Patent Application
20050181450
The present invention relates to a novel protein which binds to an IP3 receptor. This protein is useful as an IP3 indicator intracellularly and in a cell-free system.
The hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to cell surface receptor activation leads to the production of an intracellular second messenger, inositol 1,4,5-trisphosphate (IP3). IP3 induces the release of Ca2+ from intracellular Ca2+ storage organelles, mainly the endoplasmic reticulum, by binding to the IP3 receptor (IP3R). In these IP3/Ca2+ signalling cascades, the IP3 receptor works as a signal converter to convert the IP3 signal into the Ca2+ signal (1-3).
The IP3 receptor is a tetrameric intracellular IP3-gated Ca2+ release channel (3, 4). There are three distinct types of IP3 receptor in mammals (5-7). The IP3 receptor Type I (IP3R1) is highly expressed in the central nervous system, particularly in the cerebellum (8, 9). Mouse IP3R1 is composed of 2749 amino acids (5), and is divided into three functionally distinct regions: the IP3-binding domain near the N terminus, the channel-forming domain with six membrane-spanning regions close to the C terminus, and the regulatory domain separating the two regions (10, 11). Deletion mutagenesis analysis of the IP3-binding domain has shown that amino acid residues 226 to 578 of the IP3 receptor are of the minimum area required for specific and high affinity ligand binding, thus assigned to the IP3 binding core (12). The precise gating mechanism of the IP3 receptor channel triggered by IP3 remains unclear, but IP3 binding induces an undefined conformational change in the IP3 receptor, which may cause channel opening (10). Besides the channel opening, such IP3-induced conformational change has been supposed to be responsible for degradation of the IP3 receptor (13, 14).
The increase in the cytoplasmic Ca2+ concentration resulting from IP3 receptor activation regulates the activities of a variety of downstream target molecules. These downstream target molecules play key roles in many aspects of cellular responses, including fertilization, development, proliferation, secretion and synaptic plasticity (1, 2, 15). To control such a vast array of cell functions, Ca2+ signals need to be precisely regulated in terms of space, time and amplitude (2, 15). Such a complex regulation of Ca2+ signals has been partly attributed to the diversity of IP3 receptor isoform expression, assembly of heterotetrameric complexes of IP3 receptor isoforms, subcellular distributions of IP3 receptors, and regulation of IP3 receptors by Ca2+ itself, ATP and phosphorylation (3, 4, 16). IP3 receptor channels are also regulated by their interacting proteins (4, 17), including calmodulin (18, 19), FKBP12 (20-24), calcineurin (21, 23, 24, 25), ankyrin (26-28), the σ-1 receptor (28), chromogranin A and B (29-31), IRAG (32), Fyn (33), BANK (34) and the like. Moreover, a family termed CaBP has been shown to interact with the IP3 receptor in a Ca2+-dependent manner, and to directly activate the IP3 receptor in the absence of IP3 (35). The IP3 receptor has also been demonstrated to be physically coupled to its upstream or downstream signaling molecules by protein-protein interactions. For example, the IP3 receptor is coupled with group I metabotropic glutamate receptors (mGluRs) via Homer family of proteins (36), and with B2 bradykinin receptors (B2Rs) by unknown mechanism (37). Activation of mGluRs and B2Rs lead to the production of IP3 in close proximity to the IP3 receptor, resulting in efficient and specific signal propagation.
As described above, the IP3 molecule is an important second messenger that controls a variety of cell functions, and changes in its intracellular concentration may be under temporal and spatial control. To date, a method using [3H]IP3 as an indicator effective for quantifying IP3 in vitro has been developed, but such a method is not simple because of, for example, the need of radioactive isotopes. Further, unlike the case of other second messengers such as Ca2+ and cAMP, the existing methods for sequentially detecting changes in IP3 concentration within a living cell all involve monitoring transfer from the cell membrane of GFP-pleckstrin homology domain to cytoplasm. This method is not necessarily effective, because it is indirect and has problems in terms of quantitative ability, spatial resolution and the like. These are obstructions to intracellular IP3 dynamics analysis.
The present invention provides a novel IP3 receptor-binding protein that can be an effective indicator for the detection and quantification of IP3, and a method for detecting and/or quantifying IP3 using the protein. In addition, this protein works as a messenger molecule after it is released from IP3 receptor with IP3 and this protein binds to Na, bicarbonate co-transporter and PIP kinase to produce PIP2. Therefore, it works to modulate the function of PIP2 and Na+ and bicarbonate ions. Since this protein binds to the same site as IP3, and competes with IP3, this protein can be used as a modulator of IP3 induced Ca2+ release.
