buffering which in turn regulates mitochondrial metabolism and ATP production. Disruption of this Ca²⁺ balance leads to impaired cellular processes like apoptosis or necrosis and thus contributes to the pathophysiology of airway diseases. This review highlights the key role of cytoplasmic and mitochondrial Ca²⁺ signalling in regulating CRD, such as asthma and COPD. A better understanding of the dysregulation of mitochondrial Ca²⁺ homeostasis in these diseases could provide cues for the development of advanced therapeutic interventions in these diseases.

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins

Article

Full-text available

Jan 2021
INT J MOL SCI

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.

Potassium and Calcium Channel Complexes as Novel Targets for Cancer Research

Article

Full-text available

Aug 2020

The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.

Molecular Basis and Regulation of Store-Operated Calcium Entry

Chapter

Jan 2020
ADV EXP MED BIOL

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous mechanism for Ca²⁺ influx in mammalian cells with important physiological implications. Since the discovery of SOCE more than three decades ago, the mechanism that communicates the information about the amount of Ca²⁺ accumulated in the intracellular Ca²⁺ stores to the plasma membrane channels and the nature of these channels have been matters of intense investigation and debate. The stromal interaction molecule-1 (STIM1) has been identified as the Ca²⁺ sensor of the intracellular Ca²⁺ compartments that activates the store-operated channels. STIM1 regulates two types of store-dependent channels: the Ca²⁺ release-activated Ca²⁺ (CRAC) channels, formed by Orai1 subunits, that conduct the highly Ca²⁺ selective current ICRAC and the cation permeable store-operated Ca²⁺ (SOC) channels, which consist of Orai1 and TRPC1 proteins and conduct the non-selective current ISOC. While the crystal structure of Drosophila CRAC channel has already been solved, the architecture of the SOC channels still remains unclear. The dynamic interaction of STIM1 with the store-operated channels is modulated by a number of proteins that either support the formation of the functional STIM1-channel complex or protect the cell against Ca²⁺ overload.

Molecular physiology and pathophysiology of stromal interaction molecules

Article

Jan 2018

Ca ²⁺ release from the endoplasmic reticulum is an important component of Ca ²⁺ signal transduction that controls numerous physiological processes in eukaryotic cells. Release of Ca ²⁺ from the endoplasmic reticulum is coupled to the activation of store-operated Ca ²⁺ entry into cells. Store-operated Ca ²⁺ entry provides Ca ²⁺ for replenishing depleted endoplasmic reticulum Ca ²⁺ stores and a Ca ²⁺ signal that regulates Ca ²⁺ -dependent intracellular biochemical events. Central to connecting discharge of endoplasmic reticulum Ca ²⁺ stores following G protein-coupled receptor activation with the induction of store-operated Ca ²⁺ entry are stromal interaction molecules (STIM1 and STIM2). These highly homologous endoplasmic reticulum transmembrane proteins function as sensors of the Ca ²⁺ concentration within the endoplasmic reticulum lumen and activators of Ca ²⁺ release-activated Ca ²⁺ channels. Emerging evidence indicates that in addition to their role in Ca ²⁺ release-activated Ca ²⁺ channel gating and store-operated Ca ²⁺ entry, STIM1 and STIM2 regulate other cellular signaling events. Recent studies have shown that disruption of STIM expression and function is associated with the pathogenesis of several diseases including autoimmune disorders, cancer, cardiovascular disease, and myopathies. Here, we provide an overview of the latest developments in the molecular physiology and pathophysiology of STIM1 and STIM2. Impact statement Intracellular Ca ²⁺ signaling is a fundamentally important regulator of cell physiology. Recent studies have revealed that Ca ²⁺ -binding stromal interaction molecules (Stim1 and Stim2) expressed in the membrane of the endoplasmic reticulum (ER) are essential components of eukaryote Ca ²⁺ signal transduction that control the activity of ion channels and other signaling effectors present in the plasma membrane. This review summarizes the most recent information on the molecular physiology and pathophysiology of stromal interaction molecules. We anticipate that the work presented in our review will provide new insights into molecular interactions that participate in interorganelle signaling crosstalk, cell function, and the pathogenesis of human diseases.

Calcium Entry Pathways in Non-excitable Cells

Book

Jan 2016
ADV EXP MED BIOL

Juan A Rosado

Calcium entry pathways in non-excitable cells presents a concise synthesis of thoughtfully selected topics covering from the different calcium entry mechanisms in non-excitable cells to the cellular microdomains and organelles regulating the calcium entry process. Particular attention is given to the fascinating group of ion channels involved in different calcium entry pathways as well as the emerging role of these channels in human disease. Calcium entry is an essential mechanism for cellular function in non-excitable cells. In general, two main calcium entry pathways exist in non-excitable cells: one pathway, named store-operated calcium entry (SOCE) requires store depletion and the second pathway is regulated by receptor occupation, but independently on calcium store depletion. The search for the molecular components of calcium entry has identified the stromal interaction molecule 1 (STIM1), as the calcium sensor of the intracellular calcium stores, and Orai as well as TRP channels as the calcium-permeable channels located in the plasma membrane. The location, interactions and function of these channels are finely regulated by a number of scaffolding proteins, membrane microdomains and cellular organelles that fine tune the amount of calcium entering the cell. Cutting-edge and user-friendly, this volume presents relevant background information, critical analysis of the current observations and directions for future research. The book is intended for basic scientists specializing in cellular biology or ion transport, as well as for biomedical researchers.

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

Article

Full-text available

Nov 2017

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8 + T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca 2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

The axonal endoplasmic reticulum: One organelle-many functions in development, maintenance, and plasticity: Integrated Function of the Axonal ER

Article

Full-text available

Nov 2017
DEV NEUROBIOL

The endoplasmic reticulum (ER) is highly conserved in eukaryotes and neurons. Indeed, the localization of the organelle in axons has been known for nearly half a century. However, the relevance the axonal ER is only beginning to emerge. In this review we discuss the structure of the ER in axons, examining the role of ER-shaping proteins and highlighting reticulons. We analyze the multiple functions of the ER and their potential contribution to axonal physiology. First we examine the emerging roles of the axonal ER in lipid synthesis, protein translation, processing, quality control and secretory trafficking of transmembrane proteins. We also review the impact of the ER on calcium dynamics, focusing on intracellular mechanisms and functions. We describe the interactions between the ER and endosomes, mitochondria and synaptic vesicles. Finally, we analyze available proteomic data of axonal preparations to reveal the dynamic functionality of the ER in axons during development. We suggest that the dynamic proteome and a validated axonal interactome, together with state-of-the-art methodologies, may provide interesting research avenues in axon physiology that may extend to pathology and regeneration. This article is protected by copyright. All rights reserved.

Calcium Entry Pathways in Non-excitable Cells. Preface

Article

Jan 2016
ADV EXP MED BIOL

Juan A Rosado

Optical investigations reveal the effects of 2-aminoethyldiphenyl borate on STIM1 puncta formation

Article

Full-text available

Aug 2017

2-Aminoethyldiphenyl borate (2-APB) is the most commonly used pharmacological agent in the study of calcium release-activated channels (CRACs); however, its inhibitory mechanism to CRACs remains unclear. To address this issue, we systematically employed confocal imaging, dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orai1, two key components of CRACs. Imaging results support that there are two signaling pathways (Orai1-independent and Orai1-dependent) for the formation of STIM1 puncta. 2-APB could dose dependently block Orai1-independent but not Orai1-dependent STIM1 puncta formation, despite its obvious inhibition effect on store-operated Ca2+ entry (SOCE). In addition, we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization, it could completely block CAD-YFP-induced constitutive Ca2+ entry and promote the interaction between Orai1 and CAD by FRET measurements. Therefore, we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhibitory effects on Orai1 channel itself, but not the interference on puncta formation between STIM1 and Orai1.

sp(2) -Iminosugar α-Glucosidase Inhibitor 1-C-octyl-2-oxa-3-oxocastanospermine Specifically Affected Breast Cancer Cell Migration through Stim1, β1-Integrin, and FAK Signaling Pathways

