Molecules: 1,4,5-trisphosphate receptors (InsP3R) Rahul Chandrasekhar David I. Yule and Liwei Wang Department of Pharmacology and Physiology, University of Rochester. Rochester NY 14642 [email protected] Version 2.0, Octover 19, 2017 [DOI: 10.3998/panc.2017.07] Symbols: ITPR1, ITPR2, ITPR3 respectively) that share 60-70% sequence

Other Names: IP3R1, IP3R2, IP3R3 homology (47).

Originally purified and cloned from rat , 1. General: The role of InsP3R in the full length InsP3R forms a tetrameric cation pancreatic acinar and other cells selective channel in vivo (40). The three isoforms of InsP R exhibit overlapping patterns of The classical pathway linking stimulation by 3 expression, with most cells and tissues neurotransmitters and to changes in expressing more than one isoform (63). Western phosphoinositide and the subsequent blotting and quantitative PCR has revealed that InsP induced Ca2+ release was in large part 3 there is relatively equal expression of the R2 and documented by work carried out in exocrine cells, R3 isoforms in , with R1 only constituting in particular, acinar cells isolated from the ~3% of the total InsP R (63). The importance of exocrine pancreas (62). In seminal work using 3 R2 and R3 isoforms to pancreatic Ca2+ signaling permeabilized rat pancreatic acini, Streb and is most evident in studies employing InsP R colleagues demonstrated that the addition of 3 2+ InsP3 resulted in Ca release from a non- mitochondrial Ca2+ store (54). This store was later identified to be the (ER) through experiments where sub-cellular fractions from pancreatic cells were isolated and exposed 2+ to InsP3 (6, 53). Ca release is rapid, occurring as quickly as 1s following secretagogue stimulation of acinar cells. Ca2+ release is also isomer specific, with other structurally related inositol shown to be far less efficacious in 2+ releasing Ca (54). The target for InsP3 binding was later identified as the inositol 1,4,5- Figure 1: Localization of InsP3Rs in pancreatic acinar trisphosphate (InsP3R) (56). Strikingly, cells. Immunofluorescence localizations in pancreatic the secretion of digestive enzymes from lobules of InsP3Rs by confocal microscopy demonstrate pancreatic acinar cells has been shown to be the localization of InsP3R1, R2, R3 and amylase entirely dependent on the activation of InsP3Rs (A,B,C,D respectively) respectively. All 3 isoforms are and the resulting elevation in intracellular [Ca2+] predominantly localized to the apical pole of acinar cells (22). The receptor is encoded by 3 distinct directly abutting the plasma membrane (arrows in in mammalian cells (ITPR1, ITPR2, ITPR3) that panels A-C). R1 and R3 also appeared to localize to generate 3 monomeric isoforms (R1, R2, R3 perinuclear structures. (Scale bar = 10 μm). (69)

This work is subject to an International Creative Commons Attribution 4.0 license. knockout (KO) mice (22). Although single R2 or polarized, both functionally and morphologically

R3 KO mice were found to have no observable ill and InsP3Rs are predominantly expressed in ER effects or phenotypic alterations, double R2/R3 extensions in the apical regions juxtaposed to the KO mice typically gained less body weight post- acinar lumen (Figure 1) (34, 36, 42, 69). Using natally compared to single KO or WT mice. fluorescence imaging techniques, studies have

Furthermore, double KO mice tended to lose shown that stimulating InsP3 generation in these weight rapidly post weaning and typically died 4 cells results in the initiation of Ca2+ signals in the weeks after birth. These observations were apical regions, followed by the propogation attributed to the key role the R2 and R3 isoforms basally of a Ca2+ wave that is facilitated by

play in the secretion of saliva and exocytosis of peripheral InsP3Rs. Stimulation of basal

digestive enzymes from exocrine glands and the secretagogue receptors as well as uncaging InsP3 resulting inability of double KO mice to swallow or in various regions of the also confirm that 2+

properly digest dry adult food. Indeed, dou ble KO Ca signals initiate in apical regions of the mice were rescued and reached body weights pancreatic acinar cell (5, 20, 31, 58, 65). comparable to WT when a fed pre-digested wet

mash diet or an elemental diet. Pancreatic acinar 2. Structural features of the InsP3R cells from R2/R3 double KO mice were also found to accumulate zymogen granules and failed to Based on the , all three

secrete zymogen granules on stimulation with isoforms of InsP3R are predicted to have the secretagogues. The study also highlights the same general structural domains (12).