To address the above problems, we have noticed and eagerly studied IP3 receptor-binding proteins, particularly a molecule which interacts with the IP3 receptor and whose interaction with the IP3 receptor is controlled by IP3. Using an affinity column, to which a protein of the N-terminal 2217 amino acid residues corresponding to the large portion of the cytoplasm region of IP3R1 (the amino acid sequence is represented by SEQ ID NO: 7) are bound, and eluting a protein bound to the column with IP3, we have identified a novel IP3 receptor-binding protein, and named it IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate). IRBIT binds to IP3R1 in vitro and in vivo, and the subcellular localization agrees well with that of IP3R1. Further, it was also revealed that IRBIT is dissociated from IP3R1 by IP3 at a physiological concentration. Based on these results, we have considered that IRBIT is useful as an IP3 indicator, modulator of IP3-induced Ca2+ releasing activity, and modulator of PIP2 function and Na2+ and bicarbonate ions, and thus completed the present invention.
The present invention relates to the following embodiments:
What is claimed is:
The present invention will be detailed described.
A novel IP3 receptor protein, IRBIT, that we have newly cloned comprises the amino acid sequence represented by SEQ ID NO: 1. The IRBIT is composed of two reagions, the N-terminal region (amino acids 1-104) and the C-terminal region (amino acids 105-530). The C-terminal region is homologous to S-adenosylhomocysteine hydrolase but does not show enzyme activity, and the N-terminal region is an essential region for binding with the IP3 receptor (in this specification, the IP3 receptor is referred to as any subtype of the IP3 receptor including IP3R1 and IP3 receptor type II (IP3R2)). The characteristics of the protein are as follows:
Therefore, the present invention encompasses IRBIT protein, the N-terminal region (amino acids 1-104) that is essential for binding of IRBIT to the IP3 receptor, and proteins containing these amino acid sequences. In some cases, a protein containing the 1-277 amino acid sequence of IRBIT is preferred. The present invention also encompasses a protein, which is a mutant of these proteins that maintain IP3 receptor-binding activity, and comprises an amino acid sequence derived from the amino acid sequence of IRBIT, which contains a deletion, substitution or addition of at least one of amino acids, preferably 1 to 50, more preferably 1 to 20, further preferably 1 to 10, and particularly preferably 1 to 5 amino acid(s).
The present invention further encompasses genes encoding these proteins. The gene is, for example, DNA comprising the nucleotide sequence represented by SEQ ID NO: 2 encoding IRBIT protein, or comprising a nucleotide sequence of the 1-312 nucleotides of SEQ ID NO: 2 encoding the N-terminal region (amino acids 1-104) that is the IP3 receptor-binding domain of IRBIT. The present invention also includes these DNAs or DNAs hybridizing under stringent conditions to complementary strands of the DNAs. Here, the stringent conditions are referred to as, for example, conditions with a sodium concentration of 10 mM to 300 mM, and preferably 37 to 65° C., and more preferably 42° C. Alternatively, such conditions can be achieved using ECL™ direct nucleic acid labeling and detection system (Amersham Pharmacia) according to the description of the instructions included with the system. The present invention also includes a DNA comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of the 1-312 of SEQ ID NO: 2, which contains a deletion, substitution or addition of at least one of nucleotides, and encoding a protein having IP3 receptor-binding activity. Here, the number of nucleotides to be deleted, substituted or added is not specifically limited, and is preferably 1 to 100, more preferably 1 to 50, further more preferably 1 to 20, and most preferably 1 to 10. The nucleotide sequence of such a nucleic acid has homology to the nucleotide sequence represented by SEQ ID NO: 1 when calculated using BLAST or the like (for example, when the default of BLAST, specifically the parameters of initial conditions are used) of 70% or more, preferably 90% or more, further more preferably 95% or more, 96% or more, 97% or more, 98% or more or 99% or more. The present invention further encompasses RNAs corresponding to these DNAs, a vector containing these DNAs, a vector wherein a promoter is operably linked to the DNA so as to enable the expression of the above DNA, a vector wherein a DNA encoding any label for labeling the expression product (IRBIT molecule or the like) of the above DNA that is ligated in-frame to the DNA, and a host cell containing the vector (insect cells, Escherichia coli and mammalian cells are preferred, but examples are not limited thereto). Examples of the above labels may be any known labels and include, but are not limited to, labels for utilizing immunological reaction or protein-protein binding such as a histidine tag (His-tag), glutathioneS-transferase (GST) and biotin, as well as fluorescent proteins or photoproteins such as green fluorescent protein (GFP), CFP, Venus, F1AsH, ReAsH and derivatives thereof. Further, IRBIT is a protein that can be dissociated from the IP3 receptor by IP3 selectively in a concentration-dependent manner. For example, when the IP3 receptor is the IP3 receptor type I, the EC50 value for the above dissociation