Article

Feb 2017
J CELL PHYSIOL

Aberrant glycosylation changes on many glycoproteins are often related to cancer progression and metastasis. sp(2) -Iminosugar-type castanospermine analogues, inhibitors of α-glucosidases, have been reported to exhibit antitumor activity. However, their effects on cell migration and the underlying molecular mechanism are not fully understood. Here, we investigated the effect of the pseudo- C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives (CO-OCS) on breast cancer cells (MCF-7 and MDA-MB-231 cells), and MCF-10A mammary normal cell lines. We showed that CO-OCS treatment results in the drastic decrease of breast cancer cell migration without affecting cell proliferation. Furthermore, CO-OCS significantly reduced both the expression of β1-integrin, which is a crucial interacting partner of Focal Adhesion Kinase (FAK), and the phosphorylation rates of FAK and ERK1/2. CO-OCS also drastically reduced Ca(2+) entry through Store Operated Channels (SOC). Orai1 and Stim1, two N-glycosylated proteins, are involved in Store-Operated Calcium Entry (SOCE), and are essential for breast tumor cell migration. Our results showed that CO-OCS decreased the expression, at the protein level, of Stim1 without affecting that of Orai1. Moreover, cell migration and SOCE were attenuated by CO-OCS as well as when Stim1 was silenced. In contrast, in MCF-10A cells, CO-OCS slightly reduced cell migration, but was without effect on gene expression of Stim1, Orai1, β1-integrin or FAK and ERK1/2 activation. Our results provide strong evidence for a significant effect of CO-OCS on breast cancer cell migration and support that this effect was associated with β1-integrin, Stim1 and FAK signaling pathways. This article is protected by copyright. All rights reserved.

Role of STIM2 in cell function and physiopathology

Article

Jan 2017
J PHYSIOL-LONDON

An endoplasmic reticulum (ER)-resident protein that regulates cytosolic and ER free-Ca(2+) concentration by induction of store-operated calcium entry. That is the original definition of STIM2 and its function. While its activity strongly depends on the amount of calcium stored in the ER, its function goes further to intracellular signalling and gene expression. Initially undercovered by the prominent function of STIM1, STIM2 became to be vital in mice, gradually emerging as an important player in the nervous system, and cooperating with STIM1 in the immune system. STIM2 has also been proposed as a relevant player in pathological conditions related to ageing, Alzheimer and Huntington's diseases, autoimmune disorders and cancer. The discovery of additional functions, together with new splicing forms with opposite roles, clarified existing controversies about STIM2 function in SOCE. Being essential for life, but apparently not for development, new available data demonstrated a complex and still intriguing behaviour that this review summarizes, updating the current knowledge about STIM2 function. This article is protected by copyright. All rights reserved.

STIM1fl/fl Ksp-Cre Mouse has Impaired Renal Water Balance

Article

Full-text available

Jun 2016

Background/aim: STIM1 is as an essential component in store operated Ca2+ entry. However give the paucity of information on the role of STIM1 in kidney, the aim was to study the function of STIM1 in the medulla of the kidney. Methods: we crossed a Ksp-cre mouse with another mouse containing two loxP sites flanking Exon 6 of STIM1. The Ksp-cre mouse is based upon the Ksp-cadherin gene promoter which expresses cre recombinase in developing nephrons, collecting ducts (SD) and thick ascending limbs (TAL) of the loop of Henle. Results: The offspring of these mice are viable without gross morphological changes, however, we noticed that the STIM1 Ksp-cre knockout mice produced more urine compared to control. To examine this more carefully, we fed mice low (LP) and high protein (HP) diets respectively. When mice were fed HP diet STIM1 ko mice had significantly increased urinary volume and lower specific gravity compared to wt mice. In STIM1 ko mice fed HP diet urine creatinine and urea were significantly lower compared to wt mice fed HP diet, however the fractional excretion was the same. Conclusion: These data support the idea that STIM1 ko mice have impaired urinary concentrating ability when challenged with HP diet is most likely caused by impaired Ca2+-dependent signal transduction through the vasopressin receptor cascade.

Historical Overview of Store-Operated Ca2+ Entry

Chapter

May 2016
ADV EXP MED BIOL

Calcium influx is an essential mechanism for the activation of cellular functions both in excitable and non-excitable cells. In non-excitable cells, activation of phospholipase C by occupation of G protein-coupled receptors leads to the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which, in turn, initiate two Ca2+ entry pathways: Ca2+ release from intracellular Ca2+ stores, signaled by IP3, leads to the activation of store-operated Ca2+ entry (SOCE); on the other hand, DAG activates a distinct second messenger-operated pathway. SOCE is regulated by the filling state of the intracellular calcium stores. The search for the molecular components of SOCE has identified the stromal interaction molecule 1 (STIM1) as the Ca2+ sensor in the endoplasmic reticulum and Orai1 as a store-operated channel (SOC) subunit. Furthermore, a number of reports have revealed that several members of the TRPC family of channels also take part of the SOC macromolecular complex. This introductory chapter summarizes the early pieces of evidence that led to the concept of SOCE and the components of the store-operated signaling pathway.

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

Chapter

Full-text available

Feb 2013

Redox modulation of STIM-ORAI signaling

Article

Full-text available

Mar 2016
CELL CALCIUM

STIM1 and ORAI1 constitute the core machinery of the ubiquitous store-operated calcium entry pathway and loss of function in these proteins is associated with severe immune and muscular disorders. Other isoforms—STIM1L, STIM2, ORAI2 and ORAI3 exhibit varied expression levels in different cell types along with several other interaction partners and thereby play different roles to facilitate, regulate and fine-tune the calcium entry. STIM proteins convey the Ca2+ store-depletion message to the PM and thereby participate in refilling of the ER by physically interacting with the Ca2+-selective ORAI channels at the PM. STIM and ORAI are exposed to oxidative modifications in the ER, the cytosol, and at the cell surface, and redox-mediated alterations in STIM/ORAI coupling might contribute to autoimmune disorders and cancer progression. This review discusses the redox reactivity of cysteine residues in STIM and ORAI isoforms, focusing on the oxidative modifications of STIM and ORAI proteins by which STIM-ORAI signaling can be modulated.

STIM and calcium channel complexes in cancer

Article

Oct 2015
Biochim Biophys Acta

The ion Ca(2+) is a ubiquitous second messenger that mediates a variety of cellular functions. Dysfunction of the mechanisms involved in Ca(2+) homeostasis underlies a number of pathological processes, including cancer. Store-operated Ca(2+) entry (SOCE) is a major mechanism for Ca(2+) entry modulated by the intracellular Ca(2+) stores. The Ca(2+)-selective store-operated current (ICRAC) is mediated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the store-operated Ca(2+) (SOC) channel Orai1, while other non-selective cation currents (ISOC) involves the participation of members of the canonical transient receptor potential (TRPC) channel family, including TRPC1. Distinct isoforms of the key components of SOCE have been described in mammalian cells, STIM1 and 2, Orai1-3 and TRPC1-7. In cancer cells, SOCE has been reported to play an important role in cell cycle progression and proliferation, migration, metastasis and evasion of apoptosis. Changes in the expression of the key elements of SOCE and Ca(2+) homeostasis remodelling have been account to play important roles in the phenotypic changes observed in transformed cells. Despite there are differences in the expression level of the molecular components of SOCE, as well as in the relevance of the STIM, Orai and TRPC isoforms in SOCE and tumorigenesis among cancer cell types, there is a body of evidence supporting an important role for SOCE underlying the phenotypic modifications of cancer cells that propose STIM and the SOC channels as suitable candidate targets for future prognostic or therapeutic strategies.

Oocyte activation deficiency: A role for an oocyte contribution?

