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Keywords:

  • channel;
  • endoplasmic reticulum;
  • IP3;
  • IP3 receptor;
  • IRBIT

Abstract

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Inositol 1,4,5-trisphosphate (IP3) is a second messenger that induces the release of Ca2+ from the endoplasmic reticulum (ER). The IP3 receptor (IP3R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP3R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP3-binding core and N-terminal suppressor sequence of IP3R have been identified. An IP3 indicator (known as IP3R-based IP3 sensor) was developed from the IP3-binding core. The IP3-binding core’s affinity to IP3 is very similar among the three isoforms of IP3R; instead, the N-terminal IP3 binding suppressor region is responsible for isoform-specific IP3-binding affinity tuning. Various pathways for the trafficking of IP3R have been identified; for example, the ER forms a meshwork upon which IP3R moves by lateral diffusion, and vesicular ER subcompartments containing IP3R move rapidly along microtubles using a kinesin motor. Furthermore, IP3R mRNA within mRNA granules also moves along microtubules. IP3Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP3R1 activity. IP3 has been found to release Ca2+, but it also releases IRBIT (IP3R-binding protein released with IP3). IRBIT is a pseudo-ligand for IP3 that regulates the frequency and amplitude of Ca2+ oscillations through IP3R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP3Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP3R1, as revealed by cryoelectron microscopy, fits closely with these molecules.

Abbreviations used
AFM

atomic force microscopy

BDNF

brain-derived neurotrophic factor

CAPS

Ca2+-dependent activator protein for secretion

CARP

carbonic anhydrase-related protein

CCh

carbachol

CTT14aa

C-terminal cytoplasmic tail 14 amino acids

DAG

diacylglycerol

EM

electron microscopy

ER

endoplasmic reticulum

GFP

green fluorescent protein

HAP1A

huntingtin-associated protein-1A

Htt

huntingtin

IICR

IP3-induced Ca2+ release

IP3

Inositol 1,4,5-trisphosphate

IP3R

IP3 receptor

IRBIT

IP3R-binding protein released with inositol 1,4,5-trisphosphate

IRIS

IP3R-based IP3 sensor

kNBC1

kidney-type NBC1

KO

knockout

mGluR

metabotropic glutamate receptor

NBC1

Na+/HCO3− co-transporter 1

OAG

1-oleoyl-2-acyl-sn-glycerol

PKA

protein kinase A

PKC

protein kinase C

PLC

phospholipase C

pNBC1

pancreas-type NBC1

PolyQ

polyglutamine

PP

protein phosphatase

RyR

ryanodine receptor

Calcium is a metal ion that by itself is toxic to cells at high concentrations. Although the extracellular concentration of Ca2+ is on the order of 10−3 mol/L, the intracellular Ca2+ concentration is kept as low as 10−7 mol/L. Ca2+ plays important physiological roles in various cells. Two main Ca2+ mobilizing systems exist: Ca2+ influx and Ca2+ release from internal stores. Inositol 1,4,5-trisphosphate (IP3) is a second messenger produced through phosphoinositide turnover in response to many extracellular stimuli (including hormones, growth factors, neurotransmitters, neutrophins, odorants, and light and controls various Ca2+-dependent cell functions (including cell proliferation, differentiation, fertilization, embryonic development, secretion, muscular contraction, immune responses, brain functions, chemical senses, and light transduction) by inducing Ca2+ release from intracellular Ca2+ stores, such as from the ER (Streb et al. 1983; Furuichi et al. 1989, 1994; Berridge 1993). Among the various inositol phosphates and phospholipids, IP3 is unique in that it is the only molecule that has a channel as a target molecule, the IP3 receptor (IP3R) (see reviews in Biochemical Society Symposium, ed, Wakelam 2006). The function of the IP3R is regulated by phosphorylation (via Ca2+/calmodulin-dependent protein kinase II and cGMP-dependent protein kinase), suggesting that IP3R works as a cross-talk station between Ca2+ signaling and phosphorylation. IP3R blockade causes severe abnormalities in cell function at both the cellular and organismal levels. This review will describe the diverse functions of IP3R at both the molecular and functional levels. The diverse functions of IP3R may depend on the property of IP3R as a scaffold protein to form a macro signal complex. The involvement of IP3R in a new signaling pathway related to Ca2+ signaling is also described.

From P400 protein to IP3 receptor

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

We have been intensively studying P400 protein, which is deficient in mutant mice with degenerated Purkinje cells and is also decreased in mutant cerebella where the dendrites of Purkinje cells are poor and the spines on the dendrites are absent (Mikoshiba and Changeux 1978; Mikoshiba et al. 1979; Maeda et al. 1988, 1989). P400 was highly enriched in isolated Purkinje cells and in microsomal fraction in the cerebellum and was also immunolocalized to Purkinje cells (Mikoshiba et al. 1979). The absence of Ca2+ spikes in a mutant deficient in spines (Crepel et al. 1984), a hypothesis linking inositol lipids and Ca2+ signaling (Michell 1975), and the high IP3-binding activity in the cerebellum enabled us to link P400 and the IP3R. We succeeded in the solubilization and purification of P400 (Mikoshiba et al. 1979; Maeda et al. 1988) and subsequently identified it as IP3R. At that time, many researchers were attempting to identify the target molecule of IP3 to microsomes by subcellular fractionation or by purification or cDNA cloning (e.g. Prentki et al. 1984; Supattapone et al. 1988; Sudhof et al. 1991). In parallel to the purification studies, we screened our cerebellar cDNA library using specific P400 monoclonal antibodies obtained by western blot screening (which immunoprecipitate IP3-binding activity) using a λgt11 expression vector system and succeeded in obtaining the whole cDNA even before final purification. We were the first group to determine the primary sequence and identify IP3R as a transmembrane protein (Furuichi et al. 1989) (Fig. 1). An immunogold method using specific monoclonal antibodies clearly showed its localization on the ER, with the long N-terminal region of IP3R located on the cytoplasmic side (Otsu et al. 1990). Purified type-1 IP3R (IP3R1) was found to work as a Ca2+ channel when it was incorporated into a lipid bilayer (Maeda et al. 1991) or liposome (Ferris et al. 1990; Nakade et al. 1991), and the over-expression of IP3R1 cDNA-enhanced IP3-binding activity and the release of Ca2+ (Miyawaki et al. 1990). Using a function-blocking antibody, IP3R was also shown to function as a Ca2+ oscillator (Miyazaki et al. 1992). Developmental studies on the role of IP3R showed that IP3R is involved in fertilization, playing roles in both the egg (Miyazaki et al. 1992; Kume et al. 1993; Saneyoshi et al. 2002) and the sperm (Fukami et al. 2001, 2003). IP3R is also essential for the determination of dorsoventral axis formation (Kume et al. 1997; Saneyoshi et al. 2002) and cell cleavage (Muto et al. 1996). We also found that IP3R is involved in neurite extension (Takei et al. 1998) and neuronal plasticity in the cerebellum (Inoue et al. 1998) and hippocampus (Nishiyama et al. 2000; Fujii et al. 2000; Itoh et al., 2001). As IP3R plays numerous roles in various cell types and also exhibits different expression levels according to the cell type, determining how such unique and specific properties are produced was of great interest. Consequently, we intensively examined the biochemical and biophysical properties of IP3R, focusing on structure-function relationships and the identification of molecules associated with IP3R. Amazingly, numerous molecules associated with IP3R have been reported by several laboratories. Together, these molecules form a macro signaling complex capable of exerting a variety of functions. This mechanism depends on the unique structural properties of IP3R.

image

Figure 1.  Structure of inositol 1,4,5-trisphosphate (IP3) receptor and associated molecules. Upper figure illustrates IP3 receptor and protein kinase C (PKC) signaling cascade system. Both IP3 and diacylglycerol (DAG) are produced from phosphatidyl inositol bisphosphate, but both have completely different functions: IP3 releases Ca2+ from internal store and DAG activates PKC for phosphorylation of various proteins. Among the various inositol lipids and phosphates, IP3 is the only molecule that has channel as a target molecule. Lower figure shows the primary structure of IP3R. IP3R has a long N-terminal and short C-terminal with transmembrane domains near C-terminal region. IP3R1 associates with many functional molecules like scaffold protein and also with Ca2+. M1–M6: transmembrane domain located at the C-terminus, the pore: channel pore site; SI, SII, and SIII: splicing site; Branched bars at the channel domain: N-glycosylation sites; PKA: cAMP-dependent protein kinase A; PKG: cGMP-dependent protein kinase; CaM: calmodulin-binding site; ATP: ATP-binding site; FKBP: FK506-binding protein; CN: calcineurin; Htt, huntingtin; HAP1A, Htt-associated protein-1A; CytC, cytochrome c; CARP, carbonic anhydrase-related protein; IRBIT, IP3R-binding protein released with IP3. Function of each component such as N-terminal coupling domain, IP3-binding domain, internal coupling domain, transmembrane/channel/forming domain, and gatekeeper domain is described in detail in Fig. 6.

