Proteolytic fragmentation of inositol 1,4,5‐trisphosphate receptors: a novel mechanism regulating channel activity?

Abstract

Inositol 1,4,5‐trisphosphate receptors (IP₃Rs) are a family of ubiquitously expressed intracellular Ca²⁺ release channels. Regulation of channel activity by Ca²⁺, nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca²⁺ signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca²⁺‐dependent effectors. IP₃Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP₃R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP₃ binding from the channel domain, leaving an unregulated leaky Ca²⁺ pore. Contrary to this hypothesis, we demonstrate following proteolysis that N‐ and C‐termini of IP₃R1 remain associated, presumably through non‐covalent interactions. Further, we show that complementary fragments of IP₃R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP₃. While peptide continuity is clearly not necessary for IP₃‐gating of the channel, we propose that cleavage of the IP₃R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP₃R may support distinct spatiotemporal Ca²⁺ signals and activation of specific effectors. Notably, in many adaptive physiological events, the non‐apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP₃R regulation, which plays a role in varied adaptive physiological processes.

Abbreviations

IP₃

inositol 1,4,5‐trisphosphate

IP₃R

inositol 1,4,5‐trisphosphate receptor

IP₃R1

type 1 IP₃R

ER

endoplasmic reticulum

[Ca²⁺]_i

intracellular calcium concentration

Introduction

Inositol 1,4,5‐trisphosphate receptors (IP₃Rs) are a family of ligand‐gated intracellular calcium release channels that are primarily expressed on the endoplasmic reticulum (ER) membrane (Foskett et al. 2007). IP₃‐induced Ca²⁺ release controls a diverse array of cellular processes ranging from cell proliferation, differentiation, fluid secretion and motility to cell fate decisions including apoptosis and autophagy (Berridge, 1993; Joseph & Hajnoczky, 2007; Mikoshiba, 2007; Ivanova et al. 2014). It is thought that the versatility of this signal is largely a result of the spatial and temporal properties imparted by the fine control of IP₃‐induced Ca²⁺ release through regulation of IP₃R activity (Fig. 1). IP₃R functions as a signal integrator that, depending on the stimuli, encodes the specific properties of the Ca²⁺ signal necessary to activate distinct downstream effectors with fidelity, specificity and efficiency. IP₃R activity is regulated at multiple levels. For example, IP₃R can interact with an abundance of intracellular molecules including Ca²⁺, nucleotides such as ATP and cAMP, and binding proteins, including Bcl‐2, Bok and IRBIT (Bruce et al. 2003; Foskett et al. 2007; Yule et al. 2010; Schulman et al. 2013). Further, IP₃R are either homo‐ or heterotetrameric channels assembled from three receptor isoforms with additional splice variants (Nucifora et al. 1996; Joseph et al. 2000; Onoue et al. 2000). The subunit composition of the IP₃R can either dominate or contribute to the characteristics of the receptor activity (Alzayady et al. 2013 b; Chandrasekhar et al. 2015). Also, post‐translational modification, such as phosphorylation and redox modification, plays a pivotal role in regulating IP₃R activity (Fig. 1) (Betzenhauser & Yule, 2010; Yule et al. 2010; Bansaghi et al. 2014). As a further level of complexity, IP₃R type 1 (IP₃R1) has also been demonstrated to be a substrate of the cysteine proteases caspase and calpain, two enzymes often associated with cell death (Hirota et al. 1999). Through proteolytic cleavage, IP₃R monomers are processed into at least two fragments (Fig. 2); an N‐terminal fragment, which contains the IP₃ binding core and majority of the cytoplasmic region (∼210 kDa), and a C‐terminal fragment that comprises the transmembrane helices, the channel pore and the extreme C‐terminus cytoplasmic tail (∼90 kDa). To date, however, the functional consequences of the receptor fragmentation are not established and are the subject of considerable debate.

Figure 1. Schematic diagram depicting two monomers of a tetrameric IP₃R .