Article

Sep 2015
HUM REPROD UPDATE

BACKGROUND Infertility affects between 10 and 16% of couples worldwide. Twenty to 30% of cases of infertility are due to a male factor, 20–35% to a female factor, and 25–40% are due to both male and female factors. In ∼10–25% of cases, the precise underlying cause remains unclear. IVF or ICSI followed by embryo transfer can be very appropriate treatment options in cases of female tubal damage, ovulatory failure or male-factor infertility. While the use of IVF has been reported to be suitable for many infertile couples, normal IVF cycles can fail in some cases. While ICSI can represent a powerful alternative in cases of IVF failure, complete fertilization failure can still occur in 1–5% of ICSI cycles. This can be due to a variety of factors and while commonly attributed to deficiency of sperm factors, it is very likely that abnormalities in crucial oocyte factors could also play a key role.

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

Article

Full-text available

Jul 2015
CELL RES

Calcium flux through store-operated calcium entry is a major regulator of intracellular calcium homeostasis and various calcium signaling pathways. Two key components of the store-operated calcium release-activated calcium channel are the Ca(2+)-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. Following calcium depletion from the endoplasmic reticulum, STIM1 undergoes conformational changes that unmask an Orai1-activating domain called CAD. CAD binds to two sites in Orai1, one in the N terminal and one in the C terminal. Most previous studies suggested that gating is initiated by STIM1 binding at the Orai1 N-terminal site, just proximal to the TM1 pore-lining segment, and that binding at the C terminal simply anchors STIM1 within reach of the N terminal. However, a recent study had challenged this view and suggested that the Orai1 C-terminal region is more than a simple STIM1-anchoring site. In this study, we establish that the Orai1 C-terminal domain plays a direct role in gating. We identify a linker region between TM4 and the C-terminal STIM1-binding segment of Orai1 as a key determinant that couples STIM1 binding to gating. We further find that Proline 245 in TM4 of Orai1 is essential for stabilizing the closed state of the channel. Taken together with previous studies, our results suggest a dual-trigger mechanism of Orai1 activation in which binding of STIM1 at the N- and C-terminal domains of Orai1 induces rearrangements in proximal membrane segments to open the channel.Cell Research advance online publication 3 July 2015; doi:10.1038/cr.2015.80.

STIM1 Binding to Both the N' and C' Termini of Orai1 is Critical for Gating of CRAC Channels

Article

Full-text available

Jan 2015

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STIM and Orai proteins in neuronal Ca2+ signaling and excitability

Article

Full-text available

Apr 2015

Stim1 and Orai1 are ubiquitous proteins that have long been known to mediate Ca2+ release-activated Ca2+ (CRAC) current (ICRAC) and store-operated Ca2+ entry (SOCE) only in non-excitable cells. SOCE is activated following the depletion of the endogenous Ca2+ stores, which are mainly located within the endoplasmic reticulum (ER), to replete the intracellular Ca2+ reservoir and engage specific Ca2+-dependent processes, such as proliferation, migration, cytoskeletal remodelling, and gene expression. Their paralogues, Stim2, Orai2 and Orai3, support SOCE in heterologous expression systems, but their physiological role is still obscure. Ca2+ inflow in neurons has long been exclusively ascribed to voltage-operated and receptor-operated channels. Nevertheless, recent work has unveiled that Stim1-2 and Orai1-2, but not Orai3, proteins are also expressed and mediate SOCE in neurons. Herein, we survey current knowledge about the neuronal distribution of Stim and Orai proteins in rodent and human brains; we further discuss that Orai2 is the main pore-forming subunit of CRAC channels in central neurons, in which it may be activated by either Stim1 or Stim2 depending on species, brain region and physiological stimuli. We examine the functions regulated by SOCE in neurons, where this pathway is activated under resting conditions to refill the ER, control spinogenesis and regulate gene transcription. Besides, we highlighted the possibility that SOCE also controls neuronal excitation and regulate synaptic plasticity. Finally, we evaluate the involvement of Stim and Orai proteins in severe neurodegenerative and neurological disorders, such as Alzheimer’s Disease and epilepsy.

STIM1 Controls Neuronal Ca2+ Signaling, mGluR1-Dependent Synaptic Transmission, and Cerebellar Motor Behavior

Article

May 2014

In central mammalian neurons, activation of metabotropic glutamate receptor type1 (mGluR1) evokes a complex synaptic response consisting of IP3 receptor-dependent Ca(2+) release from internal Ca(2+) stores and a slow depolarizing potential involving TRPC3 channels. It is largely unclear how mGluR1 is linked to its downstream effectors. Here, we explored the role of stromal interaction molecule 1 (STIM1) in regulating neuronal Ca(2+) signaling and mGluR1-dependent synaptic transmission. By analyzing mouse cerebellar Purkinje neurons, we demonstrate that STIM1 is an essential regulator of the Ca(2+) level in neuronal endoplasmic reticulum Ca(2+) stores. Both mGluR1-dependent synaptic potentials and IP3 receptor-dependent Ca(2+) signals are strongly attenuated in the absence of STIM1. Furthermore, the Purkinje neuron-specific deletion of Stim1 causes impairments in cerebellar motor behavior. Together, our results demonstrate that in the mammalian nervous system STIM1 is a key regulator of intracellular Ca(2+) signaling, metabotropic glutamate receptor-dependent synaptic transmission, and motor coordination.

Roles of Cholesterol and PtdIns(4,5)P2 in the Regulation of STIM1-Orai1 Channel Function

Article

Mar 2023
ADV EXP MED BIOL

Calcium is one of the most prominent second messengers. It is involved in a wide range of functions at the single-cell level but also in modulating regulatory mechanisms in the entire organism. One process mediating calcium signaling involves hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by the phospholipase-C (PLC). Thus, calcium and PtdIns(4,5)P2 are intimately intertwined two second-messenger cascades that often depend on each other. Another relevant lipid associated with calcium signaling is cholesterol. Both PtdIns(4,5)P2 and cholesterol play key roles in the formation and maintenance of specialized signaling nanodomains known as lipid rafts. Lipid rafts are particularly important in calcium signaling by concentrating and localizing calcium channels such as the Orai1 channel. Depletion of internal calcium stores is initiated by the production of inositol-1,4,5-trisphosphate (IP3). Calcium depletion from the ER induces the oligomerization of STIM1, which binds Orai1 and initiates calcium influx into the cell. In the present review, we analyzed the complex interactions between cholesterol, PtdIns(4,5)P2, and the complex formed by the Orai1 channel and the signaling molecule STIM1. We explore some of the complex mechanisms governing calcium homeostasis and phospholipid metabolism, as well as the interaction between these two apparently independent signaling cascades.

Store-Operated Calcium Entry in the Cardiovascular System

Chapter

Jan 2021
ADV EXP MED BIOL

Calcium (Ca2+) is a critical regulator of cardiovascular function. The Ca2+ channels, pumps, and exchangers contributing to cytosolic Ca2+ signals governing cardiac contraction and vascular tone are well known. In addition to these Ca2+ components, store-operated calcium entry (SOCE) is a ubiquitous mechanism recently recognized underlying cardiovascular function maintenance and disease development and progression. With this review article, we hope to highlight the accumulated knowledge about the SOCE machinery and its potential contribution to cardiac and vascular function and its roles in cardiovascular pathogenesis and pathology.