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Inositol 1,4,5-trisphosphate receptor belongs to a superfamily of ion channels with six transmembrane domains but is unique in its localization near intracellular Ca2+ stores, such as on the ER, and in its dual regulation of channel opening by two second messengers, IP3 and Ca2+. A structure-function analysis of the IP3R channel is essential for understanding the fundamental principle of the generation of intracellular Ca2+ signals. Three isoforms of IP3R have been identified, each of which has a different affinity to IP3 (Blondel et al. 1994; Newton et al. 1994; Yamada et al. 1994; Yamamoto-Hino et al. 1994; Iwai et al. 2005). Biochemical analysis has revealed that the affinity of the IP3-binding core to IP3 is similar for each of the isoforms and that an IP3-binding suppressor domain linked to the N-terminal side of the binding core is responsible for the isoform-specific tuning of IP3-binding affinity (Iwai et al. 2007).

Unique structure of the IP3-binding core domain

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

As mentioned above and shown in Fig. 2, the three IP3R isoforms have different IP3-binding affinities and cooperativities (Furuichi et al. 1994; Newton et al. 1994; Iwai et al. 2005). The IP3-binding core domain is the minimum region required for specific IP3 binding and is mapped within residues 226–578 of mouse type-1 IP3R (IP3R1), a polypeptide of 2749 residues. We determined the crystal structure of the IP3-binding core domain (residues 224–604) in complex with IP3 at a resolution of 2.2 Å (Bosanac et al. 2002). 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 (Fig. 3a). Eleven amino acid residues within the IP3-binding core domain are responsible for the correct recognition of IP3 (Fig. 3b); all of these residues except Gly268 are conserved in the other IP3R isoforms (Sudhof et al. 1991; Yamamoto-Hino et al. 1994). We cloned mouse type-2 and type-3 IP3R genes (Iwai et al. 2005) and compared the IP3-binding affinities of the IP3-binding core domains among all three IP3R isoforms. The IP3-binding core domains of the three isoforms shared an approximately 70% amino acid sequence identity, and the IP3-binding affinities of the isoforms’ IP3-binding core domains were indistinguishable (Iwai et al. 2007).

image

Figure 2.  Inositol 1,4,5-trisphosphate (IP3)-binding activities of three mouse IP3 receptors (IP3Rs): IP3R1 (bsl00041), IP3R2 (bsl00066), and IP3R3 (bsl00001).

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image

Figure 3.  Structure of the inositol 1,4,5-trisphosphate (IP3) -binding core domain and the suppressor domain of mouse IP3 receptor 1. (a) Ribbon diagram of the IP3-binding core complex, comprising a beta-trefoil domain (yellow), an alpha-helical domain (green), and a hinge region (purple). The IP3 molecule (carbon in gray, phosphate in purple, and oxygen in red) lies in the space between the two domains. (b) Positioning of IP3 in IP3-binding core domain. Residues in the alpha-helical domain and the beta-trefoil domain are highlighted in green and yellow, respectively. Water molecules are shown in cyan, and the phosphate groups are shown in red. IP3 is highlighted in pink. The hinge region is shown in purple. (c) Structure of the suppressor domain. The ribbon diagram shows the head subdomain (yellow) and the arm subdomain (green) of the suppressor domain.

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N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The N-terminal 225 amino acid residues of IP3R1 function as a suppressor for IP3 binding, and the deletion of these residues results in a significant enhancement of IP3 binding (Yoshikawa et al. 1996, 1999). The suppressor domain physically interacts with the IP3-binding core domain of IP3R1 (Bultynck et al. 2004). We determined the atomic resolution structure of the suppressor domain of mouse IP3R1 using X-ray crystallography at a resolution of 1.8 Å (Bosanac et al. 2005) (Fig. 3c). The N-terminal region comprises a head subdomain, which forms a beta-trefoil fold, and an arm subdomain, which forms a helix-turn-helix structure that protrudes from the globular head subdomain. The N-terminal 604 residues of the three IP3R isoforms, containing both the suppressor domains and the IP3-binding core domains, exhibited Kd values of 49.5 ± 10.5 nmol/L (IP3R1), 14.0 ± 3.5 nmol/L (IP3R2), and 163.0 ± 44.4 nmol/L (IP3R3) (Iwai et al. 2007); these values are close to the intrinsic IP3-binding affinities estimated by analyzing the full-length IP3Rs (Iwai et al. 2005). Our results suggest that the suppression of IP3 binding generates the isoform-specific IP3-binding affinities of the three IP3R isoforms and that IP3-binding affinity is a tunable parameter, rather than a stable constant.

Site-directed mutagenesis analyses on the suppressor domain of mouse IP3R1 showed that seven conserved amino acid residues (Leu30, Leu32, Val33, Asp34, Arg36, Arg54, and Lys127) are critical for the suppression of IP3 binding (Bosanac et al. 2005). Systematic mutagenesis analyses showed that 11 type-3 specific residues (Glu39, Ala41, Asp46, Met127, Ala154, Thr155, Leu162, Trp168, Asn173, Asn176, and Val179) were critical for the type-3 receptor-specific IP3-binding affinity (Iwai et al. 2007). All these conserved and non-conserved residues, with the exception of Leu162, are located on the surface of the head subdomain of the suppressor domain, indicating that the head subdomain is responsible forisoform-specific IP3-binding affinity tuning.

Three-dimensional structure of IP3R

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

We and other groups have successfully visualized the tetrameric structure of IP3R1 using electron microscopy (EM) (Hamada et al. 2002, 2003; Jiang et al. 2002; da Fonseca et al. 2003; Serysheva et al. 2003; Sato et al. 2004). Although the structural details differed slightly among the reports, the general structure was the same. As we successfully purified IP3R1 in its native form, EM analysis revealed a reversible transition between two distinct structures with fourfold symmetry: a windmill structure and a square structure. Upon searching for suitable conditions, we fortunately found that Ca2+ promotes the transition from the square structure to the windmill structure via the relocation of four peripheral IP3-binding domains, identified by binding to heparin-gold (Hamada et al. 2002). We subsequently accumulated further biochemical and EM evidence of a structural change in the 3D architecture of IP3R1 (Fig. 4) via a process that is regulated by the physiological Ca2+ concentration (Hamada et al. 2003). The structure in the absence of Ca2+, as reconstructed using 3D EM, shows a ‘mushroom-like’ appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The ‘windmill-like’ form that occurs in the presence of Ca2+ also contains four bridges connecting the IP3-binding domain with the channel domain, verifying the Ca2+-dependence of the 3D structure of IP3R. Recently, we attempted to visualize the IP3R structure in solution dynamically using atomic force microscopy (AFM). We succeeded in the first visualization of individual IP3R particles in aqueous solution (Suhara et al. 2006).

image

Figure 4.  Model for the global structural changes that occur within inositol 1,4,5-trisphosphate (IP3) receptor 1. Proposed 3D model for the large-scale relocations of the IP3-binding domains. The plausible IP3-binding cores (blue) are liganded with IP3 (red molecules) and a 38-kDa fragment by Lys-C digestion (green).