The current model of gating suggests that the signal upon binding of IP₃ to the binding core in the N‐terminal is transmitted through an interaction between residues in the suppressor domain (blue region) to the C‐terminal channel domain to result in channel opening and Ca²⁺ release. The activity of the channel is modulated by numerous cytosolic factors ultimately encoding the particular spatial and temporal characteristics of the intracellular Ca²⁺ signal. These properties are, in turn, important for the appropriate activation of effectors.

Figure 2. Schematic diagram depicting the hypothetical result of proteolytic cleavage of IP₃R1 .

Cleavage by the cysteine proteases, calpain or caspase, results in two major peptide fragments; a large N‐terminal fragment and a C‐terminal remnant containing the channel domain. Loss of peptide continuity and/or diffusion of the cytoplasmic N‐terminal region result in an unregulated ‘leaky channel’.

In this review, we first summarize the major controversies relating to the consequences of receptor cleavage by calpain and caspase. Further, we present and discuss new findings from our lab, which suggest that proteolytic fragmentation of IP₃R does not result in disassembly of the protein and does not necessarily disable regulated Ca²⁺ release, but rather, potentially alters the regulatory properties of the channel. We propose that IP₃R fragmentation may represent a novel regulatory mechanism for IP₃R1, which may regulate cellular events in addition to cell death.

IP₃Rs are proteolytically processed by caspase and calpain

In 1999, Mikoshiba and colleagues first demonstrated that IP₃R1 harbours a canonical DEVD substrate motif for caspase cleavage in its primary sequence at amino acids 1888–1891 (Hirota et al. 1999). Consistent with the prediction, IP₃R1s were shown to be cleaved into two fragments in Jurkat cells during the process of apoptosis. These data were later confirmed in multiple cell lines including SH‐SY5Y cells, PC‐12 cells, Hela cells, DT‐40 cells and mouse oocytes during ageing, thus establishing that IP₃R1s are a bone fide caspase substrate (Haug et al. 2000; Boehning et al. 2003; Assefa et al. 2004; Verbert et al. 2008; Zhang et al. 2011). Similarly, calpain cleaves the IP₃R1 in the same region, 26 amino acids downstream of the caspase site, and generates receptor fragments with an almost identical cleavage pattern to that of caspase (Kopil et al. 2011). Importantly, calpain‐mediated IP₃R1 fragmentation was shown to occur in rat cerebellum after cardiac arrest providing in vivo evidence of IP₃R1 fragmentation in pathological conditions. However, while the idea that IP₃R1 is a substrate of both caspase and calpain is widely accepted, the functional consequences of the proteolytic cleavage remain largely unresolved. One prominent idea is the so‐called ‘leaky channel’ theory (Nakayama et al. 2004; Szlufcik et al. 2006). Accordingly, caspase and/or calpain activated during the cell death process cleave the IP₃R1 into two prominent fragments. The theory then posits that disruption of receptor continuity following proteolysis abolishes the tight regulation of the channel by IP₃ binding in the N‐terminal fragment and this results in essentially an unregulated, constitutively active ‘leaky’ ER channel formed solely from the C‐terminal fragment (Fig. 2). The obvious consequence is that cytosolic calcium is progressively elevated as calcium leaks from the intracellular store. This event could conceivably recruit additional active caspase and calpain, generate more fragmented IP₃R1s, and thereby establishe a fatal feed‐forward cycle that can efficiently amplify the death signal. In this scenario the fundamental function of IP₃R1 fragmentation is to globally and continuously increase the intracellular calcium concentration ([Ca²⁺]_i) and thereby accelerate the execution of apoptosis.