inability of R1 to rescue double R2/R3 KO mice Specifically, the InsP3R monomer is (22). conventionally divided into 3 functional domains: an N-terminal ligand binding domain (LBD), a The fidelity and specificity of the Ca2+ signal coupling and modulatory domain and a C-terminal required for exocytosis is thought to be largely transmembrane domain (TMD) that contains the determined by the differential expression, channel (21) (Figure 2A). To date, the majority of localization and modulation exhibited by the 3 structural insights were from the investigations of

InsP3R isoforms (21). For the most part , InsP3Rs R1, including the recent structures of R1 revealed are predominantly localized to the ER, although by cryo-EM (Figure 2B and 2C) and x-ray

the golgi, nucleus, plasma membrane, crystallography (26). Therefore, in this review, the peroxisomes and endolysosomal vesicles have locations of common functional modules in

also been reported to express small levels of InsP3R, which are also present in R2 and R3 at

InsP3Rs. Pancreatic acinar cells are highly homologous regions, are described using the

Figure 2: Illustration of R1 depicting location of suppressor domain, ligand binding core, regulation and coupling domain and channel domain (A). Structure of R1 revealed by Cryo-electron microscopy at near atomic resolution of 4.7 A (B and C). (B) is a side view along membrane plane of a R1 monomer. (C) is a tetrameric R1 viewed from cytosol. Each monomer of the tetramer are color-coded. Figures are adapted from (19) 2

sequence of R1. InsP3 binding is increased InsP3 binding affinity, deletion of the SD mediated by the ‘core’ ligand binding domain, also results in the loss of channel activity, which constitutes amino acids (AA) 224-578 of the indicating that the SD is required for inducing 2+ LBD. This region contains 10 conserved positively InsP3R activation and Ca release. The pivotal

charged and residues (3 critical, role of SD for InsP3R activation was also strongly R265, K508, R511) that are thought to supported by Cryo-EM structure of allosterically coordinate the negatively charged homotetrameric R1 solved at near-atomic 3- PO4 groups of InsP3 in a binding pocket (67). By resolution (19). The Serysheva group showed that using concatenated subunits (1, 18), our group the SD domain forms inter-subunit interactions

has recently reported that InsP3 molecules must with ARM2, ARM3, β-TF2 and CTD domains of be bound to each monomer of the four subunits to adjacent subunits, which may underlie a direct

open the channel and activate InsP3R (2). This functional coupling between the N- and C-

gating mechanism ensures the fidelity of InsP3 terminus of the receptor (19). Based on induced release and may provide hydropathy plots, the TMD is similar in structure to mechanistic insight into diseases where mutations that of RyRs and voltage gated K+, Na+ and Ca2+

have an impact on InsP3 binding to the receptor. channels and constitutes 6 putative

The three isoforms have differing InsP3 binding transmembrane regions (TM1-6) (16). The TMD is affinities that are regulated by the first 223 AA of responsible for the ER targeting (44, 46) and for

the LBD, termed the suppressor domain (SD). the oligomerization of the InsP3R into tetramers,

Specifically, competitive InsP3 binding assays which occurs co-translationally (30). Lastly, TM5 using GST fusion constructs encoding AA 1-604 and 6 forms the pore through which Ca2+ is demonstrate that R2 has an 11-fold greater conducted (48). The loop between TM5 and 6

affinity (Kd) for InsP3 compared to R3 (14 nM vs. contains a selectivity filter (GVGD; similar to the 163 nM), with R1 having an intermediate affinity super family of cation selective channels) that (49.5 nM) (29). Similar studies in pancreatic acini provides some degree of cation selectivity to the 2+ revealed a Kd of 6 nM (24), comparable to that of InsP3R (21). However, it is poorly Ca selective R2, while permeabilized Ca2+ release assays in and allows conduction of monovalent cations 2+ + acinar cells demonstrated an EC50 for InsP3 of 0.8 (Ca :K = 6:1). In fact, it is believed that 2+ µM (45) (Figure 3).Deletion of the suppressor “functional” Ca selectivity of InsP3R is primarily

domain (SD) results in the loss of distinct InsP3 determined by virtue of SERCA being a highly affinities between the isoforms and a 10-100 fold selective Ca2+ pump and Ca2+ being by the far the

increase in InsP3 affinity (29). Despite an most abundant cation in the ER. To “gate” and

Figure 3: (A) Permeabilized cell Ca2+ release assay in acinar cells. Isolated pancreatic acini were loaded with furaptra prior to permeabilization with β-escin. ER is loaded through SERCA upon application of Ca2+ containing buffer containing MgCl2, and ATP, as indicated by the increase in fluorescence ratio. Removal of MgCl2 deactivates

SERCA and (B) addition of varying [InsP3] releases from stores as indicated by the decrease in fluorescence ratio.