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Unfortunately, the spatial resolution of the AFM technique does not enable fine conformational changes produced by ligands like IP3 or ATP to be detected. Thus, high-resolution structures in the presence of IP3, Ca2+, or other allosteric ligands must be captured using cryo-EM and X-ray crystallography techniques. Early studies provided information on structural changes using static specimens, but dynamic changes in the IP3R structure could not be visualized. To observe dynamic conformational changes in aqueous solutions and in living cells, real-time changes must be monitored using AFM or photometric analysis. As the cryo-EM analysis revealed a highly ordered 3D architecture of a tetrameric IP3R constructed in living cells via unknown mechanisms, further study is needed to determine the biogenetic processes involved in the birth of tetrameric IP3R channels in living cells.

To clarify the structural basis for channel gating via IP3 and Ca2+, we further analyzed the precise 3D structure of the Ca2+-free form of tetrameric IP3R using cryo-EM with a helium cooled specimen stage and an automatic particle selection system. Our structural analysis at a resolution of 15 Å revealed a multi-porous architecture and L-shaped densities as IP3-binding domains that were assigned according to the X-ray structure (Fig. 5). We proposed a model that explained a mechanism for the regulation of Ca2+ release by co-agonists, Ca2+ and IP3 (Sato et al. 2004).

image

Figure 5.  Inositol 1,4,5-trisphosphate (IP3) receptor contains multiple cavities and L-shaped ligand-binding domains. The ribbon diagram of the IP3-binding domain with bound IP3 (a) was based on the X-ray structure (1N4K). The arrows indicate the changes that were made to the crystal structure of the IP3-binding domain to obtain a better fit with the L-shaped density in the 3D map of the unliganded IP3 receptor 1. The figure is color-coded, with the alpha-helical domain shown in purple, the beta-sheet domain shown in green, the bound IP3 molecule shown in blue, the Ca-I site (residues E246, E425, D426, and E428) shown in yellow, and the Ca-II site (E283, E285, D444, and D448) shown in red. The fit of the modified crystal structure into the density map of the whole receptor is shown, with the IP3-binding domain represented by both a ribbon diagram and a space-filled model (b). The ‘N’ and ‘C’ denote the N- and the C-terminuses of the domain, respectively.

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Gating mechanism of the IP3R channel

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The structure of IP3Rs has traditionally been divided into three functional domains: the N-terminal ligand-binding domain; the modulatory/coupling domain; and the C-terminal transmembrane/channel-forming domain (Furuichi et al. 1989, 1994). When the C-terminal channel-forming domain or the caspase 3-cleaved form of mouse IP3R1 was expressed in HeLa and COS-7 cells, both ATP and thapsigargin failed to induce an increase in cytosolic Ca2+ (Nakayama et al. 2004). The entry of Ca2+ after store depletion (store-operated Ca2+ entry) was normally observed in these cells, indicating that the Ca2+ stores of cells expressing the above truncated IP3R were nearly empty in the resting state and that these proteins continuously leaked Ca2+. We propose that the channel-forming domain of IP3R1 is necessary to keep the channel closed.

To understand the mechanism of the channel’s IP3-induced gating, we analyzed the channel properties of deletion mutants retaining both the IP3-binding domain and the channel-forming domain of IP3R1 (Uchida et al. 2003). We found that mutants lacking the N-terminal 223 residues, corresponding to the IP3-binding suppressor domain, or residues 651–1130 did not exhibit any measurable Ca2+ release activity in response to the addition of IP3. These two mutants retained their IP3-binding activity and the normal folding structure of at least the C-terminal channel-forming domain. These results suggest that residues in the regions of 1–223 and 651–1130 are critical for the functional coupling between IP3 binding and channel opening. We proposed a novel five-domain structural model in which conformational changes in the IP3-binding core domain caused by IP3 binding are transmitted through both the N-terminal coupling/suppressor domain and the internal coupling domain to the C-terminal tail, triggering channel opening (Fig. 6). The unique gating machinery comprised of the N-terminal ligand-binding domain and the C-terminal channel-forming domain may account for the generation of the characteristic behavior of IP3R channels, such as quantal Ca2+ release without desensitization.

image

Figure 6.  Five-domain structural model of mouse inositol 1,4,5-trisphosphate (IP3) receptor 1. The structure of IP3 receptor 1 is divided into five functional domains: an N-terminal coupling/suppressor domain, an IP3-binding core domain, an internal coupling domain, a transmembrane/channel-forming domain, and a gatekeeper domain. The signal for IP3 binding is transferred through both the N-terminal and internal coupling domains to the gatekeeper domain, which triggers a conformational change in the activation gate formed within the transmembrane/channel-forming domain.

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Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Inositol 1,4,5-trisphosphate sensors have been developed by a few groups using genetic and other techniques (Tanimura et al. 2004; Sato et al. 2005; Matsu-ura et al. 2006; Remus et al. 2006). For example, we developed an IP3R-based IP3 sensor (IRIS) that was based on the ligand-binding domain of the IP3R and a green fluorescent protein (GFP) variant (Fig. 7) (Matsu-ura et al. 2006). The IP3 sensors showed IP3-dependent reductions in fluorescence resonance energy transfer (FRET) between a variant of yellow fluorescent protein Venus and an enhanced cyan fluorescent protein fused to the IP3-binding domain. IRIS-1 exhibited a maximal response of 25.1 ± 8.0% after the addition of an excess amount of IP3, with an apparent IP3 sensitivity of 549 ± 62 nmol/L. The IP3 sensitivity of IRIS-1 was not influenced by the addition of 1 μmol/L Ca2+. We created various IP3 sensors with different IP3 sensitivities, dynamic ranges, and reaction rates by modifying the IP3-binding domain.

image

Figure 7.  Inositol 1,4,5-trisphosphate (IP3) receptor-based IP3 sensor (IRIS-1) IP3 binding to IRIS-1 decreases FRET between ECFP and Venus. IRIS is the personification of the rainbow in Greek mythology.

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We used these IP3 sensor proteins to monitor the spatiotemporal dynamics of cytosolic IP3 in single HeLa cells exposed to extracellular stimuli. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. The IP3 concentration did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with little or no oscillations during cytosolic Ca2+ oscillations (Fig. 8). The results were unexpected because neither a constant elevation nor a simple mirroring of [Ca2+] changes was found in regard to the actual [IP3] dynamics during Ca2+ oscillations. These results indicate that Ca2+-induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity of Ca2+ spike generation progressively decreases during Ca2+ oscillations.

image

Figure 8.  Inositol 1,4,5-trisphosphate (IP3) dynamics during the first and subsequent Ca2+ spikes in metabotropic glutamate receptor 5a-expressing HeLa cells. Time courses of changes in the emission of Indo-1 (−F/Fbase) (a) and IP3 receptor-based IP3 sensor-1 (R/Rbase) (b) during the first four Ca2+ spikes evoked by 100 μmol/L of glutamate. The broken horizontal lines indicate the baseline levels. The vertical lines indicate the times of the onset (open circle) and the end (closed circle) of the abrupt [Ca2+] rises. The open arrowheads and the closed arrowheads indicate the times of onset of the increases in the Indo-1 and IP3 receptor-based IP3 sensor-1 signals, respectively. (See Matsu-ura et al. 2006 for further details.).