Evidence supporting the involvement of leaky IP₃R channels in apoptosis

Some evidence in support of this idea comes from DT40‐3KO cells, a chicken B‐lymphocyte cell line where all three isoforms of IP₃R have been genetically deleted (Sugawara et al. 1997; Assefa et al. 2004; Verbert et al. 2008). This cell line has been widely used in characterizing the activity of IP₃Rs because it allows expression of IP₃R constructs on an unambiguously null genetic background. Notably, it has been demonstrated that DT40‐3KO is relatively more resistant to programmed cell death than cells expressing IP₃R1, indicating a potential role for IP₃R1 in mechanisms underlying apoptosis (Jayaraman & Marks, 1997; Assefa et al. 2004; Khan et al. 2007). Interestingly, the sensitivity to apoptosis in DT40‐3KO cells expressing IP₃R1 is completely abolished by mutation of the caspase cleavage sequence into a motif rendered non‐cleavable (Assefa et al. 2004). The most direct evidence in support of the general concept comes from measurement of Ca²⁺ signals following ectopic expression in DT40‐3KO cells of various constructs corresponding to different isolated regions of IP₃R1. For example, a construct encoding only the caspase‐cleaved C‐terminal fragment (Δ1–1891), essentially the channel domain ‘stump’, was shown to express appropriately in the ER and enhance Ca²⁺ leak from intracellular stores. Interestingly, no marked reduction in the ER Ca²⁺ content was observed in these experiments, presumably as the sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) pumping activity largely counteracted the ER leak. The expression of the fragment was also associated with augmentation of both the rate and the extent of apoptosis triggered by staurosporine (Assefa et al. 2004; Verbert et al. 2008). Additional reports generally consistent with the idea of the fragment functioning as a leaky channel include reports that ectopic expression of the channel domain in mouse oocytes decreased the ER Ca²⁺ content and induced apoptosis (Zhang & Fissore, 2014).

Similarly, a construct encoding the corresponding calpain‐cleaved C‐terminal channel stump (Δ1–1917 IP₃R1), when expressed in both the mouse neuroblastoma cell line and primary cortical neurons reduced the ER Ca²⁺ content and promoted cell death following challenge with detrimental stimuli (Kopil et al. 2011, 2012). While these data provide good evidence that expression of C‐terminal fragments of IP₃R can result in Ca²⁺ leak from the ER, caveats related to the interpretation of these data include that the channel domain was probably overexpressed to various degrees in these studies, and importantly that the channel C‐terminal fragments were expressed in the absence of the N‐terminal cytoplasmic domain of IP₃R.

Evidence inconsistent with leaky channels leading to cell death

While the leaky channel theory provides an attractive potential correlation linking IP₃R1 fragmentation and unregulated Ca²⁺ signalling to apoptosis, some observations are not necessarily consistent with this overall scenario. First, clues as to the consequences of fragmentation by intracellular proteases may be offered by earlier data investigating the structure of IP₃R by subjecting purified IP₃Rs to limited digestion in vitro with the protease trypsin (Joseph et al. 1995; Yoshikawa et al. 1999). A low concentration of trypsin cleaves IP₃R1 at four distinct and reproducible sites, resulting in five predictable fragments. These data have been interpreted to indicate that the overall structure of the protein is tightly folded resulting in globular domains with the proteolysis occurring at solvent‐exposed cleavage sites between domains. Four of the resultant fragments are derived from cleavage of the cytosolic portion of IP₃R1, while the fifth is the membrane‐associated channel domain, similar to the C‐terminal stump produced by caspase and calpain. Of note, all four tryptic cytosolic fragments were shown to remain associated with the fifth C‐terminal fragment by co‐immunoprecipitation (Joseph et al. 1995; Yoshikawa et al. 1999). Provocatively, IP₃R1 in cerebellar microsomes exposed to trypsin or caspase retained significant IP₃‐induced calcium release, although it should be noted that a small portion of intact IP₃Rs might remain in the preparation (Yoshikawa et al. 1999). These data suggest that while peptide continuity is clearly disrupted following proteolytic processing by trypsin and caspase, IP₃R1s, at least initially, retain general domain structure presumably through non‐covalent protein–protein interactions. Perhaps, most importantly, these data also provide early evidence that IP₃Rs are not necessarily simply disabled by proteolysis.