Stoichiometry of the interactions between endogenous Orai1 and STIM1 during store-operated Ca2+ entry

Thesis

Sep 2021

Yihan Shen

Ca2+ is a ubiquitous intracellular messenger. It regulates a variety of cellular activities, ranging from muscle contraction, neuronal transmission, secretion and cell growth to apoptosis. Store-operated Ca2+ entry (SOCE) is a major pathway of Ca2+ signalling and exists in almost all metazoans. SOCE is activated by loss Ca2+ from the endoplasmic reticulum (ER), which causes stromal interaction molecule 1 (STIM1) to accumulate at junctions between the ER and plasma membrane (PM). Within these membrane contact sites (MCS), STIM1 forms puncta that trap and activate Orai1 Ca2+ channels in the PM, allowing Ca2+ to flow into the cytoplasm and slowly replenish Ca2+ in the ER. Elucidating the binding stoichiometry of STIM1 and Orai1 is essential for understanding Orai1 gating and mechanisms of SOCE. Most previous studies of SOCE used cells overexpressing STIM1 and/or Orai1, which might perturb their behaviours. This study used a HeLa cell line in which one copy of the endogenous STIM1 gene was tagged with EGFP using CRISPR/Cas9 gene-editing to understand the stoichiometry and dynamics of STIM1 and Orai1 in SOCE. I confirmed that SOCE was normal in STIM1-EGFP HeLa cells and that the tagged and untagged versions of STIM1 mixed freely and interacted with Orai1. Total internal reflection fluorescence (TIRF) imaging analyses indicated that there was only a modest increase in the average size of STIM1 puncta after store depletion. Stepwise photobleaching analyses revealed that there was an average of 14.5 STIM1 molecules within each punctum in cells with empty Ca2+ stores. Orai1 was immunostained and the fluorescence intensity distributions of the Orai1 puncta were minimally affected by store depletion. Furthermore, the fluorescence intensities of Orai1 that colocalized with STIM1 puncta were similar to those remote from them. Only a small proportion (26%) of STIM1 colocalized with Orai1 at MCS identified by MAPPER, a fluorescent marker constitutively present in the ER-PM junctions. I conclude that each SOCE complex comprises a small cluster of STIM1 and is likely to include no more than one active Orai1 channel. The presence of a single Orai1 channel within each SOCE junction is estimated to be enough to account for observed SOCE-mediated Ca2+ signals, but it contradicts suggestions that STIM1 promotes clustering of Orai1 within MCS.

Arachidonic Acid (Leukotriene C4) Regulated Calcium Channel

Chapter

Jan 2020

The activation of phospholipase C (PLC)-coupled receptors triggers calcium (Ca²⁺) entry across the plasma membrane (PM) through pathways that are dependent and independent on endoplasmic reticulum (ER) Ca²⁺ store depletion. The former, which is mediated by the Ca²⁺ release activated Ca²⁺ (CRAC) channel, and the latter, which is mediated by the arachidonic acid regulated Ca²⁺ (ARC) channel both mediate highly selective Ca²⁺ influxes that activate distinct physiological processes in a variety of cell types. These channels are molecularly composed of homologs of the ORAI proteins: CRAC channels mainly require ORAI1 with ORAI2 and ORAI3 acting as negative modulators while ARC channels require both the long translational variant of ORAI1, ORAI1α, and ORAI3. The ER transmembrane protein, Stromal interacting molecule 1 (STIM1), is also necessary for the activation of both channels. Hence, both channels have relatively similar biophysical and pharmacological properties; albeit, there are important nuances that differentiate them. Defining features of ARC channels include activation by receptor-generated arachidonic acid (AA) and its metabolite leukotriene C4 (LTC4), and positive regulation by PKA through phosphorylation of T389 residue on STIM1. The physiological relevance of ARC channels is emerging especially in regard to pancreatic cells and vascular smooth muscle cells, which emphasizes the importance of studying this channel.

Review: Structure and Activation Mechanisms of CRAC Channels

Chapter

Jan 2020
ADV EXP MED BIOL

Ca²⁺ release activated Ca²⁺ (CRAC) channels represent a primary pathway for Ca²⁺ to enter non-excitable cells. The two key players in this process are the stromal interaction molecule (STIM), a Ca²⁺ sensor embedded in the membrane of the endoplasmic reticulum, and Orai, a highly Ca²⁺ selective ion channel located in the plasma membrane. Upon depletion of the internal Ca²⁺ stores, STIM is activated, oligomerizes, couples to and activates Orai. This review provides an overview of novel findings about the CRAC channel activation mechanisms, structure and gating. In addition, it highlights, among diverse STIM and Orai mutants, also the disease-related mutants and their implications.

Molecular Mechanisms of Gastrointestinal Signaling

Chapter

Jun 2018

Menizibeya Osain Welcome

The epithelial cells of the gastrointestinal (GI) tract communicate with each other and with cells of other organs via a complex network of highly regulated movement of ions and biomolecules. The molecules ensure regulated activity of cells, tissues, and organs of the GI system and the body as whole. The regulated movement and subsequent activities of the biomolecules released from one cell to the target are made possible by receptive substances (receptors) localized on the membrane of the target cells or intracellular organelles, or in the cytosol. This process, which is referred to as cell-to-cell communication or cellular signaling, ensures the regulated functioning of the cells and tissues of the GI system and the whole organism. This chapter is dedicated to the mechanism of cell-to-cell communication and signaling in normal and relates it to how disease develops. Basic mechanisms of GI epithelial cell signaling and gut nutrient receptor sensing (GI chemosensation) are discussed.

From Stores to Sinks: Structural Mechanisms of Cytosolic Calcium Regulation

Chapter

Jan 2017
ADV EXP MED BIOL

All eukaryotic cells have adapted the use of the calcium ion (Ca2+) as a universal signaling element through the evolution of a toolkit of Ca2+sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca2+between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca2+levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca2+sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca2+can subsequently signal the opening surface channels which permit the movement of Ca2+from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca2+sinks by taking up Ca2+in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IP3Rs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca2+depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca2+from the extracellular space to the cytosol; uptake of Ca2+into the matrix through the mitochondrial Ca2+uniporter (MCU) further shapes the cytosolic Ca2+levels. Recent structural elucidations of these key Ca2+toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca2+levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IP3R, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.

The STIM-orai pathway: STIM-orai structures: Isolated and in complex

Chapter

Sep 2017
ADV EXP MED BIOL

Considerable progress has been made elucidating the molecular mechanisms of calcium (Ca²⁺) sensing by stromal interaction molecules (STIMs) and the basis for Orai channel activity. This chapter focuses on the available high-resolution structural details of STIM and Orai proteins with respect to the regulation of store-operated Ca²⁺ entry (SOCE). Solution structures of the Ca²⁺-sensing domains of STIM1 and STIM2 are reviewed in detail, crystal structures of cytosolic coiled-coil STIM fragments are discussed, and an overview of the closed Drosophila melanogaster Orai hexameric structure is provided. Additionally, we highlight structures of human Orai1 N-terminal and C-terminal domains in complex with calmodulin and human STIM1, respectively. Ultimately, the accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.

Structural perturbations induced by Asn131 and Asn171 glycosylation converge within the EFSAM core and enhance stromal interaction molecule-1 mediated store operated calcium entry

Article

Nov 2016
Biochim Biophys Acta

A major intracellular calcium (Ca(2+)) uptake pathway in both excitable and non-excitable eukaryotic cells is store-operated Ca(2+) entry (SOCE). SOCE is the process by which endoplasmic reticulum (ER)-stored Ca(2+) depletion leads to activation of plasma membrane Ca(2+) channels to provide a sustained increase in cytosolic Ca(2+) levels that mediate a plethora of physiological processes ranging from the immune response to platelet aggregation. Stromal interaction molecule-1 (STIM1) is the principal regulator of SOCE and responds to changes in ER stored Ca(2+) through luminal sensing machinery composed of EF-hand and SAM domains (EFSAM). The EFSAM domain can undergo N-glycosylation at Asn131 and Asn171 sites; however, the precise role of EFSAM N-glycosylation in the Ca(2+) sensing mechanism of STIM1 is unclear. By establishing a site-specific chemical approach to covalently linking glucose to EFSAM and examining α-helicity, thermal stability, three dimensional atomic-resolution structure, Ca(2+) binding affinity and oligomerization, we show that N-glycosylation of the EFSAM domain enhances the properties that promote STIM1 activation. This augmentation occurs through changes in structure localized near the Asn131 and Asn171 sites that together permeate through the protein core and lead to decreased Ca(2+) binding affinity, reduced stability and enhanced oligomerization. Congruently, Ca(2+) influx via SOCE in HEK293 cells co-expressing Orai1 and STIM1 was diminished when N-glycosylation was blocked by introducing Asn131Gln and Asn171Gln mutations. Collectively, our data suggests that N-glycosylation enhances the EFSAM destabilization-coupled oligomerization in response to ER Ca(2+) depletion thereby augmenting the role of STIM1 as a robust ON/OFF regulator of SOCE.