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IP3Rs form macro signal complexes

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The expression, localization, and function of IP3Rs are regulated by various cellular proteins. Cytochrome c was recently found to bind to the C-terminal region (Boehning et al. 2003). In addition, huntingtin (Htt) -associated protein-1A (HAP1A) was found to bind to the C-terminus of IP3R1 (Tang et al. 2003; Bezprozvanny and Hayden 2004). IP3R1 activation by IP3 is sensitized by the polyglutamine (polyQ) expansion of Htt caused by Huntington disease. Protein phosphatases (PPI and PP2A) (DeSouza et al. 2002; Tang et al. 2003), RACK1 (Patterson et al. 2004), ankyrin (Bourguignon et al. 1993), and chromogramin have been reported to bind to the channel region and regulate channel activity (Yoo et al. 2000). Carbonic anhydrase-related protein (CARP) has been found to bind to a central part between the IP3-binding core and the channel region and to regulate channel activity (Hirota et al. 2003). Homer (Tu et al. 1998) binds to the N-terminal suppressor region of IP3R. The physiological relevance of these interactions is expected to be important, but the details are not yet clear. The interactions of various proteins are described in further detail below.

Interactions among the N-terminal suppressor region, the IP3-binding core region, and a cysteine at the C-terminus near the pore region are known to be important for the gating of the IP3R channel pore. Therefore, these areas are regarded as hot spots for channel gating. Most of the above-mentioned associated proteins bind to these hot spot areas.

Homer

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Homer associates with the N-terminal suppressor region of IP3R (Tu et al. 1998). The proline-rich Homer ligand has been associated with group 1 metabotropic glutamate receptors (mGluR1) and IP3R, and these receptors co-immunoprecipitate as a complex with Homer from brain tissue (Tu et al. 1998). In cerebellar Purkinje cells, some IP3R1 appears to be part of multimeric, junctional Ca2+ signaling networks, the composition of which has been shown to include plasma membrane Ca2+ ATPase, mGluR1, and Homer 1b/x (Sandona et al. 2003). Homer also associates with ryanodine receptors type-1(RyR1) and regulates the function of IP3R (Feng et al., 2002). As Homer associates with shank, NMDA receptors, and TrpC channels, IP3R may dynamically interact with these channels to exert a variety of neural functions.

Protein 4.1N regulates translocation and lateral diffusion of IP3R1

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

We identified protein 4.1N as a molecule that binds to the C-terminal cytoplasmic tail of IP3R1 using a yeast-two hybrid system. Protein 4.1N associated with IP3R1 in both subconfluent and confluent MDCK cells, a well-studied and tightly polarized epithelial cell line. Both 4.1N and IP3R1were predominantly translocated from the cytoplasm (and the nucleus) to the basolateral membrane domain when the MDCK cells grew from subconfluence to confluence, whereas several other endogenous or exogenously expressed ER marker proteins, like calnexin, calreticulin, calsequestrin, and sarcoplasmic/ER calcium-ATPase, remained present in the cytoplasm in confluent MDCK cells. The localization of IP3R1 at the basolateral membrane domain was determined by its 4.1N-binding region and could be blocked by a fragment of the IP3R1-binding region of 4.1N. These data suggest that 4.1N serves to regulate IP3R1 subcellular localization (Zhang et al. 2003). In one strain of mutant mice that lacks myosin-Va, a kind of myosin motor, type-1 IP3R is dislocated from the Purkinje cell spines to the dendrites, and the mice exhibit impaired long-term depression, similar to the phenotype of type-1 IP3R-null knockout (KO) mice. This finding suggests that the precise spatiotemporal regulation of IP3R expression is critical for physiological development. Therefore, 4.1N and other cytoskeletal proteins may play important roles in the subcellular localization of IP3Rs. The role of 4.1N in lateral diffusion is described in the section on IP3R trafficking.

ER luminal redox sensor, ERp44

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The dysfunction of the ER, a locus of intracellular Ca2+-signaling and quality control for protein biogenesis, has recently been implicated in apoptosis and neurodegenerative diseases like Alzheimer or Huntington disease. We focused on the interior of the ER and searched for proteins that bind to the luminal domain of IP3R to search for the specific molecular event that occurs on the luminal side of the ER. We successfully found that ERp44, an ER luminal protein belonging to the thioredoxin family, directly interacts with the luminal region of IP3R; this interaction is dependent on pH, the Ca2+ concentration, and the redox state. We also clarified that the subtype-specific binding of ERp44 inhibited the Ca2+-release activity of IP3R1 in vivo. Mutagenesis experiments revealed that cysteine residues in the luminal region were required for the inhibition of IP3R1 by ERp44. Thus, we suggested that the ERp44/IP3R1 system may act as a molecular sensor monitoring the environment in the ER lumen and transmitting signals from the lumen of the ER to the cytosolic space in living cells (Higo et al. 2005) (Fig. 9).

image

Figure 9.  Endoplasmic reticulum (ER) luminal environment-dependent regulation of inositol 1,4,5-trisphosphate (IP3) receptor 1 (IP3R1) by ERp44.

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Huntingtin-associated protein-1a

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Huntington disease is caused by polyQ expansion of the Htt-protein. HAP1A associates with the IP3R1 carboxy-terminus. The IP3R1-HAP1A-Htt ternary complex is formed both in vitro and in vivo. In planar lipid bilayer reconstitution experiments, IP3R1 activation by IP3 is sensitized by the polyQ expansion of Htt caused by Huntington disease. These results suggest a novel molecular link between Htt and IP3R1-mediated neuronal Ca2+ signaling (Tang et al. 2003). PolyQ expansion in the amino-terminal region of Htt causes the binding and activation of IP3R1 in both planar lipid bilayers and medium spiny striatal neurons (Bezprozvanny and Hayden 2004). This important finding suggests that Parkinson disease is also linked to IP3R1/Ca2+ signaling.

Carbonic anhydrase-related protein

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Carbonic anhydrase-related protein (CARP) has been identified as a Purkinje cell-specific gene (Nograd et al. 1997). CARP comprises 291 amino acids and a central carbonic anhydrase motif, but lacks carbonic anhydrase activity (Kato 1990). CARP binds to the modulatory domain of IP3R1 (amino acids 1387–1647) (Hirota et al. 2003). CARP inhibits IP3 binding to IP3R1 by reducing IP3R1s affinity for IP3. As the sensitivity of Purkinje cells to IP3-induced Ca2+ release (IICR) is relatively low, this phenomenon could be caused by the co-expression of CARP with IP3R in Purkinje cells. This study suggests that the neuronal plasticity of Purkinje cells in the cerebellum differs from that of other neurons, such as those in the hippocampus, at a molecular level.

Protein phosphatases, PP1and PP2A

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Protein kinase A (PKA) and protein phosphatases (PP1 and PP2A) are components of the IP3R macro signal complex (DeSouza et al. 2002; Tang et al. 2003). Association with PP1 facilitates the dephosphorylation of PKA-phosphorylated PP2A and inhibits the activity of recombinant IP3R1 reconstituted into planar lipid bilayers. The PKA phosphorylation of IP3R1 increases the sensitivity of IP3Rs (DeSouza et al. 2002; Tang et al. 2003). Phosphorylation and dephosphoylation will be important subjects in future studies on IP3R-related Ca2+ signaling.

Ankyrin

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Ankyrin is known to link various transmembrane proteins to actin through spectrin or fodrin. IP3R has been shown to bind to ankyrin (Bourguignon et al. 1993, 1995), as does RyR (Bourguignon and Jin 1995); ankyrin also binds the synthetic peptide 2546-GGVGDVLRKPS-2556 of mouse IP3R1 (Bourguignon and Jin 1995). However, the ankyrin-binding site on IP3R is thought to be on the luminal side of the ER (Yoshikawa et al. 1992; Michikawa et al., 1994) and to differ in sequence from the ankyrin-binding site on RyR1 (Furuichi et al. 1989). Further detailed analyses are needed, but ankyrin may associate with the IP3R and may be involved in the regulation of Ca2+ signaling.

Chromogranin A and B

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Chromogranin A and B are major proteins present inside secretory granules, and act as high-capacity, low-affinity Ca2+ storage proteins. Purified IP3R interacts directly with chromogranin A and B at an intravesicular pH of 5.5.