Further inconsistences with the idea that Ca²⁺ leak through cleaved IP₃Rs is a causative event in mediating apoptosis have also been reported. Joseph and colleagues reported that expression of a pore mutant IP₃R1 in DT40‐3KO cells that cannot conduct Ca²⁺ is still competent to increase the activation of caspase and augment cell death (Khan et al. 2007). These data suggest that calcium leak per se from the IP₃R1s or indeed fragmented IP₃R1s is not indispensable for IP₃R1‐dependent apoptosis. In addition, IP₃R1s may not be the initial caspase target during apoptosis (Elkoreh et al. 2012). The strong apoptotic stimulus staurosporine results in the generation of IP₃R1 fragments only after almost complete cleavage of poly ADP‐ribose polymerase, a biomarker of apoptosis, while ‘physiological’ stimuli such as Trail, tumour necrosis factor and UV lead to little IP₃R1 fragmentation even though caspase is clearly activated. This finding was confirmed in MCF‐7 cells by Boehning and colleagues (Akimzhanov et al. 2013). Furthermore, in this latter study, an increase of IP₃ production was detected in cells treated with staurosporine and scavenging of IP₃ production substantially decreased cell death. These data imply that IP₃ signalling, but not necessarily IP₃R1 fragmentation, plays a major role in apoptosis. Given the earlier data that IP₃R fragments remain associated following trypsin‐induced digestion in vitro and possibly retain some function, perhaps the major conceptual difficulty with the leaky channel theory is the assumption that the cytosolic IP₃R1 fragments immediately dissociate after caspase or calpain cleavage in vivo and thus exert no further influence over channel function.

Re‐evaluation of the functional consequences of IP₃R proteolysis: no evidence for leaky channels

Because of the somewhat contradictory interpretation of data relating to the functional consequences of proteolytic processing of IP₃R1s, we have recently revisited these issues in DT40‐3KO expressing IP₃R1 (Alzayady et al. 2013 a). First, we investigated a central assumption of the leaky channel hypothesis, namely that the ‘soluble’ N‐terminal fragment of IP₃R1 is free to dissociate from the C‐terminal channel domain after proteolytic fragmentation leaving an unregulated channel stump. Our initial experiments demonstrated that, following 12 h treatment with staurosporine, IP₃R fragmentation occurs resulting in products consistent with caspase and calpain cleavage profiles (Fig. 3 A). Strikingly, however, both N‐terminal and C‐terminal fragments remained in membrane fractions and not in the cytosolic fraction (Fig. 3 B). Further strong support for the concept that the major fragments remained associated was provided by analysis of fragmented IP₃R1s following separation on native, non‐denaturing gels. These experiments revealed that the majority of fragmented IP₃R1s, remarkably, migrate at the same molecular weight as the full‐length receptor and thus suggest it largely retains tetrameric architecture after proteolytic fragmentation during apoptosis, at least at this time point (Fig. 3 C). These data also indicate that if the C‐terminal fragment is retained in isolation in the ER membrane, it must occur at a later time point. This would necessitate a multistep process involving more than the initial cleavage event, but also disruption of the interaction between receptor fragments followed by the subsequent removal of cytosolic fragments from the complex.

Figure 3. Exposure of DT40‐3KO cells expressing rat IP₃R1 to staurosporine results in the fragmentation of the protein .

A, Western blot of protein separated by SDS‐PAGE probed with an antibody recognizing the N‐terminus of IP₃R1. In B, the N‐terminal fragment remains associated with the membrane fraction. C, Western blot of protein separated under non‐denaturing conditions demonstrates that the majority of the fragmented receptor retains tetrameric structure. Adapted from Alzayady et al. (2013 a) with permission.

