In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-A crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.
The inositol 1,4,5-trisphosphate (IP 3 ) 3 receptors (IP 3 Rs) function as IP 3 -gated Ca 2ϩ release channels located on intracellular Ca 2ϩ stores, such as the endoplasmic reticulum (1). Mammalian IP 3 R family consists of three isoforms (IP 3 R1, IP 3 R2, and IP 3 R3), and they form homotetrameric or heterotetrameric channels (2). There is evidence of a functional difference among the three isoforms of IP 3 R in terms of their IP 3 sensitivity (3-5) and cooperativity with respect to IP 3 binding (5). The intrinsic association constants of mouse IP 3 R1, IP 3 R2, and IP 3 R3 expressed in Sf9 cells are estimated to be 3.5 ϫ 10 7 , 1.7 ϫ 10 8 , and 3.4 ϫ 10 6 (M Ϫ1 ), respectively (5). IP 3 R2 exhibits both negative and positive cooperativity, whereas IP 3 R3 exhibits negative IP 3 binding cooperativity (5). This diversity of responsiveness to IP 3 observed among the three IP 3 R isoforms may contribute to the generation of the different degree of IP 3 sensitivity of the Ca 2ϩ store. The molecular basis of the isoform-specific IP 3 -binding affinity, however, is not well understood.The IP 3 -binding domain of IP 3 R1 is composed of two functional domains, the amino-terminal suppressor domain and the carboxyl-terminal IP 3 -binding core domain (6). The IP 3 -binding core domain is the minimum region required for specific IP 3 binding and is mapped within residues 226 -578 of mouse IP 3 R1, a polypeptide of 2749 residues (6). The amino-terminal 225 amino acid residues of IP 3 R1 function as the suppressor for IP 3 binding, and deletion of these residues results in significant enhancement of IP 3 binding (6). The atomic resolution structures of both the suppressor domain (7) and the IP 3 -binding core domain (8) of mouse IP 3 R1 were solved by x-ray crystallography to 1.8-and 2.2-Å resolution, respectively. The IP 3 -binding core domain comprises two asymmetric domains, the -domain and ␣-domain. A highly positive-charged pocket is created at the interface of these two domains to which an IP 3 molecule binds. The 11 amino acid residues in the IP 3 -binding core domain of IP 3 R1 are responsible for the coordination of IP 3 (8), and all of them except Gly-268 are conserved in other isoforms. The suppressor domain contains a -trefoil fold and a helix-turn-helix structure inserted between two -strands of the -trefoil fold (7). The conserved 7 amino acid residues, which are clustered on one side of the suppressor domain, were found to be critical for the suppression of IP 3 binding (7). These structural and functional analyses of the IP 3 -binding domain have been carried out mainly using the type-1 isoform, and the suppression ability of the amino-terminal regions of IP 3 R2 and IP 3 R3 has not been well characterized.
Inositol 1,4,5-trisphosphate (IP 3 ) 1 is a second messenger generated by the phosphatidylinositol signaling cascade response to hormones, neurotransmitters, and growth factors (1) and causes Ca 2ϩ release from intracellular stores by binding to the IP 3 receptor (IP 3 R), which is an IP 3 -gated intracellular Ca 2ϩ release channel. Ca 2ϩ release in the cytoplasm occurs in complex spatial and temporal patterns, such as Ca 2ϩ waves and Ca 2ϩ oscillations, and regulates many cellular responses, including fertilization, muscle contraction, secretion, cell growth, differentiation, apoptosis, and synaptic plasticity (1).The IP 3 R family consists of three isoforms (IP 3 R1, IP 3 R2, and IP 3 R3) (2), and the primary structure of all three IP 3 R types has been determined in rat (3-5) and human (6 -9). Only a single type of IP 3 R was identified in frog (10), fly (11), starfish (12), lobster (13), and nematode (14). Three isoforms have been found to exist in the mouse (15, 16), but the primary structure of mouse IP 3 R2 and IP 3 R3 has not yet been determined.IP 3 -gated Ca 2ϩ release channels are composed of four subunits (17). The expression levels of the three IP 3 R isoforms are different in each tissue, but most tissues contain multiple isoforms (18). Heterotetrameric channels were detected as well as homotetrameric channels (19), indicating that the structural diversity of the IP 3 -gated channels is greater than the number of genes. There is evidence of functional differences among the three types of IP 3 R in terms of their IP 3 sensitivity (20, 21) and the modulatory effects on them from cytoplasmic Ca 2ϩ (22), ATP (22), calmodulin (23), and cAMP-dependent protein kinase (24). Additional diversity of IP 3 -gated channels is produced by alternative splicing, and alternative splicing segments, designated SI, SII, and SIII, have been found in the mouse (25), rat (26), and human (9) IP 3 R1. These findings suggest that the channels composed of different isoforms possess distinctive functions, but little is known about the physiological significance and exact functional differences arising from the heterotetrameric assembly of IP 3 R.In many cells, the Ca 2ϩ increase triggered by IP 3 is propagated throughout the cytoplasm; however, the diffusion of free Ca 2ϩ is spatially restricted because many immobile or slowly diffusing Ca 2ϩ -binding proteins are present in the cytoplasm (27). Propagation of Ca 2ϩ is thought to be mediated by regenerative Ca 2ϩ release through IP 3 Rs, which are regulated by cytoplasmic Ca 2ϩ (28). Positive feedback regulation of IP 3 R by Ca 2ϩ enables the Ca 2ϩ released by one receptor to excite its
These results are consistent with the results of our previous clinical studies. It is thus suggested that in-vivo disposition may be predicted from in-vitro results using recombinant transporters.