Selective activation of distinct Orai channels by STIM1

Article

Nov 2016

Trevor J Shuttleworth

An adenovirus vector-mediated RNA interference inhibits efficiently rat stim1 expression in vascular smooth muscle cells and rat carotid artery

Article

Jun 2016
RES J BIOTECHNOL

RNA interference (RNAi) is a general mechanism to knockdown a target gene expression but the efficient delivery of siRNA into cells is important for successful application of RNAi. In this study, we demonstrated an efficient and specific knockdown of stim1 (an important protein required for calcium influx and cells proliferation) in vascular smooth muscle cells and rat carotid artery which indicated a promising application of this adenovirus system in vascular physiological functions. Two target sequences for stim1 were chemically synthesized and recombinant adenoviruses for RNAi-mediated stim1 were produced. Rat aortic VSMCs were primarily cultured and transfected by an adenoviral vectors. Angioplasty of the rat left carotid artery was performed by using a balloon embolectomy catheter and transfected by an adenoviral vectors. Stim1 protein level was measured by western blot. An adenovirus vector for stim1 knockdown was successfully constructed. At 1, 3 and 5 day after infection with Ad-si/stim1 at MOI of 10 pfu/cells, the protein levels of stim1 was decreased significantly with compared to the Ad-empty group. The infection of Ad-si/stim1 virus (3 day after infection at 10 puf/cells of MOI) resulted in a robust decrease in store-operated Ca2+ entry (SOCE). Ad-mediated siRNA infected the rat carotid artery efficiently and inhibit the expression of stim1 significantly. This study develops a model system to study the function of stim1 in vivo and in vitro and the siRNA expression and specific gene knockdown can be achieved by adenoviral vector in vivo and in vitro.

Analyse der Rolle von TRP Proteinen für die Aktivierung humaner CD4+-Zellen mittles siRNA Technologie

Thesis

Jan 2008

Anna Silvia Wenning

TRP CHANNELS AND THE CALCIUM ENTRY SIGNALPLEX

Article

Sep 2014

Juan A Rosado

The molecular mechanisms of STIM/Orai communications. A Review in the Theme: STIM and Orai Proteins in Calcium Signaling

Article

Jan 2016

Ca(2+) entry into the cell via store-operated Ca(2+) release activated Ca(2+) (CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, secretion and cell death. These store-operated Ca(2+) channels open following depletion of intracellular Ca(2+) stores and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an ER-located Ca(2+) sensor, while Orai forms a highly Ca(2+) selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography thereby establishing a portray of key mechanistic steps in the CRAC channel signaling cascade. The focus is laid on the activation of the STIM proteins, the following coupling of STIM1 to Orai1, and the consequent, structural rearrangements that gate the Orai channels into the open state to allow Ca(2+) permeation into the cell.

Store-Operated Calcium Channels

Article

Full-text available

Sep 2015

Store-operated cofor calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca2+ sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca2+ from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca2+ depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease.

The STIM-Orai Pathway

Chapter

Dec 2012

Considerable progress has been made in elucidating the molecular mechanism of calcium (Ca2+) sensing by stromal interaction molecules (STIM). This chapter focuses on the biophysical, biochemical and structural properties of the luminal region of STIM1 and STIM2, with a structural emphasis on the conserved EF-hand and SAM domains (EF-SAM). The current data suggests that STIM molecules are in a quiescent, non-signaling state via the auto-inhibition of EF-SAM oligomerization. Upon destabilization of the intramolecular EF-hand:SAM association in response to Ca2+-depletion, homotypic EF-SAM protein-protein interactions initiate an allosteric cascade that culminates in translocated STIM1 oligomers recruiting and opening Orai1 plasma membrane Ca2+ channels. The regulatory distinctions observed for STIM1 and STIM2 are associated with differences in Ca2+ affinity, the forces promoting the EF-hand:SAM interaction and the hydrophobic makeup of the SAM domains.

Galpha 12 and Galpha 13 Regulate Extracellular Signal-regulated Kinase and c-Jun Kinase Pathways by Different Mechanisms in COS7 Cells

Article

Full-text available

Aug 1996

Michel P. Faure

Many growth factors and agonists for G protein-coupled receptors activate mitogen-activated protein (MAP) kinase pathways, including the extracellular signal-regulated kinase (ERK) pathway and the c-Jun kinase (JNK) pathway. Transient transfection of dominant negative and constitutively active pathway components in COS-7 cells shows that two G protein subunits, Gα12 and Gα13, inhibit the ERK pathway and stimulate the JNK pathway. Constitutively active (GTPase-deficient) Gα12 and Gα13 both inhibit ERK pathway activation by epidermal growth factor. A Gα13/αz chimera, which responds to stimulation by Gi-coupled receptors, mediates inhibition of ERK via such a receptor, the dopamine-2 receptor. In addition, expression of a dominant negative mutant of the GTPase, Cdc42, blocks activation of the JNK pathway by Gα12 and Gα13 but does not alter inhibition of ERK activation by the same Gα proteins; conversely, mutationally activated Cdc42 stimulates the JNK pathway but has no effect on the ERK pathway. Our results show that different mechanisms mediate two effects of Gα12 and Gα13: the ERK pathway inhibition is mediated at the level of MAP kinase kinase in a Ras- and Raf-independent fashion, whereas the JNK pathway stimulation is mediated by Cdc42.

Structure of the C-terminal sterile α-motif (SAM) domain of human p73α

Article

Full-text available

Apr 2001

p73 is a homologue of the tumour suppressor p53 and contains all three functional domains of p53. The α-splice variant of p73 (p73α) contains near its C-terminus an additional structural domain known as the sterile α-motif (SAM) that is probably responsible for regulating p53-like functions of p73. Here, the 2.54 Å resolution crystal structure of this protein domain is reported. The crystal structure and the published solution structure have the same five-helix bundle fold that is characteristic of all SAM-domain structures, with an overall r.m.s.d. of 1.5 Å for main-chain atoms. The hydrophobic core residues are well conserved, yet some large local differences are observed. The crystal structure reveals a dimeric organization, with the interface residues forming a mini four-helix bundle. However, analysis of solvation free energies and the surface area buried upon dimer formation indicated that this arrangement is more likely to be an effect of crystal packing rather than reflecting a physiological state. This is consistent with the solution structure being a monomer. The p73α SAM domain also contains several interesting structural features: a Cys-X-X-Cys motif, a 310-helix and a loop that have elevated B factors, and short tight inter-helical loops including two β-turns; these elements are probably important in the normal function of this domain.

Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression

Article

Full-text available

Aug 2001
EMBO J

TEL is a transcriptional repressor that is a frequent target of chromosomal translocations in a large number of hematalogical malignancies. These rearrangements fuse a potent oligomerization module, the SAM domain of TEL, to a variety of tyrosine kinases or transcriptional regulatory proteins. The self-associating property of TEL–SAM is essential for cell transformation in many, if not all of these diseases. Here we show that the TEL–SAM domain forms a helical, head-to-tail polymeric structure held together by strong intermolecular contacts, providing the first clear demonstration that SAM domains can polymerize. Our results also suggest a mechanism by which SAM domains could mediate the spreading of transcriptional repression complexes along the chromosome.

pEF-BOS, a powerful mammalian expression vector

Article

Full-text available

Oct 1990

Full textFull text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (159K), or click on a page image below to browse page by page. 5322

Biogenesis of the vitronectin receptor in human endothelial cell: Evidence that the vitronectin receptor and GPIIb-IIIa are synthesized by a common mechanism

Article

Full-text available

Jun 1989

Human endothelial cells express a membrane glycoprotein alpha beta heterodimer similar to the human platelet glycoprotein IIb-IIIa complex (GPIIb-IIIa). This noncovalently associated complex is the vitronectin receptor (VNR). These two receptors belong to the cytoadhesin family and share the same beta subunit. They express different recognition specificities: platelet GPIIb-IIIa is a receptor for fibrinogen, fibronectin, and von Willebrand factor (vWF), whereas VNR is a receptor for vitronectin, and is possibly a receptor for fibrinogen and vWF. We analyzed the biosynthesis of the endothelial cell VNR. Our data show that VNR alpha is a two-chain protein which is biosynthesized as a single-chain precursor: the pro-VNR alpha. Pro-VNR alpha forms a complex with VNR beta, and this association occurs prior to the Golgi-mediated processing of the oligosaccharide side chains. Mature VNR beta is glycosylated by not fully processed oligosaccharide side chains because it remains endoglycosidase H (endo H) sensitive, even when the complex is expressed on the cell surface. This characteristic appears as a common feature for the members of the cytoadhesin family. These results indicate that although VNR and GPIIb-IIIa are biosynthesized in different cells, their expression is controlled by similar mechanisms, providing further support for the concept that the cytoadhesin family constitutes a distinct group of adhesion receptors.