To investigate the functional consequences of IP₃R1 fragmentation, we constructed dual promoter vectors encoding various combinations of complementary N‐ and C‐terminal fragments of IP₃R1 designed based on the in vivo caspase and calpain, and in vitro trypsin, cleavage products and stably expressed the fragments in DT40‐3KO cells (Alzayady et al. 2013 a). The rationale was to mimic the generation of fragmented IP₃Rs in a controlled setting with no full‐length endogenous IP₃Rs. Intriguingly, both co‐immunoprecipitation and native gel assay show that every pair of complementary IP₃R1 fragments expressed in DT40‐3KO cells associates and efficiently assembles into tetrameric IP₃R1s (Alzayady et al. 2013 a). Stable expression of either C‐terminal fragments alone or complementary N and C‐terminal fragments did not result in an elevated basal [Ca²⁺] or decrease in ER store content. Although Ca²⁺ leak was not measured directly, and thus we cannot formally exclude a minor leak, adequately compensated for by SERCA activity, these data are not consistent with significant increase of basal [Ca²⁺]_i or depletion of ER store mediated by the leak through C‐terminal fragments, at least at the expression level achieved in this study. More importantly, all complemented IP₃Rs were functionally regulated by IP₃ as stimulation of cells expressing these constructs with Gαq‐coupled protease‐activated receptor 2 (PAR2) agonist resulted in robust Ca²⁺ release (Fig. 4). These results show unequivocally, that peptide continuity per se is not necessary for IP₃‐mediated calcium release and it therefore follows that the IP₃R protein is not simply disabled by protease activity. These results are also entirely consistent with conceptually similar experiments performed on ryanodine receptors (RyRs), which share a similar general domain structure to IP₃Rs (George et al. 2004). Again, overexpression of the C‐terminal of RyR containing the channel pore resulted in a leaky channel that was unregulated by ligands. However, co‐expression of the N‐terminal portion of RyR reconstituted normal regulated Ca²⁺ release. Further experiments showed that functional IP₃R channels could also be assembled from complementary IP₃R2 and IP₃R3 fragments and even ‘chimeric’ fragments contributed by different subtypes (Alzayady et al. 2013 a). These data establish a general rule that peptide continuity is not required for gating IP₃‐mediated IP₃R activation, and in total strongly suggest that contrary to being a leaky intracellular Ca²⁺ channel, IP₃R1s are still at least minimally regulated after receptor fragmentation and are competent to gate Ca²⁺ release by IP₃ (Fig. 5).

Figure 4. Ectopic expression of IP₃R1 fragments corresponding to those produced by caspase and calpain proteolysis results in functional Ca²⁺ release channels .

In A, the Gαq‐coupled protease‐activated receptor (PAR) agonist trypsin can stimulate Ca²⁺ release from DT40‐3KO cells expressing IP₃R1, but not the parent DT40‐3KO cells. In B, DT40‐3KO cells were transfected singly, or with a combination of the C‐ terminal or N‐terminal fragments corresponding to those produced by caspase cleavage. PAR agonist stimulation resulted in robust Ca²⁺ release in cells expressing both fragments. In C, DT40‐3KO cells were transfected singly, or with a combination of the C‐terminal or N‐terminal fragments corresponding to those produced by calpain cleavage. PAR agonist stimulation resulted in robust Ca²⁺ release in cells expressing both fragments as monitored by an increase in 340/380 fluorescence ratio in cells loaded with Fura‐2. Adapted from Alzayady et al. (2013 a) with permission.

Figure 5. Schematic diagram depicting our working hypothesis based on data in Figs 3 and 4 .

Following proteolytic cleavage of IP₃R1, the N and C terminal fragments remain associated through non‐covalent interactions, possibly prominently involving the N‐ and C‐termini. The complemented receptor is not disabled, and can be gated by IP₃ following agonist stimulation. We speculate that disruption of peptide continuity may, however, impact the spatio‐temporal characteristics of the Ca²⁺ signal produced by the cleaved protein.

Proteolytic fragmentation of IP₃R: a novel mode of regulation?