The presence of chromogranin A in IP3R-reconstituted liposomes significantly enhances IICR activity, and increases the amount of IP3 that is bound to the reconstituted IP3R (Yoo et al. 2000). In lipid bilayer experiments, chromogranin A has also been shown to increase the release activity of IP3R by increasing the probability of channel opening and the mean open time. In addition, secretory granules of bovine adrenal medullary chromaffin cells contain three isoforms of IP3R, as shown using immunogold EM. The expressed cDNA of bovine IP3R2 and IP3R3 co-immunoprecipitated with chromogranin A and B in NIH3T3 or COS-7 cells, where IP3R was also detected. This finding indicates that the three isoforms of IP3R form complexes with chromogranin A and B.

RACK1 and cytochrome c

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

RACK1 was originally found to bind the phosphorylated active forms of PKC (Ron et al. 1994) and to act as a scaffold protein, bringing activated PKC into contact with its various substrates (Chang et al. 2002; Birikh et al. 2003; Ceci et al. 2003). RACK1 binds IP3R and regulates Ca2+ release by enhancing IP3Rs-binding affinity for IP3 (Patterson et al. 2004). This work may be important for demonstrating a close interaction between IP3R/Ca2+ signaling and PKC phosphorylation.

Cytochrome c binds to IP3Rs and amplifies apoptosis (Boehning et al. 2003). The addition of 1 nmol/L of cytochrome c blocks the Ca-dependent inhibition of IP3R function. Cytochrome c translocates to the ER and binds IP3R early during apoptosis. This results in a sustained, oscillatory increase in cytosolic Ca2+. These Ca2+ events are linked to the coordinated release of cytochrome c from all the mitochondria, possibly augmenting cytochrome c release and amplifying the apoptotic signal. This research is important for linking mitochondria and IP3Rs on the ER during apoptosis.

Four intracellular trafficking pathways involving IP3Rs

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The ER contains intracellular Ca2+ stores and is composed of a continuous meshwork of membrane extending throughout the cell. The molecular mechanism responsible for the generation of ‘localized’ Ca2+ signals from the ‘widely distributed’ ER in neurons remains to be elucidated. The localization of IP3R to specific regions in the cell is now considered an important factor in the spatial regulation of Ca2+ release.

mRNA transport as a component of the mRNA granule

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The selective transport and localization of certain types of mRNA and the subsequent local protein synthesis in neuronal dendrites are now considered fundamental mechanisms in synaptic plasticity. Many dendritic mRNAs are transported as a component of ribonucleoprotein complexes called ‘mRNA granules’ that contain ribosomes, other components of the translational machinery, and various mRNA-binding proteins (Knowles et al. 1996; Tiedge and Brosius 1996; Torre and Steward 1996). Staufen (Kiebler et al. 1999) is a well-known component of mRNA granules. We cloned an RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) (Mizutani et al. 2000) and found that SYNCRIP is a component of mRNA granules in rat hippocampal neurons. mRNA granules labeled with fluorescent protein-tagged SYNCRIP were transported bidirectionally within the dendrite at approximately 0.05 μm/s in a microtubule-dependent manner. The 3′-untranslated region of IP3R1 mRNA was co-transported with SYNCRIP, suggesting that IP3R1 mRNA is delivered into the dendrites via mRNA granule transport (Bannai et al. 2004a). This result also raises the possibility that the amount of IP3R can be modified by the local translation of IP3R1 mRNA.

Although the translation of IP3R mRNA is activity dependent, it is not clear whether IP3R mRNA is translated within the dendrite in an activity-dependent manner. Movement of IP3R mRNA is of a great importance to the physiology of the IP3R. This does not reflect trafficking of the receptor but rather greatly affects the site of synthesis of IP3R.

Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Endoplasmic reticulum vesicles and mitochondria were recently shown to exchange Ca2+ bidirectionally, leading the researchers to propose that communication between the perisynaptic ER vesicles and mitochondria can shape intracellular Ca2+ signals and modulate synaptic and integrative neural activities in respiratory neurons (Mironov and Symonchuk 2006). If this is true in hippocampal neurons, the neuronal signal that modifies the dynamics of vesicular ER subcompartments should be sought, as this would provide another clue to understanding the physiological function of vesicular subcompartments in the ER. The relationship between spatially regulated Ca2+ signals (e.g. dendritic Ca2+ wave) and the localization of vesicular ER subcompartments would be an interesting topic to investigate.

To visualize the dynamics of the ER membrane in the dendrites of living neurons, we expressed fluorescent protein-tagged ER proteins in cultured mouse hippocampal neurons and monitored their movements using time-lapse microscopy. We found that subcompartments of the ER form in relatively large vesicles that are capable of taking up and releasing Ca2+, similar to the reticular ER. These vesicular subcompartments of the ER moved rapidly along the dendrites in both anterograde and retrograde directions at a velocity of 0.2–0.3 μm/s. The depletion of microtubules, the over-expression of dominant-negative kinesin, and kinesin depletion by antisense DNA reduced the number and velocity of the moving vesicles, suggesting that kinesin may drive the transport of vesicular subcompartments of the ER along microtubules in the dendrites. The rapid transport of Ca2+-releasable subcompartments of the ER might contribute to the rapid supply of fresh ER proteins to the distal part of the dendrite, or to the spatial regulation of intracellular Ca2+ signaling (Bannai et al. 2004b).

Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Inositol 1,4,5-trisphosphate receptor 1 plays an important role in neuronal functions; however, the dynamics of IP3R1 on the ER membrane and its regulation in living neurons remains unknown. GFP-tagged IP3R1 was expressed in cultured rat hippocampal neurons and its lateral diffusion was observed using the fluorescence recovery after photobleaching technique. GFP-tagged IP3R1 gradually migrated into the photobleached area from both ends. This apparent diffusive nature allowed us to measure the effective diffusion constant by fitting our observations to a diffusion model. The diffusion constant of IP3R1 was 0.26 ± 0.10 μm2/s. The depletion of actin filaments increased the diffusion constant of IP3R1, suggesting that the diffusion of IP3R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP3R1 (Zhang et al. 2003) and contains an actin-spectrin binding region, was responsible for this actin-induced regulation of the IP3R1 diffusion constant. The diffusion of IP3R3, an IP3R isoform that lacks the ability to bind to 4.1N, was not dependent on actin filaments, but became dependent on actin filaments after the addition of a 4.1N-binding sequence (C-terminal cytoplasmic tail 14 amino acids; CTT14aa). The over-expression of another 4.1N-binding site of IP3R1, cytoplasmic tail middle 1 (Maximov et al. 2003), also increased the diffusion of IP3R1. However, IP3R1-ΔCTT14aa (IP3R1 lacking the CTT14aa) did not bind to 4.1N and its diffusion constant was larger than that of full-length IP3R1. Taking together, these results, we concluded that 4.1N serves as a linker protein between IP3R1 and actin filaments and that the CTT14aa region of IP3R1 is involved in the regulation of IP3R1 diffusion (Fig. 10). This actin filament-dependent regulation of IP3R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling (Fukatsu et al. 2004, 2006).

image

Figure 10.  Schematic model of regulatory mechanism for inositol 1,4,5-trisphosphate (IP3) receptor 1 (IP3R1) diffusion. (a) Protein 4.1N binds spectrin-actin filaments and the C-terminal cytoplasmic tail 14 amino acids (CTT14aa) region of IP3R1. Therefore, IP3R1 does not diffuse freely. (b) IP3R3 does not bind to 4.1N and is able to diffuse freely.