Effects of inhibitors of N-linked oligosaccharide processing on the biosynthesis and function of insulin and insulin-like growth factor-I receptors

Article

Full-text available

May 1988

We have used specific inhibitors of oligosaccharide processing enzymes as probes to determine the involvement of oligosaccharide residues in the biosynthesis and function of insulin and insulin-like growth factor-I receptors. In a previous study (Duronio, V., Jacobs, S., and Cuatrecasas, P. (1986) J. Biol. Chem. 261, 970-975) swainsonine was used to inhibit mannosidase II, resulting in the production of receptors containing only hybrid-type oligosaccharides. These receptors had a slightly lower molecular weight and were much more sensitive to endoglycosidase H, but otherwise behaved identically to normal receptors. In this study, we used two compounds that inhibit oligosaccharide processing at earlier steps: (i) N-methyl-1-deoxynojirimycin (MedJN), which inhibits glucosidases I and II and yields glucosylated, high mannose oligosaccharides, and (ii) manno-1-deoxynojirimycin (MandJN), which inhibits mannosidase I and yields high mannose oligosaccharides. In the presence of MandJN, HepG2 cells synthesized receptors of lower molecular weight, which were cleaved into alpha and beta subunits and were able to bind hormone and autophosphorylate. These receptors were as sensitive to endoglycosidase H as receptors made in the presence of swainsonine. In the presence of MedJN, receptors of only slightly lower molecular weight than normal were synthesized and were shown to contain some glucosylated high mannose oligosaccharides. These receptors were able to bind hormone and retained hormone-sensitive autophosphorylation activity. In both cases, the incompletely processed receptors could be detected at the cell surface by cross-linking of iodinated hormone and susceptibility to trypsin digestion, although less receptor was present in cells treated with MedJN. Studies of receptor synthesis using pulse-chase labeling showed that the receptor precursors synthesized in the presence of MedJN were cleaved into alpha and beta subunits at a slower rate than normal receptors or those made in the presence of MandJN. Inhibition of oligosaccharide processing had no effect on the association of the receptor subunits into disulfide-linked oligomeric complexes.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine

Article

Full-text available

Sep 1995

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Thyroid Hormone Modulates the Interaction between Iron Regulatory Proteins and the Ferritin mRNA Iron-responsive Element

Article

Full-text available

Jun 1996

The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T3) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T3 augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T3 with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T3 augmented an iron-induced increase in CAT activity by approximately 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T3-receptors may be necessary for this T3-induced response, because the effect could not be reproduced by the addition of T3 directly to cytoplasmic extracts and was absent in CV-1 cells which lack T3-receptors. We conclude that T3 can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T3 to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.

Identification of stromal cell products that interact with pre-B cells

Article

Full-text available

Sep 1996

Our understanding of lympho-hematopoietic microenvironments is incomplete, and a new cloning strategy was developed to identify molecules that bind to B lineage lymphocyte precursors. A cell sorting procedure was used for initial enrichment of cDNAs from stromal cell mRNA that contained signal sequences and were therefore likely to encode transmembrane or secreted proteins. A second step involved expression of the library as soluble Ig fusion proteins. Finally, pools representing these proteins were screened for the ability to recognize pre-B cells. This approach resulted in the cloning of biglycan, syndecan 4, collagen type I, clusterin, matrix glycoprotein sc1, osteonectin, and one unknown molecule (designated SIM). The full-length cDNA of SIM revealed that it is a type I transmembrane protein, and its intracellular domain has weak homology with myosin heavy chain and related proteins. Staining of established cell lines and freshly isolated hematopoietic cells with the Ig fusion proteins revealed distinct patterns of reactivity and differential dependence on divalent cations. Biglycan-, sc1-, and SIM-Ig fusion proteins selectively increased interleukin 7-dependent proliferation of pre-B cells. Overexpression of the entire SIM protein affected the morphology of 293T cells, while expression of just the extracellular portion was without effect. Thus, a series of stromal cell surface molecules has been identified that interact with blood cell precursors. Three of them promoted the survival and/or proliferation of pre-B cells in culture, and all merit further study in relation to lympho-hematopoiesis.

Ligand Activation of ELK Receptor Tyrosine Kinase Promotes Its Association with Grb10 and Grb2 in Vascular Endothelial Cells

Article

Full-text available

Oct 1996

ELK is a member of the Eph-related tyrosine kinase family that includes receptors signaling axonal guidance, neuronal bundling, and angiogenesis. We recently identified ELK expression in human renal microvascular endothelial cells and sought to identify intracellular proteins through which it signals responses. The cytoplasmic domain of ELK was used as "bait" in a yeast two-hybrid screen to identify interactive proteins expressed from a randomly primed embryonic murine library (E9.5-10.5). Among interactive products of 76 cDNAs characterized, 10 nonidentical, overlapping clones encoded the SH2 domain of the recently reported Grb10 adapter protein, and an additional 3 encoded Grb2. A self-phosphorylated recombinant, baculovirus-expressed GST-ELKcy fusion protein bound Grb10 and Grb2 from human renal microvascular endothelial cell extracts, while the unphosphorylated fusion form did not. Site-directed mutation identified Tyr-929 as a putative phosphorylation site required for Grb10, but not Grb2, interaction in yeast and recombinant protein assays. The ELK ligand, LERK-2/Fc, stimulated tyrosine phosphorylation of ELK, and recruitment of Grb10 and Grb2 to endothelial ELK receptors recovered by wheat germ agglutinin lectin and immunoprecipitation. These findings define ligand-activated interaction between ELK and the SH2 domains of Grb2 and the newly identified Grb10 protein that shares homology with a Caenorhabditis elegans gene product implicated in neural cell migration.

Mutant Isoforms of the Anti-Mullerian Hormone Type II Receptor Are Not Expressed at the Cell Membrane

Article

Full-text available

Dec 1996

Anti-Müllerian hormone, a member of the transforming growth factor beta superfamily, produces early regression of Müllerian ducts in the male fetus through binding to a serine/threonine kinase receptor, homologous to type II receptors of the transforming growth factor beta (TGF-beta) family. A splice mutation of this receptor, described in a patient with abnormal retention of Müllerian derivatives, generates two mutant isoforms, one lacking the second exon and the other bearing an insertion of 12 bases between exons 2 and 3. Using hemagglutinin-tagged recombinant receptors, we have visualized wild type and mutant receptors in COS cells by Western blotting and immunoprecipitation. The 82-kDa, endoglycosidase H-insensitive, mature form of the wild type receptor is reduced to 68 kDa by N-glycosidase F treatment. Mutant receptor isoforms, 73 and 63 kDa for the long and short form, respectively, are sensitive to endoglycosidase H, suggesting that they are retained in the endoplasmic reticulum. Indeed, only the wild type receptor was expressed on the cell surface and bound iodinated anti-Müllerian hormone. These results provide a biological explanation for the failure of the mutant receptor to induce Müllerian regression.