ATP enhances Ca2؉ release from inositol (1,4,5)-trisphosphate receptors (InsP 3 R). However, the three isoforms of InsP 3 R are reported to respond to ATP with differing sensitivities. Ca 2؉ release through InsP 3 R1 is positively regulated at lower ATP concentrations than InsP 3 R3, and InsP 3 R2 has been reported to be insensitive to ATP modulation. We have reexamined these differences by studying the effects of ATP on InsP 3 R2 and InsP 3 R3 expressed in isolation on a null background in DT40 InsP 3 R knockout cells. We report that the Ca 2؉ -releasing activity as well as the single channel open probability of InsP 3 R2 was enhanced by ATP, but only at submaximal InsP 3 levels. Further, InsP 3 R2 was more sensitive to ATP modulation than InsP 3 R3 under similar experimental conditions. Mutations in the ATPB sites of InsP 3 R2 and InsP 3 R3 were generated, and the functional consequences of these mutations were tested. Surprisingly, mutation of the ATPB site in InsP 3 R3 had no effect on ATP modulation, suggesting an additional locus for the effects of ATP on this isoform. In contrast, ablation of the ATPB site of InsP 3 R2 eliminated the enhancing effects of ATP. Furthermore, this mutation had profound effects on the patterns of intracellular calcium signals, providing evidence for the physiological significance of ATP binding to InsP 3 R2.
3 -induced conformational change to the open state. We also found that GFP-IP 3 R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligandinduced clustering of a ligand-gated channel protein.Many extracellular stimuli are received by trimeric G protein-or tyrosine kinase-coupled receptors and relayed to signaling molecules including phospholipase C (PLC), 1 which leads to production of a second messenger, inositol 1,4,5-trisphosphate (IP 3 ) (1). IP 3 in turn activates an internal membrane receptor/Ca 2ϩ channel, the IP 3 receptor (IP 3 R), and induces Ca 2ϩ release from internal Ca 2ϩ stores (mostly endoplasmic reticulum (ER)). This IP 3 -Ca 2ϩ pathway is particularly important because it is located at the intersection of many signaling pathways and is therefore thought to play a central role in cellular signaling.Rather recently, evidence has been accumulating that the subcellular localization of IP 3 R is tightly controlled and changes according to circumstances (2-4). In addition, IP 3 R is known to form large clusters on the ER (5). Very importantly, theoretical modeling (6 -8) predicted that IP 3 R clustering affects both elementary events (e.g. Ca 2ϩ blips and puffs) and the formation of complex cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] C ) patterns (e.g. Ca 2ϩ oscillations and waves). Particularly Shuai and Jung (6) predicted that if IP 3 concentration is relatively low (i.e. physiological situation), there is an optimal extent of clustering of IP 3 receptor for effective elevation of cytosolic calcium concentration. When the clusters of IP 3 receptor would become very large, the whole calcium signals elicited by low IP 3 concentrations would be significantly reduced. Experimental verification of this hypothesis has not been performed mainly because the molecular mechanism of IP 3 R clustering remains totally unknown, and thus it is impossible to inhibit or artificially modulate it. Wilson et al. (5) suggested that IP 3 R clustering was triggered by increased [Ca 2ϩ ] C since not only Ca 2ϩ -mobilizing agonists but a Ca 2ϩ ionophore (ionomycin) and an inhibitor of sarco/ endoplasmic Ca 2ϩ -ATPase (thapsigargin) induced it. They also showed that it is independent of ER vesiculation (9). Later it was shown that two small GTP-binding proteins, Cdc42 and Rac, are involved in agonist-induced IP 3 R clustering but that was because they augmented Ca 2ϩ signals as a whole and not because they influenced intracellular trafficking (10). In short, the detailed molecular mechanism of IP 3 R clustering remains largely unknown, and it is important to know it to understand precise roles of IP 3 R clustering. In this study we simultaneously imaged the dynamics of enhanced green fluorescent protein (GFP)-tagged IP 3 R type 1 (GFP-IP 3 R1) and [Ca 2ϩ ] C by using fluorescent Ca 2ϩ indicators. Based on the results of the mutational and pharmacological analyses, we concluded that IP 3 R clustering is initiated by a conformational c...
The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca(2+) channel protein that plays crucial roles in generating complex Ca(2+) signalling patterns. IP(3)R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells' responsiveness to an IP(3)-generating agonist ATP and Ca(2+) leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca(2+) concentration. Interestingly, store-operated (-like) Ca(2+) entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca(2+) stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca(2+). We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca(2+) concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca(2+) stores.
Our findings suggest that aberrant CADM1 and 4.1B expression is involved in progression of breast cancer, especially in invasion into the stroma and metastasis.
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