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Cluster formation of IP3Rs on ER membranes

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Inositol 1,4,5-trisphosphate receptor forms clusters on ER membranes when the cytosolic Ca2+ level is elevated. We monitored the dynamics of GFP-tagged IP3R1 in living cells (Tateishi et al. 2005) and found that the time course for IP3R1 clustering, evoked by IP3-generating agonists, did not correlate with cytosolic Ca2+ changes but seemed compatible with the cytosolic IP3 concentration. IP3 production alone induced IP3R1 clustering in the absence of a significant increase in Ca2+, but elevated Ca2+ without IP3 production did not induce IP3R1 clustering. IP3R1 mutants that did not undergo an IP3-induced conformational change failed to form clusters. These results suggested that an IP3-induced conformational change in the receptor, but not a Ca2+-induced conformational change, was essential for the induction of IP3R1 clustering on ER membranes. We also found that GFP-IP3R1 clusters colocalized with ERp44 (Fig. 11), a luminal ER protein that inhibits its channel activity (Higo et al. 2005).

image

Figure 11.  Colocalization of green fluorescent protein-inositol 1,4,5-trisphosphate (IP3) receptor 1 (IP3R1) clusters and ERp44. Top: The green fluorescent protein-IP3R1 cluster induced by 100 μmol/L of ATP stimulation is colocalized with DsRed2-ERp44 in COS-7 cells. Bottom: The same stimulation does not change the morphology of the ER. Scale bars: 10 μm.

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The sizes and shapes of the stimulus-induced clusters differed among the three types of IP3R, and IP3R2 formed clusters even in resting cells (Iwai et al. 2005). IP3 binding-deficient IP3R2 and IP3R3 mutants failed to form stimulus-induced clusters, suggesting that the IP3 binding step is involved in cluster formation in all three types of IP3R. Because IP3R2 possesses the highest intrinsic affinity for IP3 binding among the three IP3R isoforms, the cytosolic IP3 concentration in resting cells may be sufficient to induce IP3R2 accumulation. Interestingly, the size of IP3R2 clusters in resting cells was compatible to that of elementary Ca2+ events, Ca2+ puffs. Because IP3R2 forms clusters in resting cells, IP3R2 may contribute to the formation of IP3R clusters from which Ca2+ puffs originate.

We found a novel alternative splicing segment, SIm2, at 176–208 of IP3R2 (Iwai et al. 2005). IP3R2 SIm2 had neither IP3-binding activity nor Ca2+ releasing activity. The long form (IP3R2) was dominant, but the short form (IP3R2 SIm2) was detected in all the tissues that were examined. IP3R2 SIm2 did not form clusters in either resting or stimulated cells. The co-expression of IP3R2 SIm2 prevented stimulus-induced IP3R clustering, suggesting that IP3R2 SIm2 functions as a negative coordinator of stimulus-induced IP3R clustering. The expression of IP3R2 SIm2 in CHO-K1 cells significantly reduced ATP-induced Ca2+ entry, but not Ca2+ release, suggesting that the novel splice variant of IP3R2 specifically influences the dynamics of the sustained phase of Ca2+ signals. The functional coupling between IP3R clustering on the ER membrane and the activation of the Ca2+ entry pathway should be an interesting topic to investigate.

We plan to examine the working hypothesis that the lateral diffusion of IP3R on the ER membrane is modified by a stimulus that induces IP3R clustering. Lateral diffusion will be analyzed using Quantum Dots, a single molecule imaging technique. Although computer simulation studies have shown that stimulus-coupled IP3R clustering might reduce IP3R channel activity, the consequences and functions of IP3R clustering remain an enigma. To address this question, we plan to examine whether ERp44, a protein that binds to IP3R in the ER lumen (Higo et al. 2005), inhibits the channel activity of clustered IP3R1. Analyzing the contribution of IP3R clustering to Ca2+-influx would also be an interesting issue.

In summary, four methods of intracellular trafficking involving IP3R have been described: (i) as mRNA, (ii) in vesicular subcompartments of the ER, (iii) by means of lateral diffusion on the ER membrane, and (iv) by receptor clustering on the ER membrane (Fig. 12). Our next goal is to examine the relationship between these intracellular IP3R dynamics and spatiotemporal Ca2+ patterns. We are also interested in the IP3 dynamics that are critical for determining Ca2+ signaling patterns. Our new IP3 probe, ‘IRIS,’ will also be a powerful tool for revealing the relationship between intracellular IP3 dynamics and the spatiotemporal Ca2+ patterns that contribute to higher brain functions.

image

Figure 12.  Four methods of intracellular inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) trafficking. (a) As mRNA on mRNA granules. (b) In vesicular subcompartments of the endoplasmic reticulum (ER). (c) By lateral diffusion on the ER membrane. (d) By receptor clustering on the ER membrane. Movement of IP3R mRNA does not reflect trafficking of IP3R but greatly affects the site of IP3R synthesis.

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New signaling pathway

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

IRBIT, a pseudo ligand of IP3R

Binding partners to the cytoplasmic region of IP3R1 (residues 1–2217) were screened using a high-salt extract of crude rat brain microsomes with IP3 elution, and a novel protein, IP3R-binding protein released with IP3 (IRBIT) was identified. IRBIT, consisting of 530 amino acids, has a domain homologous to S-adenosylhomocysteine hydrolase near the C-terminus and a 104-amino acid appendage containing multiple potential phosphorylation sites near the N-terminus. In vitro binding experiments showed that the N-terminal region of IRBIT is essential for its interaction with IP3R1 and that IRBIT binds to the IP3-binding core domain. IP3 dissociated IRBIT from IP3R1 with an EC50 of ∼0.5 μmol/L, i.e. it was 50-times more potent than other inositol polyphosphates, such as inositol 1,3,4,5-tetrakisphosphate and inositol 1,4-bisphosphate. Alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP3 and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context (Ando et al. 2003). These results suggest that IRBIT has at least two biological functions: a function in an IP3R-bound state and a function after its release from IP3R. The state that IRBIT assumes is determined by the intracellular IP3 concentration.

Effect of IRBIT on the IP3-binding activity and the IP3-induced Ca2+ release activity of IP3R

The phosphorylation sites of IRBIT that are essential to IP3R binding were determined. Using [3H]IP3-binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells, IRBIT was found to suppress the activation of IP3R by competing with IP3. The phosphorylation of four Ser residues (Ser68, 71, 74, and 77) of IRBIT was essential for the interaction with IP3R. Ten out of the 12 amino acids residues essential for IP3 recognition of IP3R participated in the binding to IRBIT. Based on these results, we proposed that IRBIT may act as an endogenous ‘pseudoligand’ in its IP3R-bound state (Ando et al. 2006). To determine whether IRBIT suppresses IP3R-mediated Ca2+ release in intact cells, we utilized RNA interference to suppress the expression of IRBIT in HeLa cells. IRBIT suppressed the expression of IRBIT in HeLa cells but had no effect on the expression of IP3Rs. The depletion of IRBIT by siRNA IRBIT resulted in an increase in the number of cells that responded to the threshold dose, of ATP stimulation. Therefore, IRBIT regulates IICR.

IRBIT works as a messenger to regulate acid-base balance

To reveal the functions of IRBIT after its release from IP3R, we screened for target molecules of IRBIT. Na+/HCO3− co-transporter 1 (NBC1) was identified as an IRBIT-binding protein. Of the two major splicing variants of NBC1, pancreas-type NBC1 (pNBC1) and kidney-type NBC1 (kNBC1), IRBIT was found to bind specifically to pNBC1 but not to kNBC1. IRBIT binds to the N-terminal pNBC1-specific domain, and its binding depends on the phosphorylation of multiple serine residues of IRBIT. Also, an electrophysiological analysis in Xenopus oocytes revealed that pNBC1 requires the co-expression of IRBIT to manifest substantial activity comparable with that of kNBC1, which displays substantial activity independently of IRBIT. These results strongly suggest that pNBC1 is the target molecule of IRBIT and that IRBIT plays an important role in pH regulation through pNBC1. Also, our findings raise the possibility that regulation through IRBIT enables NBC1 variants to have different physiological roles (Shirakabe et al. 2006). As many hereditary human diseases involve NBC1, IRBIT is likely involved in the pathogenesis of these diseases.