GOK: A gene at 11p15 involved in rhabdomyosarcoma and rhabdoid tumor development

Article

Full-text available

Nov 1997

The expression of GOK, a gene recently identified at 11p15.5, was studied in breast cancer, rhabdomyosarcoma, and rhabdoid tumor cell lines. In these neoplasms, deletions at 11p15 and suppression of tumorigenicity induced by a normal human chromosome 11 were previously demonstrated. Whereas breast cancer cell lines express readily detectable levels of GOK mRNA, expression is absent in rhabdomyosarcoma and rhabdoid tumor cell lines. This is in contrast with the high expression of GOK in skeletal muscle, the normal tissue of origin of rhabdomyosarcomas, suggesting that down-regulation of GOK expression could be involved in tumor development. In agreement with this hypothesis, transfection of GOK cDNA into G401 derived from a rhabdoid tumor and RD cells derived from a rhabdomyosarcoma that do not express detectable levels of GOK mRNA, induced cell death. Because GOK expression is not compatible with growth of these tumor cells, these results support the hypothesis that loss of GOK expression plays a role in tumor establishment or progression and suggest that GOK may act as a recessive tumor suppressor gene in rhabdomyosarcomas and rhabdoid tumors. On the contrary, transfection of GOK cDNA into the breast cancer cell line HBL100 produced no detectable effects, indicating that the growth-suppressive effect of GOK in RD and G401 cells was specific. Because rhabdomyosarcomas have been observed in cases of Beckwith-Wiedemann syndrome, a genetic disorder linked to 11p15, a role of GOK in this disease cannot be excluded.

The crystal structure of an Eph receptor SAM domain reveal a mechanism for modular dimerization

Article

Full-text available

Feb 1999
Nat Struct Biol

The sterile alpha motif (SAM) domain is a novel protein module of approximately 70 amino acids that is found in a variety of signaling molecules including tyrosine and serine/threonine protein kinases, cytoplasmic scaffolding and adaptor proteins, regulators of lipid metabolism, and GTPases as well as members of the ETS family of transcription factors. The SAM domain can potentially function as a protein interaction module through the ability to homo- and hetero-oligomerize with other SAM domains. This functional property elicits the oncogenic activation of chimeric proteins arising from translocation of the SAM domain of TEL to coding regions of the betaPDGF receptor, Abl, JAK2 protein kinase and the AML1 transcription factor. Here we describe the 2.0 A X-ray crystal structure of a SAM domain homodimer from the intracellular region of the EphA4 receptor tyrosine kinase. The structure reveals a mode of dimerization that we predict is shared amongst the SAM domains of the Eph receptor tyrosine kinases and possibly other SAM domain containing proteins. These data indicate a mechanism through which an independently folding protein module can form homophilic complexes that regulate signaling events at the membrane and in the nucleus.

Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain

Article

Full-text available

Sep 1999

p73 and p63 are two recently cloned genes with homology to the tumor suppressor p53, whose protein product is a key transcriptional regulator of genes involved in cell cycle arrest and apoptosis. While all three proteins share conserved transcriptional activation, DNA-binding and oligomerization domains, p73 and p63 have an additional conserved C-terminal region. We have determined the three-dimensional solution structure of this conserved C-terminal domain of human p73. The structure reveals a small five-helix bundle with striking similarity to the SAM (sterile alpha motif) domains of two ephrin receptor tyrosine kinases. The SAM domain is a putative protein-protein interaction domain found in a variety of cytoplasmic signaling proteins and has been shown to form both homo- and hetero-oligomers. However, the SAM-like C-terminal domains of p73 and p63 are monomeric and do not interact with one another, suggesting that this domain may interact with additional, as yet uncharacterized proteins in a signaling and/or regulatory role.

Monomeric Structure of the Human EphB2 Sterile Motif Domain

Article

Full-text available

Jan 2000

The sterile alpha motif (SAM) domain is a protein module found in many diverse signaling proteins. SAM domains in some systems have been shown to self-associate. Previous crystal structures of an EphA4-SAM domain dimer (Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. (1999) Nat. Struct. Biol. 6, 44-49) and a possible EphB2-SAM oligomer (Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Science 283, 833-836) both revealed large interfaces comprising an exchange of N-terminal peptide arms. Within the arm, a conserved hydrophobic residue (Tyr-8 in the EphB2-SAM structure or Phe-910 in the EphA4-SAM structure) is anchored into a hydrophobic cleft on a neighboring molecule. Here we have solved a new crystal form of the human EphB2-SAM domain that has the same overall SAM domain fold yet has no substantial intermolecular contacts. In the new structure, the N-terminal peptide arm of the EphB2-SAM domain protrudes out from the core of the molecule, leaving both the arm (including Tyr-8) and the hydrophobic cleft solvent-exposed. To verify that Tyr-8 is solvent-exposed in solution, we made a Tyr-8 to Ala-8 mutation and found that the EphB2-SAM domain structure and stability were only slightly altered. These results suggest that Tyr-8 is not part of the hydrophobic core of the EphB2-SAM domain and is conserved for functional reasons. Cystallographic evidence suggests a possible role for the N-terminal arm in oligomerization. In the absence of a direct demonstration of biological relevance, however, the functional role of the N-terminal arm remains an open question.

Effect of N-Linked Glycosylation on Glycopeptide and Glycoprotein Structure

Article

Full-text available

Jan 2000
CURR OPIN CHEM BIOL

Asparagine-linked glycosylation is an enzyme-catalyzed, co-translational protein modification reaction that has the capacity to influence either the protein folding process or the stability of the native glycoprotein conjugate. Advances in both glycoconjugate chemical synthesis and glycoprotein expression methods have increased the availability of these once elusive biopolymers. The application of spectroscopic methods to these proteins has begun to illuminate the various ways in which the saccharide affects the structure, function and stability of the proteins.

Intracellular Functions of N-Linked Glycans

Article

Full-text available

Apr 2001

N-linked oligosaccharides arise when blocks of 14 sugars are added cotranslationally to newly synthesized polypeptides in the endoplasmic reticulum (ER). These glycans are then subjected to extensive modification as the glycoproteins mature and move through the ER via the Golgi complex to their final destinations inside and outside the cell. In the ER and in the early secretory pathway, where the repertoire of oligosaccharide structures is still rather small, the glycans play a pivotal role in protein folding, oligomerization, quality control, sorting, and transport. They are used as universal “tags” that allow specific lectins and modifying enzymes to establish order among the diversity of maturing glycoproteins. In the Golgi complex, the glycans acquire more complex structures and a new set of functions. The division of synthesis and processing between the ER and the Golgi complex represents an evolutionary adaptation that allows efficient exploitation of the potential of oligosaccharides.

Unstimulated human CD4 lymphocytes express a cytoplasmic immature form of the common cytokine receptor gamma-chain

Article

Full-text available

Aug 2001

As a component of various cytokine receptors, common cytokine receptor gamma-chain (gamma(c)) is essential in the development of the immune system and plays an important role in different stages of inflammatory and immune responses. Here we establish that resting CD4 T cells and the Jurkat CD4 T cell line do not express the mature form of gamma(c) (64 kDa) recognized by mAb Tugh4. However, these cells constitutively transcribe the corresponding gamma(c) gene. This apparent paradox was solved by the demonstration that polyclonal anti-gamma(c) Abs detected endoglycosidase-H-sensitive immature forms of gamma(c) (54-58 kDa) expressed by quiescent CD4 T lymphocytes and Jurkat cells. Immature gamma(c) is characterized as an intracellular component localized in the endoplasmic reticulum. Pulse-chase analysis shows that the immature gamma(c) is rapidly degraded after synthesis. After activation of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the endoglycosidase-H-resistant mature form of gamma(c) is detectable at the cell surface and in the endosomal compartment. For the first time, our results demonstrate that a cytokine receptor chain may be constitutively produced as an immature form. Furthermore, this supports the notion that expression of the functional form of gamma(c) may require intracellular interactions with lineage- or subset-specific molecular partners.