Peptide continuity and thus communication of information through the entire length of the IP₃R1 peptide chain is clearly not required for IP₃ gating of IP₃R1. In hindsight, perhaps this is not totally unexpected in light of data that suggest that the conformational change on binding of IP₃ occurring in the N‐terminal binding core is communicated to opening of the channel pore in the C‐terminus by a direct physical interaction between the two distant domains (Boehning & Joseph, 2000; Bhanumathy et al. 2012). Nevertheless, although IP₃ binding is obviously necessary for Ca²⁺ release, the overall spatiotemporal characteristics of the Ca²⁺ signal are dictated by numerous regulatory inputs to the receptor. The structural and molecular basis underlying regulatory input by the majority of other important modulators, such as Ca²⁺, ATP, phosphorylation and binding partners is much less well defined and may indeed rely on short‐distance allosteric modulation of the channel activity that is dependent on peptide continuity. A particular form of regulation might depend on modulation of IP₃ binding or perhaps direct regulation of gating or channel activity – in this scenario the regulatory motif would probably reside in the same fragment as the target of its action. This leads us to speculate that proteolytic fragmentation may constitute a form of IP₃R1 regulation in itself. Theoretically, this might occur as IP₃R fragmentation results in an alteration (either loss or gain) of a particular set of modulatory inputs regulating channel activity. This change in receptor properties, might in turn be manifested as a distinct Ca²⁺ release profile, transformed in terms of peak amplitude, duration of signal or frequency of oscillations (Fig. 5). An added level of regulation might occur as the degree of proteolysis is tuned by other cellular input. Of interest, recent evidence suggests that association of Bok, a pro‐apoptotic Bcl‐2 family member interacts with IP₃R1 and protects the channel from caspase‐3‐mediated cleavage (Schulman et al. 2013). If only a subset of IP₃Rs were subject to proteolytic processing at a particular site, it could be envisioned that any alterations in properties might even occur in a local subcellular region. Given that it is firmly established that particular spatiotemporal Ca²⁺ release profiles determine the activation of specific downstream pathways, IP₃R1 fragmentation may constitute a novel cellular switch resulting ultimately in signalling to different Ca²⁺‐dependent effectors and entraining distinct cellular outcomes.

Consistent with this idea, cleavage of an increasing number of proteins by caspase and calpain has been shown to alter, rather than abolish, activity. Examples of substrates where activity is altered include the nuclease CAD (Larsen et al. 2010), interleukins (Kuranaga & Miura, 2007) and calcineurin (Mukerjee et al. 2000). Thus, the idea of regulation by proteolysis is an emerging theme (Balcerzak et al. 1995; Sordet et al. 2002; Neumar et al. 2003; Fernando et al. 2005; Connolly et al. 2014; Buffolo et al. 2015). Much work is needed to firmly establish this concept and ongoing work in our laboratory is focusing on determining how IP₃R fragmentation alters both the overall spatiotemporal characteristics of the Ca²⁺ signal and the biophysical properties of the IP₃R at the single channel level. We envision that this information may provide clues as to how the distinct downstream effectors are specifically activated by fragmented IP₃R1s. Since, IP₃R2 and IP₃R3 are also subject to proteolytic cleavage, it will also be important to determine if the activity of these subtypes is altered by cleavage (Wojcikiewicz et al. 1999; Diaz & Bourguignon, 2000).

Future perspective: not just cell death. Are fragmented IP₃R1s involved in physiological activities of caspase and calpain?

In addition to a potential general role in apoptosis, an exciting possibility is that proteolytic fragmentation of IP₃R1s by caspase and calpain may, in fact, be involved in many other physiological events. In recent years, growing evidence suggests that the activity of these two cysteine proteases may also be important for numerous cell events other than cell death (Balcerzak et al. 1995; Sorimachi et al. 1997; Sordet et al. 2002; Neumar et al. 2003; Fernando et al. 2005; Kuranaga & Miura, 2007; Larsen et al. 2010; Zadran et al. 2010; Connolly et al. 2014; Buffolo et al. 2015). Indeed, protease activity has been implicated in adaptive events as diverse as cytoskeletal remodelling (Helfer et al. 2006), synaptic plasticity (Zadran et al. 2010), cell migration (Geisbrecht & Montell, 2004), maturation and differentiation (Sordet et al. 2002; Fernando et al. 2005; Kuranaga & Miura, 2007). In these cases, low level and often local protease activity is necessary, but the substrates of the proteases and therefore the underlying mechanisms are not well defined. Because of the slow turnover of IP₃Rs, fragmentation may provide an attractive means of conferring a long‐term modification of the activity of IP₃Rs suitable for initiating an adaptive response. Therefore, the possible occurrence and potential functional consequences of fragmented IP₃Rs in the aforementioned adaptive events are worthy of further investigation.