Collectively, we found that the IP3 sensitivity of IP3R is regulated by IRBIT functioning as a pseudoligand in the resting state and that when the IP3 concentration is increased, IP3R functions not only as a Ca2+ releasing channel but also as an IRBIT-releasing channel, suggesting that IP3R serves as a signaling center for both Ca2+- and IRBIT-mediated signaling pathways (Figs 13 and 14). Therefore, the signaling pathway may be modified as follows: [extracellular signal IP3-IP3R (i) Ca2+ release, (ii) IRBIT release – NBC1 activation, and (iii) IRBIT release – Ca2+ oscillation modification].

image

Figure 13.  Inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) -binding protein released with inositol 1,4,5-trisphosphate (IRBIT) functions as a pseudoligand for IP3R in the resting state and as a third messenger released from IP3R in the presence of second messenger IP3. IRBIT regulates activity through direct competition for the binding site on IP3 receptors. Once IP3 reaches a threshold concentration, it displaces IRBIT from the binding site. (p = phosphate group, indicating the phosphorylation status; green dots = calcium ions).

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image

Figure 14.  Inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) -binding protein released with inositol 1,4,5-trisphosphate (IRBIT) activates Na+/HCO3− co-transporter 1 (NBC1). IRBIT is bound to the IP3-binding core of the cytoplasmic domain of IP3R under normal conditions (left), after which IRBIT associates with pancreas-type NBC1 to stimulate ion transport across the plasma membrane (top); (yellow circle = phosphate).

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Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Recent studies have indicated that Na,K-ATPase, in addition to being the key regulator of intracellular Na+ and K+ concentrations, can, in the presence of ouabain – a specific inhibitor of Na,K-ATPase, trigger intracellular Ca2+ oscillations. We investigated the molecular mechanism of ouabain-calcium signal transduction. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and IP3R. Ouabain significantly enhanced the FRET between Na,K-ATPase and IP3R. Partial truncation of the N-terminal tail of the Na,K-ATPase catalytic α-subunit abolished the Ca2+ oscillations and the downstream activation of nuclear factor-kappa B. The N-terminal tail of the Na,K-ATPase catalytic α-subunit (αNTT) directly bound to the IP3-ligand binding domain of IP3R. Three amino acid residues (LKK, conserved in most species and most α-isoforms) are essential for binding. In wild-type cells, a low concentration of ouabain triggers low-frequency calcium oscillations that activate nuclear factor-kappa B and protect the cells from apoptosis. These effects are suppressed in cells that over-express a peptide corresponding to αNTT but not in cells that over-express a peptide corresponding to αNTTδLKK. These data indicate that Na,K-ATPase, together with ouabain, regulates intracellular Ca2+ oscillations and subsequent cellular functions via a direct interaction with the IP3-ligand binding domain of IP3R (Fig. 15) (Miyakawa-Naito et al. 2003; Zhang et al. 2006).

image

Figure 15.  Ouabain triggers intracellular Ca2+ oscillations via a direct interaction between the N-terminal tail of Na,K ATPase and the inositol 1,4,5-trisphosphate (IP3) -ligand binding domain of IP3 receptor (IP3R).

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Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

The stimulation of various cell surface receptors leads to the production of IP3 and DAG through phospholipase C (PLC) activation, and IP3 and DAG in turn trigger Ca2+ release through IP3Rs and PKC activation, respectively. The amount of IP3 produced is particularly critical for the spatiotemporally coordinated Ca2+-signaling patterns. Recently, we reported a novel signal cross-talk between DAG and the IP3-mediated Ca2+-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of PKC activity and extracellular Ca2+. OAG-induced Ca2+ oscillation was completely abolished by the depletion of Ca2+ stores or the inhibition of PLC and IP3Rs, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IICR. In addition, the intracellular accumulation of endogenous DAG via a DAG-lipase inhibitor greatly increased the number of cells responding to low doses of agonist stimulation. Our findings indicate a novel physiological function of DAG, i.e. the amplification of Ca2+ signaling through the enhancement of IP3 production via its positive feedback effect on PLC activity (Hisatsune et al. 2005).

Physiological functions of three IP3R isoforms

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

In over 10 years, many laboratories have established that IP3R plays a crucial role in higher brain functions, such as behavior and learning and memory (Matsumoto et al. 1996; Fujii et al. 2000; Nishiyama et al. 2000). Long-term potentiation (Fujii et al. 2000) in the hippocampus was enhanced in IP3R1-deficient mice, while long-term depression in the cerebellum was lost (Inoue et al. 1998).

Therefore, IP3R1 is involved in brain function. IP3R1 is also involved in early development, i.e. in neurite formation and dendritic extension (Takei et al. 1998). These reports were based on findings using IP3R-null KO mice or a specific antibody against IP3R1, 18A10.

The application of antagonists like 2-aminoethoxydiphenyl borate (Maruyama et al. 1997) and Xestospongin – an extract from sponges, and IP3 sponge, a peptide sequence that traps IP3 are also useful for determining the functions of IP3Rs (Iwasaki et al. 2002). However, how IP3R contributes to brain function and development remains largely unclear. Mutant mice lacking the three types of IP3R (Matsumoto et al. 1996; Futatsugi et al. 2005) revealed ‘unexpected’ physiological functions of IP3Rs in various tissues when the ‘abnormal’ phenotypes of the mutant mice were compared with the ‘normal’ phenotypes of wild-type mice. The discovery that IP3Rs are involved in exocrine function is one such important finding.

Role of IP3R2, 3 in exocrine secretion

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

We found that IP3R2-R3 double KO (KO) mice exhibited growth abnormalities (Futatsugi et al. 2005). Despite a normal caloric intake, the double mutants were hypoglycemic and lean, suggesting that IP3R2-R3 KO mice had difficulties digesting nutrients. We examined saliva secretion after the subcutaneous injection of pilocarpine, which mimics cholinergic stimulation, and found that salivation was severely impaired in the IP3R2-R3 KO mice. Because salivation in the salivary gland acinar cells is triggered by an increase in the cytosolic Ca2+ concentration [Ca2+]i, we measured the [Ca2+]i in salivary gland cells isolated from IP3R KO mice. Cholinergic stimulation increased the [Ca2+]i in IP3R2 or IP3R3 single KO mice, but the effect was severely diminished in IP3R2-R3 KO mice. Histological analysis of the pancreatic tissues also revealed abnormalities in the IP3R2-R3 KO mice (Futatsugi et al. 2005). The pancreatic acinar cells of the IP3R2-R3 KO mice contained an abnormal accumulation of unreleased zymogen granules. Further examination of IP3R2-R3 KO mice showed that cholinergically mediated [Ca2+]i increase and the secretion of pancreatic amylase was abolished in the double mutants (Fig. 16).

image

Figure 16.  Defective pancreatic exocrine function in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) IP3R2-R3 double knockout (KO) mice. Upper figure: Amylase release from dissociated pancreatic acinar cells stimulated by carbachol (CCh) for 30 min. Release is represented as a percentage of the initial intracellular amylase content (amylase activity in unstimulated cells). The value of each point is the average ± SEM of each genotype (= 6–8). Western blot analysis of amylase expression in the pancreas. Ten microgram of protein was probed with anti-amylase antibody. Amylase content of acinar cells of IP3R2-R3 double KO mice is the same as wild-type mice. Middle figure: Full secretory vesicles are visible in the cytoplasm of pancreatic acinar cells in IP3R2-R3 double KO mice (d, e, f, and g), while wild-type and single KO mice have cytoplasmic areas without vesicles (a, b, and c). Lower figure: Ca2+ signaling is impaired in IP3R2-R3 double KO mice.