Identification and characterization of the STIM (stromal interaction molecule) gene family: Coding for a novel class of transmembrane proteins

Article

Full-text available

Sep 2001

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

Biogenesis of the vitronectin receptor in human endothelial cell: evidence that the vitronectin receptor and GPIIb-IIIa are synthesized by a common mechanism

Article

May 1989

Human endothelial cells express a membrane glycoprotein alpha beta heterodimer similar to the human platelet glycoprotein IIb-IIIa complex (GPIIb-IIIa). This noncovalently associated complex is the vitronectin receptor (VNR). These two receptors belong to the cytoadhesin family and share the same beta subunit. They express different recognition specificities: platelet GPIIb-IIIa is a receptor for fibrinogen, fibronectin, and von Willebrand factor (vWF), whereas VNR is a receptor for vitronectin, and is possibly a receptor for fibrinogen and vWF. We analyzed the biosynthesis of the endothelial cell VNR. Our data show that VNR alpha is a two-chain protein which is biosynthesized as a single-chain precursor: the pro-VNR alpha. Pro-VNR alpha forms a complex with VNR beta, and this association occurs prior to the Golgi- mediated processing of the oligosaccharide side chains. Mature VNR beta is glycosylated by not fully processed oligosaccharide side chains because it remains endoglycosidase H (endo H) sensitive, even when the complex is expressed on the cell surface. This characteristic appears as a common feature for the members of the cytoadhesin family. These results indicate that although VNR and GPIIb-IIIa are biosynthesized in different cells, their expression is controlled by similar mechanisms, providing further support for the concept that the cytoadhesin family constitutes a distinct group of adhesion receptors.

Solution structure of the receptor tyrosine kinase EphB2 SAM domain and identification of two distinct homotypic interaction sites

Article

Oct 1999
PROTEIN SCI

The sterile alpha motif (SAM) is a protein interaction domain of around 70 amino acids present predominantly in the N‐ and C‐termini of more than 60 diverse proteins that participate in signal transduction and transcriptional repression. SAM domains have been shown to homo‐ and hetero‐oligomerize and to mediate specific protein‐protein interactions. A highly conserved subclass of SAM domains is present at the intracellular C‐terminus of more than 40 Eph receptor tyrosine kinases that are involved in the control of axonal pathfinding upon ephrin‐induced oligomerization and activation in the event of cell‐cell contacts. These SAM domains appear to participate in downstream signaling events via interactions with cytosolic proteins. We determined the solution structure of the EphB2 receptor SAM domain and studied its association behavior. The structure consists of five helices forming a compact structure without binding pockets or exposed conserved aromatic residues. Concentration‐dependent chemical shift changes of NMR signals reveal two distinct well‐separated areas on the domains' surface sensitive to the formation of homotypic oligomers in solution. These findings are supported by analytical ultracentrifugation studies. The conserved Tyr932, which was reported to be essential for the interaction with SH2 domains after phosphorylation, is buried in the hydrophobic core of the structure. The weak capability of the isolated EphB2 receptor SAM domain to form oligomers is supposed to be relevant in vivo when the driving force of ligand binding induces receptor oligomerization. A formation of SAM tetramers is thought to provide an appropriate contact area for the binding of a low‐molecular‐weight phosphotyrosine phosphatase and to initiate further downstream responses.

SAM: A novel motif in yeast sterile and Drosophila polyhomeotic proteins

Article

Sep 1995

Christopher P. Ponting

Single copies of an approximately 65-70 residue domain are shown to be present in the sequences of 14 eukaryotic proteins, including yeast byr2, STE11, ste4, and STE50, which are essential participants in sexual differentiation. This domain, named SAM (sterile alpha motif), appears to participate in other developmental processes because it is also present in Drosophila polyhomeotic gene product and related homologues, which are thought to regulate determination of segmental specification in early embryogenesis. Its appearance in byr2 and STE11, which are MEK kinases, and in proteins containing pleckstrain homology, src homology 3, and discs-large homologous region domains, suggests possible participation in signal transduction pathways.

EF-hands reach out

Article

Feb 1996
Nat Struct Biol

Robert H. Kretsinger

The difference in amino-acid sequence and inferred molecular mechanisms between the pair of EF-hands in BM-40 and those of other EF-hand proteins force us to reconsider the evolutionary and functional relationships among the members of this diverse family of proteins.

Rechsteiner M, Rogers SW.. PEST sequences and regulation by proteolysis. Trends Biochem Sci 21: 267-271

Article

Aug 1996
TRENDS BIOCHEM SCI

In 1986, we proposed that polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction. For much of the past decade there were only sporadic experimental tests of the hypothesis. This situation changed markedly during the past two years with a number of papers providing strong evidence that PEST regions do, in fact, serve as proteolytic signals. Here, we briefly review the properties of PEST regions and some interesting examples of the conditional nature of such signals. Most of the article, however, focuses on recent experimental support for the hypothesis and on mechanisms responsible for the rapid degradation of proteins that contain PEST regions.

Molecular Cloning of a Novel Human Gene (D11S4896E) at Chromosomal Region 11p15.5

Article

Nov 1996

A novel gene (GOK) has been cloned from human chromosome region 11p15.5 that is believed to contain a gene or genes associated with a number of pediatric malignancies, including Wilms tumor. A 4-kb cDNA has been cloned and it encodes a predicted protein of approximately 84 kDa that could be translated in vitro. Computer analysis predicted that the protein had a signal peptide and may contain a transmembrane helix. Restriction mapping by pulsed-field electrophoresis indicates that GOK is located 1.7 kb telomeric of RRM1, and both genes are transcribed in the same direction. GOK displays high evolutionary conservation: cloning and partial sequencing of a mouse genomic clone revealed 90% identity with the human gene at both the nucleotide and the predicted amino acid levels.

Neutralising Antibodies to the Granulocyte Colony-stimulating Factor Receptor Recognise both the Immunoglobulin-like Domain and the Cytokine Receptor Homologous Domain

Article

Feb 1997

To define regions of the granulocyte colony-stimulating factor (G-CSF) receptor that are important for ligand binding, neutralising monoclonal antibodies to the human receptor have been produced. Eleven antibodies recognised six different receptor epitopes. Antibodies from three of the epitope groups were able to detect the receptor by western blotting but did not inhibit G-CSF binding. The other three antibody groups inhibited G-CSF binding either completely (groups 1 and 2) or partially (group 3). All the antibodies inhibited proliferation of BA/F3 cells expressing the G-CSF receptor to varying extents. By using human-marine chimeric receptors, the binding sites of the antibodies were mapped to the immunoglobulin-like domain (groups 1 and 3), the cytokine receptor homologous domain (group 2) or the fibronectin type III domains (groups 4 to 6). These results show that the immunoglobulin-like and cytokine receptor homologous domains of the receptor are important for ligand binding and subsequent signalling.

Oligomeric Structure of the Human EphB2 Receptor SAM Domain

Article

Mar 1999

The sterile alpha motif (SAM) domain is a protein interaction module that is present in diverse signal-transducing proteins. SAM domains are known to form homo- and hetero-oligomers. The crystal structure of the SAM domain from an Eph receptor tyrosine kinase, EphB2, reveals two large interfaces. In one interface, adjacent monomers exchange amino-terminal peptides that insert into a hydrophobic groove on each neighbor. A second interface is composed of the carboxyl-terminal helix and a nearby loop. A possible oligomer, constructed from a combination of these binding modes, may provide a platform for the formation of larger protein complexes.

A Web-Based Tool for the Study of enetically Mobile Domains

Article

Feb 2000

SMART (a Simple Modular Architecture Research Tool) allows the identification and annotation of genetically mobile domains and the analysis of domain architectures (http://SMART.embl-heidelberg.de ). More than 400 domain families found in signalling, extracellular and chromatin-associated proteins are detectable. These domains are extensively annotated with respect to phyletic distributions, functional class, tertiary structures and functionally important residues. Each domain found in a non-redundant protein database as well as search parameters and taxonomic information are stored in a relational database system. User interfaces to this database allow searches for proteins containing specific combinations of domains in defined taxa.

STIM1: A novel phosphoprotein located at the cell surface

Article

Sep 2000
Biochim Biophys Acta

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Stromal interaction molecule 1 (STIM1), a transmembrane protein with growth suppressor activity, contains an extracellular SAM domain modified by N-linked glycosylation

Abstract

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