(C) Concentration response analysis for InsP3. EC50 for InsP3 is 0.8 µM (10) 3 open the channel, . The CT tail (last 160 AA) and irrespective of [InsP3], implying that ATP is the large (1700 AA) but less conserved required for maximal activity of R1 and R3 (8, 9). modulatory domain contains putative binding sites Each isoform also differs in its affinity for ATP, for the numerous modulators of InsP3R activity with R2 having 3 fold and 10 fold higher affinities (68). These modulators, which include Ca2+, ATP than R1 and R3, respectively (8). ATP binding and PKA, all contribute in distinct ways to the was originally believed to occur at Walker A like differential Ca2+ release profiles encoded by the 3 motifs (G-X-G-X-X-G) that exist in each isoform. isoforms. One such motif, called ATPB, is conserved across all 3 isoforms. Additionally, R1 contains 2 other sites: ATPA and ATPC, the latter only being found 3. Modulation of the InsP3R in the S2- variant which is expressed in peripheral Cytosolic Ca2+ is the most important regulator of tissues (59). Recently, mutagenesis studies have

InsP3Rs, modulating activity in a biphasic manner demonstrated that only the ATPB site in R2 is (14). Numerous putative Ca2+ binding sites have important in mediating the modulatory effects of been identified, and Ca2+ has been shown to ATP (45) and this regulation is important for induce dramatic conformational changes in R1 (3, defining the sensitivity of Ca2+ release in 4, 25, 49, 50). For the most part, studies have pancreatic acinar cells (24). In contrast, the ATPB shown that in the presence of InsP3, low to sites in R1 and R3, in addition to the ATPA and optimal [Ca2+] (300 nM) stimulates channel activity ATPC sites in R1, play no role in modulating 2+ while higher cytosolic [Ca ] inhibits it (14). To InsP3R activity through ATP. explain this biphasic regulation, Foskett and collaborators have proposed a model in which Further, another level of receptor regulation 2+ InsP3R activity is regulated by two distinct Ca results from the subunit composition of InsP3R. binding sites: a stimulatory and an inhibitory site. Most cells express more than one isoform of

Under resting conditions, the inhibitory site has a InsP3R; as a result, different isoforms of InsP3R higher affinity for Ca2+ than the stimulatory site. display overlapping expression patterns (63). Accordingly, this site is occupied at resting Extensive studies with various experimental conditions and inhibits InsP3R activity. The techniques, including cross-linking (15, 43), binding of InsP3 decreases the inhibitory sites’ immunostaining (27, 33, 34, 69), and co- affinity for Ca2+, thereby allowing Ca2+ to bind to immunoprecipitation (1, 64), have consistently the stimulatory site and positively regulating demonstrated that InsP3R can be hetero- channel activity. The downstroke of the biphasic tetrameric channels formed from at least two 2+ curve is the result of Ca binding to the inhibitory isoforms of InsP3R. An obvious question is what is sites due to cytosolic [Ca2+] being elevated the contribution of each subunit in a hetero- beyond 300 nM (57). In effect, they suggest that tetramer? Our lab showed that an individual 2+ Ca is only an essentially InsP3R co-agonist, with subtype can either contribute equally, or dictate 2+ 2+ InsP3s sole role being simply to modulate Ca the Ca release properties of the hetero- sensitivities (37). tetrameric InsP3R, depending on the presence and the concentrations of key regulators (ATP Cytosolic ATP has also been shown to and Ca2+) (17). In the context of the pancreas, R2 differentially modulate the activity of all 3 isoforms and R3 are the predominant isoforms and form in an allosteric manner. This mode of regulation is hetero-tetramers accounting for the major thought to link the metabolic status of the cell to proportion of the InsP3R (63). A hetero-tetramer the Ca2+ release. Specifically, studies show that containing two R2 and two R3 is predicted to R2 is only modulated by ATP at sub-maximal exhibit blended channel activities under optimal

[InsP3] while R1 and R3 activity is affected conditions; in contrast, R2 in the same hetero-