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These results demonstrate that IP3R2 and IP3R3 are the major Ca2+ release channels responsible for secretagogue-induced Ca2+ signaling in pancreatic and salivary acinar cells and subsequent digestive enzyme secretion. As type-2 and type-3 IP3Rs are possibly involved in the secretory processes of other various exocrine tissues, such as sweat glands and lacrimal glands (Futatsugi et al. 2005), studying the molecular mechanisms of secretion in tissue regions where the secretory processes remain poorly understood will be important. IP3R2-R3 KO mice show symptoms of dry mouth and dry eye (unpublished observations) that are similar to the characteristics of Sjogren’s syndrome. An analysis of sera from patients with Sjogren’s syndrome revealed that anti-IP3R antibodies were present at a rate of close to 50%, as detected using western blotting (Miyachi et al. 2007). Antibodies against IP3Rs are also present in more than 30% of sera samples from patients with systemic rheumatic disease (Miyachi et al. 2007), suggesting that IP3Rs play an important role in exocrine secretion and that immunoscreening could be used as a diagnostic tool for Sjogren’s syndrome and systemic rheumatic diseases.

Role of IP3R1 in brain-derived neurotrophic factor production

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

We found that cultured Purkinje cells from IP3R1 KO (IP3R1KO) mice exhibited abnormal dendritic morphology (Fig. 17) (Hisatsune et al. 2006). Interestingly, despite the huge amount of IP3R1 expression in Purkinje cells, the IP3R1 in granule cells, not in Purkinje cells, was responsible for the shape of the Purkinje cell dendrites. We also found that brain-derived neurotrophic factor (BDNF) application rescued the dendritic abnormality of IP3R1KO Purkinje cells and that the increase in BDNF expression in response to activation of the AMPA receptor and the mGluR was impaired in IP3R1KO cerebellar granule cells. In addition, we observed abnormalities in the dendritic morphology of Purkinje cells and in the ultrastructure of parallel fiber-Purkinje cell synapses in IP3R1KO mice in vivo. These results indicated that the activation of AMPA receptor and mGluR increases BDNF expression through IP3R1-mediated signaling in cerebellar granule cells, contributing to the dendritic outgrowth of Purkinje cells intercellularly, possibly by modifying the parallel fiber-Purkinje cell synaptic efficacy. Recently, Furuichi’s group reported a new protein, known as Ca2+-dependent activator protein for secretion 2 (CAPS2), is found to be involved in BDNF production in the cerebellum (Sadakata et al. 2004). It would be interesting to know how IP3R and CAPS cooperate with regard to the secretion of BDNF. CAPS-deficient mice exhibit symptoms similar to human psychological disease. Conditioned IP3R1 KO mice should help us to understand the real function of IP3R1 in higher brain functions in greater detail.

image

Figure 17.  (a) Abnormal dendritic morphology of inositol 1,4,5-trisphosphate (IP3) receptor 1-knockout (IP3R1KO) Purkinje cells in vivo. Purkinje cells in acute cerebellar slices from wild-type (WT) and IP3R1KO mice (P17–P20) were loaded with intracellular solution containing fluorescein using a patch pipette to obtain. After 15 min, the dendritic morphology of the Purkinje cells was visualized using a two-photon microscope. Typical images of the dendritic morphology of WT and IP3R1KO Purkinje cells reconstructed from an image stack of serial focal planes are shown (upper panels). The lower panels show magnified views of the areas indicated by the red squares in the upper panels. Right panel: a schematic model of the regulation of dendritc morphology in Purkinje cells by IP3R1 expressed in granule cells. Brain-derived neurotrophic factor (BDNF) production in granule cells by AMPAR stimulation is augmented by the co-stimulation of metabotropic glutamate receptor (mGluR5) through IP3R1-mediated signaling. Then, BDNF production increases the probability of transmitter release from the pre-synaptic terminals of granule cells in an autocrine fashion, leading to an increase in the excitability of Purkinje cells and the subsequent branching of Purkinje cell dendrites.

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Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Several lines of evidence show that synaptic activity regulates the level of IP3R1 expression in neurons. The effect of chronic activity blockade on the localization and level of IP3R1 expression was studied in cultured hippocampal neurons. The chronic blockade of NMDA receptors (NMDARs), one of the major Ca2+-permeable ion channels, increased the number of neurons that expressed a high level of IP3R1 without any apparent changes in its intracellular localization. The up-regulation of IP3R1 depended on transcription and protein synthesis and required cAMP-dependent protein kinase activity. Moreover, although most of the control hippocampal neurons did not respond to mGluR stimulation, chronically NMDAR-blocked neurons with high IP3R1 expression levels became sensitive to mGluR stimulation. These findings suggest that chronic NMDAR blockade increases IP3R1 expression and enhances sensitivity to mGluR stimulation. The change in the IP3R1 expression level in response to alterations in synaptic activity may be an important determinant of the sensitivity of Ca2+ stores to G protein-coupled receptor stimulation and may help to maintain intracellular Ca2+ homeostasis in hippocampal neurons (Cai et al. 2004).

Conclusions

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References

Inositol 1,4,5-trisphosphate receptor is an intracellular signaling center that forms a macro signal complex based on its properties as a scaffold protein (Fig. 18). Biophysical and biochemical studies of the molecular properties of IP3Rs have helped us to realize that various mechanisms contribute to the fine-tuning of IP3R-mediated Ca2+ signal. These mechanisms equip different isoforms, assemble various IP3R-associated molecules, or dynamically change the subcellular localization of the signal. The discovery of numerous binding partners suggests that IP3Rs form a macro signal complex and function as a center of multiple signaling cascades. The diversity of Ca2+ signaling patterns and/or subcellular distribution mechanisms of IP3Rs are most likely a product of the components of the IP3Rs-signaling complex, which can differ from cell to cell, and even from subcellular space to subcellular space.

image

Figure 18.  Summary of the roles of inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ signaling. IP3 receptor (IP3R) is a Ca2+ channel that enables the release of Ca2+ from the endoplasmic reticulum (ER). Released Ca2+ plays various roles in cell function. In addition, IP3R works as a scaffold protein associating directly with various functional molecules to form a macro signal complex. The associated molecules also exert various functions. Altogether, IP3R functions as a signaling center inside the cell. The molecules indicated in the parentheses associate directly with IP3R.

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Our ultimate goal is to understand the precise role of IP3R-mediated Ca2+ signaling in recognition, learning and memory, and consciousness. By focusing our study on the regulation of IP3R-mediated Ca2+ signaling using the various mechanisms described in this review, we expect to elucidate the molecular basis of IP3R function in numerous brain developmental processes and brain functions. To achieve this goal, the study of IP3Rs needs to remain diverse, especially when looking at the involvement of IP3Rs in signaling complexes.

References

  1. Top of page
  2. Abstract
  3. From P400 protein to IP3 receptor
  4. Unique structure of the IP3-binding core domain
  5. N-terminal IP3-binding suppressor domain is responsible for isoform-specific IP3 binding
  6. Three-dimensional structure of IP3R
  7. Gating mechanism of the IP3R channel
  8. Development of IP3 sensor, IRIS, and monitoring of IP3 dynamics in living cells
  9. IP3Rs form macro signal complexes
  10. Homer
  11. Protein 4.1N regulates translocation and lateral diffusion of IP3R1
  12. ER luminal redox sensor, ERp44
  13. Huntingtin-associated protein-1a
  14. Carbonic anhydrase-related protein
  15. Protein phosphatases, PP1and PP2A
  16. Ankyrin
  17. Chromogranin A and B
  18. RACK1 and cytochrome c
  19. Four intracellular trafficking pathways involving IP3Rs
  20. mRNA transport as a component of the mRNA granule
  21. Kinesin-dependent transport of IP3R in vesicular ER subcompartments along microtubules
  22. Lateral diffusion of IP3R1 regulated by actin filaments and 4.1N
  23. Cluster formation of IP3Rs on ER membranes
  24. New signaling pathway
  25. Na,K-ATPase binds to IP3R and regulates Ca2+ oscillation
  26. Amplification of Ca2+ signaling by diacylglycerol-mediated IP3 production
  27. Physiological functions of three IP3R isoforms
  28. Role of IP3R2, 3 in exocrine secretion
  29. Role of IP3R1 in brain-derived neurotrophic factor production
  30. Activity-dependent gene expression of IP3R1 controls mGluR-induced Ca2+ dynamics in hippocampal neurons
  31. Conclusions
  32. References
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