Neural development may be an attractive initial model to investigate this general concept. The broad importance of each protein in development is indicated by the knockout of either IP₃R1 or caspase in mice, which results in perinatal lethality with severe neurodevelopmental abnormalities (Matsumoto et al. 1996; Matsumoto & Nagata, 1999). Ca²⁺ release through IP₃Rs is reported to be important for neurite sprouting and arborization (Takei et al. 1998; Fiedler & Nathanson, 2011). Although the substrate(s) are not defined, caspase activity is significantly increased during differentiation of neurospheres and pharmacological inhibition alters the expression pattern of neuro‐specific genes resulting in a reduction in neurite extension (Fernando et al. 2005). Moreover, similar results were obtained in a model of PC‐12 cell differentiation, suggesting a general physiological role of caspase in neural differentiation (Vaisid et al. 2005). In summary, there is clear evidence suggesting the activities of caspase and IP₃R1 are both essential for neural development. It will be exciting to investigate whether fragmented IP₃R1s appear and contribute to these events.

Skeletal muscle myoblast differentiation may be another process where IP₃R1 fragmentation may occur and potentially play a role. Elevated caspase and calpain activity have been shown to be important for the transformation of myoblasts to myotubes, highlighting a non‐apoptotic role of the proteases in this process (Balcerzak et al. 1995; Larsen et al. 2010). While the expression and function of IP₃Rs in differentiated myotubes are still debated (Moschella et al. 1995; Powell et al. 2001; Tjondrokoesoemo et al. 2013), there is consistent evidence showing that all three isoforms of IP₃R are expressed in the myoblast (Arnaudeau et al. 2006; Blaauw et al. 2012; Antigny et al. 2014). Of note, during myoblast differentiation, IP₃R1 expression level is increased, and this corresponds with cells exhibiting spontaneous oscillatory calcium signals (Antigny et al. 2014). In addition, evidence from knockdown and overexpression strategies indicates the degree of myoblast differentiation is directly related to the expression level of IP₃R1. This strongly suggests that IP₃R1s play an essential role in myoblast differentiation. In total, myoblast differentiation provides a physiological platform for a potential cross talk among caspase, calpain and IP₃R1. Nevertheless, in common with the case of neural development, establishing a causal link will require demonstrating that IP₃Rs are an important substrate of caspase and/or calpain, and its proteolytic fragmentation is necessary for particular step(s) in myoblast differentiation.

Concluding remarks

IP₃R1 fragmentation may be a relatively common event, underlying many other physiological events including differentiation of macrophages, osteoblasts and stem cells (Sordet et al. 2002; Mogi & Togari, 2003; Kang et al. 2004; Fujita et al. 2008; Connolly et al. 2014). All these events have common features, including expression of IP₃R1 and non‐cell‐death‐related caspase and calpain activities. In summary, accumulating evidence suggests that proteolytic fragmentation by caspase and calpain may be a novel regulatory mechanism for IP₃R1. Evidence from the literature warrants further studies to investigate the potential role of IP₃R1 fragmentation in both physiological and pathological conditions.

Additional information

Competing interests

None declared.

Funding

This work was supported by NIH (National Institute of Dental and Craniofacial Research) grants DE19245 and DE14756.

Biography

Liwei Wang (left) is a senior graduate student and Kamil Alzayady (right) a Research Assistant Professor working in the Department of Pharmacology and Physiology at The University of Rochester with David Yule (PhD). The lab is interested in how a ubiquitous message, changes in intracellular [Ca²⁺], activate diverse physiological processes with specificity. A central tenet of our work is that fine tuning of Ca²⁺ release through inositol 1,4,5‐trisphosphate receptors plays major roles in shaping the spatio‐temporal dynamics of Ca²⁺ signals which in turn is pivotal for the fidelity of the signal.