4 tetramer can dictate the channel activities under A multitude of commercially available isoform suboptimal conditions (17). specific antibodies can be utilized to probe for

each isoform of the InsP3R. Additionally, there are Region-specific proteolysis is a novel form of antibodies that detect all 3 isoforms regulation for all three isoforms of InsP3R. R1 has  InsP3R1 Rabbit polyclonal (H-80) from Santa been well demonstrated to be a substrate for the Cruz (sc-28614); epitope corresponding to AA intracellular cysteine , caspase and 1894-1973 in the cytosolic modulatory domain (28, 32). However, the functional  InsP3R1, UC Davis/NIH NeuroMab Facility, consequences of receptor fragmentation are a clone L24/18; epitope against AA 2680-2749 subject of considerable debate. Recent evidence in C-terminus strongly suggests that region-specific proteolysis  InsP3R3 Goat polyclonal (C-20) from Santa modifies R1 channel regulation (51, 60, 61). Cruz (sc-7277); epitope corresponding to C- Specifically, stimulation of caspase or calpain terminus 2+ fragmented R1 results in Ca signals with distinct  InsP3R3 purified mouse monoclonal from BD temporal characteristics and can activate Transduction Laboratories (Cat: 610313) alternative signaling pathways (51, 61). Further  InsP3R1/2/3 Rabbit polyclonal (H-300) from studies are necessary to guage if R2 and R3 are Santa Cruz (sc-28613); epitope corresponding subject to similar regulation. to AA 2402-2701 in C-terminus of InsP3R-2 (human) Lastly, protein A (PKA) mediated phosphorylation has been shown to directly B. cDNA 2+ increase InsP3R Ca flux and single channel Accession number: activity of R1 and R2 albeit by phosphorylation at InsP3R1: rat, 55925609 different residues. Specifically, studies show that InsP3R2: mouse, 60593031 PKA mediated phosphorylation at S1598 and InsP3R3: rat, 6981109 S1755 on R1 and S937 on R2 significantly enhance Ca2+ release and single channel activity C. Cell Lines 2+ (7, 11, 68). Conversely, no effects on Ca release SHSYFY Neuroblastoma: 99% InsP3R1 (63) through R3 have been observed after PKA AR42J Rat pancreatoma: 86% InsP3R2; 12% stimulation, despite evidence that R3 is InsP3R1; 2% InsP3R3 (63) phosphorylated by PKA at 3 sites in vivo (52, 68). RINM5F Mouse insulinoma: 96% InsP3R3; 4% Thus far, no single channel studies have been InsP3R1(63) performed on R3 to rule out any PKA mediated effects on channel activity. DT40 triple knockout cell lines: Kurosaki and colleagues have generated a DT40 chicken B

InsP3Rs are also regulated and bound by lymphocyte cell line with the endogenous numerous other and accessory , InsP3Rs stably knocked out (55). These triple including cGMP protein kinase (PKG), Akt kinase, knockout cells can subsequently be stably FK506 binding protein, , CaBP, IRBIT, transfected with constructs encoding Bcl-2/XL, cytochrome C, RACK and Erp44 (21). individual mammalian InsP3R isoforms, allowing the study of activity and regulation of 4, Tools available to study the each isoform in isolation. These cell lines are available on request from our laboratory. InsP3R HEK triple knockout cell line: David Yule and A. Antibodies colleagues have generated HEK triple 5

knockout cell line with the endogenous Agonists include , trypsin and

InsP3Rs stably knockout (2). In addition to cholecystokinin. DT40 triple knockout cell line, HEK triple knockout cell line provides an alternative Antagonists

mammalian InsP3R null background system Heparin (66); Caffeine (13); 2- for research. Aminoethoxydiphenyl borate (2-APB) (38, 41); Xestospongin (23) D. Mouse Lines

InsP3R-1 knockout mice (39) F. Techniques used to study InsP3R function

InsP3R-2 (35) and R-3 single and double See review by Betzenhauser MJ., Wagner knockout mice (22) LE., Won JW and Yule DI. (2008) Studying isoform-specific inositol 1,4,5-trisphosphate E. Agonists/Antagonists/ receptor function and regulation. Methods 46, Agonists pp: 177-182. (10)

Commercially available InsP3: D-myo-inositol Betzenhauser, Matthew J., Won, Jong Hak, 1,4,5-trisphoshate hexapotassium salt; Enzo Park, Hyungseo and Yule, David I. (2011). Life Sciences (cat: ALX-307-00) Measurement of Intracellular Calcium Cells are often stimulated with Gq coupled Concentration in Pancreatic Acini. The

GPCR agonists to stimulate InsP3 production. Pancreapedia: Exocrine Pancreas Knowledge Base, DOI:10.3998/panc.2011.34

5. References

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