Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum

Calreticulin is a 46-kDa Ca2+-binding chaperone found across a diverse range of species. The protein is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage capacity. Calreticulin is also an important molecular chaperone involved in "quality control" within secretory pathways. The protein contains structurally and functionally unique domains with specialized functions. Studies on calreticulin knockout mice indicate that the protein is essential in early cardiac development. The protein also plays an important role in autoimmunity and cancer.     

Calreticulin, Ca2+-binding chaperon of the endoplasmic reticulum

The endoplasmic reticulum (ER) plays a vital role in many cellular processes, including Ca2+ storage and release. Calreticulin is a Ca2+-binding chaperon residing in ER. The protein is a key component of the quality control pathways in ER. In the ER lumen, calreticulin performs two major functions, works as a chaperon and regulates Ca2+ homeostasis. In cardiac muscle, calreticulin plays an important role in cardiac development and pathology.     

Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.     

Dissecting physical structure of calreticulin,an intrinsically disordered Ca2+-buffering chaperone from endoplasmic reticulum

Calreticulin (CALR) is a Ca²⁺ binding multifunctional protein that mostly resides in the endoplasmic reticulum (ER) and plays a number of important roles in various physiological and pathological processes. Although the major functions ascribed to CALR are controlling the Ca²⁺ homeostasis in ER and acting as a lectin-like ER chaperon for many glycoproteins, this moonlighting protein can be found in various cellular compartments where it has many non-ER functions. To shed more light on the mechanisms underlying polyfunctionality of this moonlighting protein that can be found in different cellular compartments and that possesses a wide spectrum of unrelated biological activities, being able to interact with Ca²⁺ (and potentially other metal ions), RNA, oligosaccharides, and numerous proteins, we used a set of experimental and computational tools to evaluate the intrinsic disorder status of CALR and the role of calcium binding on structural properties and conformational stability of the full-length CALR and its isolated P- and C-domains.     

Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum

Protein disulfide isomerase (PDI) is a protein-thiol oxidoreductase that catalyzes the oxidation, reduction and isomerization of protein disulfides. In the endoplasmic reticulum PDI catalyzes both the oxidation and isomerization of disulfides on nascent polypeptides. Under the reducing condition of the cytoplasm, endosomes and cell surface. PDI catalyzes the reduction of protein disulfides. At those locations, PDI has been demonstrated to participate in the regulation of reception function, cell-cell interaction, gene expression, and actin filament polymerization. These activities of PDI will be discussed, as well as its activity as a chaperone and subunit of prolyl 4-hydroxylase and microsomal triglyceride transfer protein.     

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.     

On the roles of Ca2+ diffusion, Ca2+ buffers, and the endoplasmic reticulum in IP3-induced Ca2+ waves.

We have investigated the effects of Ca2+ diffusion, mobile and stationary Ca2+ buffers in the cytosol, and Ca2+ handling by the endoplasmic reticulum on inositol 1,4,5-trisphosphate-induced Ca2+ wave propagation. Rapid equilibration of free and bound Ca2+ is used to describe Ca2+ sequestration by buffers in both the cytosol and endoplasmic reticulum (ER) lumen. Cytosolic Ca2+ regulation is based on a kinetic model of the inositol 1,4,5-trisphosphate (IP3) receptor of De Young and Keizer that includes activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane and SERCA Ca2+ pumps in the ER. Diffusion of Ca2+ in the cytosol and the ER and the breakdown and diffusion of IP3 are also included in our calculations. Although Ca2+ diffusion is severely limited because of buffering, when conditions are chosen just below the threshold for Ca2+ oscillations, a pulse of IP3 or Ca2+ results in a solitary trigger wave that requires diffusion of Ca2+ for its propagation. In the oscillatory regime repetitive wave trains are observed, but for this type of wave neither the wave shape nor the speed is strongly dependent on the diffusion of Ca2+. Local phase differences lead to waves that are predominately kinematic in nature, so that the wave speed (c) is related to the wavelength (lambda) and the period of the oscillations (tau) approximately by the formula c = lambda/tau. The period is determined by features that control the oscillations, including [IP3] and pump activity, which are related to recent experiments. Both solitary waves and wave trains are accompanied by a Ca2+ depletion wave in the ER lumen, similar to that observed in cortical preparations from sea urchin eggs. We explore the effect of endogenous and exogenous Ca2+ buffers on wave speed and wave shape, which can be explained in terms of three distinct effects of buffering, and show that exogenous buffers or Ca2+ dyes can have considerable influence on the amplitude and width of the waves.     

Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum

We have identified a novel endoplasmic reticulum (ER)-resident protein, named "calumin", which is expressed in various tissues. This protein has a molecular mass of approximately 60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment, and a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of binding Ca2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and neonatal lethality, fluorometric Ca2+ imaging detected insufficient Ca2+ contents in intracellular stores and attenuated store-operated Ca2+ entry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin plays an essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision.     

Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

Recent data have revealed an unexpected role of Bcl-2 in modulating the steady-state levels and agonist-dependent fluxes of Ca(2+) ions. Direct monitoring of endoplasmic reticulum (ER) Ca(2+) concentration with recombinant probes reveals a lower state of filling in Bcl-2-overexpressing cells and a higher leak rate from the organelle. The broader set of indirect data using cytosolic probes reveals a more complex scenario, as in many cases no difference was detected in the Ca(2+) content of the intracellular pools. At the same time, Ca(2+) signals have been shown to affect important checkpoints of the apoptotic process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the data on the effect of Bcl-2 on [Ca(2+)](er), (ii) the functional significance of the Ca(2+)-signalling alteration and (iii) the current insight into the possible mechanisms of this effect.     

Modulation of active Ca2+ uptake by the islet-cell endoplasmic reticulum.

The possible effects of calmodulin and cyclic AMP on active Ca2+ uptake by the islet-cell endoplasmic reticulum were investigated. Neither calmodulin nor cyclic AMP affected the rate of active Ca2+ uptake, or the steady-state filling capacity of the endoplasmic reticulum when measured in the absence of oxalate. Consistent with these results, calmodulin did not activate the Ca2+-stimulated ATPase activity associated with this cell fraction. During the course of these experiments., it was unexpectedly discovered that the rate of Ca2+ uptake, as well as the steady-state Ca2+ filling capacity of the endoplasmic reticulum, were markedly increased by unidentified factor(s) in the cytosol. This effect could be demonstrated by reconstitution of the membranes in cytosol, or by direct addition of fresh or dialysed cytosol to the Ca2+ uptake assays. The degree of activation by the cytosol indicates that the endoplasmic reticulum may play a prominent role in controlling beta-cell Ca2+ concentrations and that the unidentified activator(s) present in the cytosol may be involved in regulation of this function.     

Ca2+ regulation of interactions between endoplasmic reticulum chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.     

ERp57, a multifunctional endoplasmic reticulum resident oxidoreductase

ERp57 is a 58-kDa thiol oxidoreductase and a member of the protein disulfide isomerase (PDI)-like family. ERp57 is highly similar to other PDI family members in terms of amino acid sequence and structural/functional domain organization; however, it possesses some distinctive structural features that dictate its unique functions in the cell. This protein plays an important role in endoplasmic reticulum quality control of newly synthesized glycoproteins, is critical in major histocompatability complex (MHC) class I assembly and regulates gene expression. Studies on ERp57-deficient mice indicate that the protein is critical during embryonic development. The protein has been implicated in human pathologies including cancer and Alzheimer's disease.     

Activation of the endoplasmic reticulum Ca2+ pump of pancreatic acini by Ca2+ mobilizing hormones

Stimulation of the pancreatic acinar cells with Ca2+ mobilizing hormones increased the ATP-dependent Ca2+ uptake into the ER of permeabilized cells. Activation of the ER Ca2+ pump resulted in increased apparent affinity for Ca2+ from 0.26 to 0.09 uM and Vmax from 2.68 to 5.74 nmoles/mg prot./min. The apparent affinity of the pump for VO4 = was dependent on [Ca2+]. Activation of the pump also decreased apparent affinity for VO4 = from 12 to 32 uM at [Ca2+] of 0.138 uM. These findings suggest that pump activation is due to acceleration of the rate of the conformational transition between the VO4 = (E2) and Ca2+ (E1) sensitive forms of the pump.     

Release of Ca2+ from the endoplasmic reticulum contributes to Ca2+ signaling in Dictyostelium discoideum

Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted D. discoideum proteome revealed three putative Ca2+ pumps and four putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in D. discoideum.     

Cloning of a sea urchin sarco/endoplasmic reticulum Ca2+ ATPase

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a vesicular integral membrane protein, is the best-characterized member of the P-type ion translocating ATPase superfamily. Here we describe the cloning and structural analysis of a sea urchin SERCA (suSERCA) cloned from testis cDNA. The approximately 112 kDa suSERCA is 1022 amino acids with approximately 70% identity and 80% similarity to all known mammalian SERCA isoforms. suSERCA shares all the structural features of mammalian SERCAs, including domains: A, actuator; N, nucleotide-binding; and P, phosphorylation, and also 10 transmembrane helices. Like human SERCA2, the suSERCA has a possible 11th transmembrane segment in its extreme C-terminus. The alignment of three sequences (suSERCA, human SERCA2, and rabbit SERCA1a) shows that the Ca2+ binding residues and kinks (required to form the ion-binding pocket) are 100% conserved. The annotated suSERCA gene consists of 24 exons separated by 23 introns and is approximately 30 kb. Western blots show that suSERCA is present in sea urchin eggs and testis, but not in mature spermatozoa. Treatment of live sperm with SERCA inhibitors has no effect on intracellular calcium, suggesting the absence of SERCA in sea urchin spermatozoa.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.