is approximately 0.5 μM. This is higher than a Kd value (Kd=83 to 100 nM: see 60, 61) during the binding of IP3R1 to IP3 measured by IP3 binding assay according to a conventional method. However, the difference may be due to a difference in buffer conditions (this assay has been performed under conditions ranging from pH 8.0 to 8.3 and with low ion intensity) based on the fact that the binding affinity of IP3 with the IP3 receptor is largely dependent on pH and ion intensity (62-64). Studies according to a method using a surface plasmon resonance biosensor, which is different from a conventional method, showed a Kd value of 336 nM, when the N-terminal region of IP3R1 (amino acids 1-604) was measured under physiological conditions (pH 7.4 and 150 mM NaCl) (64), suggesting that the affinity in this case was approximately 7.5-fold lower than the Kd value determined by a conventional assay. This value is also close to the EC50 of IRBIT (approximately 0.5 μM: measured under conditions of pH 7.4 and 100 mM NaCl) required for its dissociation from the interaction with IP3R1 (In the present invention, GST fusion protein was used for the convenience of the experiment, and this protein is referred to as GST-EL in this specification). Taken together with these findings, it can be considered that IRBIT is dissociated from the IP3 receptor by binding of IP3 to the IP3 receptor. This is supported by data, such as the results of measurement made by Luzzi et al. using a detector cell/capillary electrophoresis system, wherein the intracellular IP3 concentration was tens of nM before stimulation, and then increased to few μM after stimulation (50). As described above, the EC50 of IP3 required for the dissociation of IRBIT from IP3R1 is approximately 0.5 μM, and this value is within the fluctuation range of the intracellular IP3 concentration before and after stimulation. Thus, it is strongly suggested that IRBIT is dissociated from the IP3 receptor following IP3 production induced by extracellular stimulation.
With the characteristic of IRBIT that it is dissociated from the IP3 receptor by IP3 in a concentration-dependent manner, a protein containing at least IRBIT or its IP3 receptor-binding domain can be used as a novel IP3 indicator. Specifically, for example, using IRBIT, a sample is allowed to contact with an IRBIT-IP3 receptor complex (the IP3 receptor in this case is not necessarily a complete molecule, but may be a fragment containing at least a region to which both IP3 and IRBIT can bind (hereinafter, referred to as IP3BD)). Then, the amount of IRBIT that has been dissociated from the IP3 receptor is quantified, or conversely the amount of IRBIT-IP3 receptor/IP3BD complex remaining after contact with IP3 is quantified, so as to make it possible to quantitatively detecting IP3. The same result is obtained when a protein containing at least the IP3 receptor-binding domain of IRBIT is used.
Quantitative detection of free IRBIT or an IRBIT-IP3 receptor complex can also be performed by immunological techniques.
The term “antibody” in the present specification includes complete antibody molecules, fragments retaining antigen-binding ability derived from the complete antibody, and derivatives thereof. Antibodies against IRBIT may be monoclonal or polyclonal antibodies, both of which can be obtained by any general method that involves immunizing animals with an IRBIT molecule or a peptide (a part of the IRBIT molecule). A purification method of these antibody molecules and a purification method which involves cleaving into antibody fragments having binding affinity for IRBIT are also known by persons skilled in the art. Any appropriate method can be selected from these methods, as desired. Persons skilled in the art can readily detect free IRBIT using these antibody molecules. Persons skilled in the art can also readily perform a method for detecting an IRBIT-IP3 receptor/IP3BD complex using the antibody molecules. For example, IP3R1 is previously bound to a solid phase support (beads, assay plate surfaces or the like), and then IRBIT bound via IP3R1 to the solid phase can be detected.
Further, IRBIT is labeled with an appropriate molecule, and then the labels are detected, so that free IRBIT or an IRBIT-IP3 receptor/IP3BD complex can be quantitatively detected. Examples of known molecules for labeling proteins include radioactive labels, immuno-labels, dye labels, fluorescent labels and luminescent labels. Labeling can be performed by binding any one of these labeling molecules to an IRBIT molecule by any known method according to the labeling molecule. Persons skilled in the art can also readily detect signals derived from the labels used herein based on the known art.
First, a complex (referred to as an IRBIT-IP3 receptor/IP3BD complex) comprising a protein that contains at least IRBIT or the IP3 receptor-binding domain thereof and the IP3 receptor or IP3BD is prepared. Since IRBIT forms a complex with the IP3 receptor intracellularly, it can also be purified from natural cells (cerebellum neurons and the like) expressing the IP3 receptor. Alternatively, from cells (Escherichia coli, Sf9 insect cells or the like) forced to express an IRBIT or a protein containing at least the IP3 receptor-binding domain of the IRBIT and a protein containing IP3 receptor molecules or IP3BD, a microsome fraction can be fractionated by a biochemical fractionation method. Further alternatively, after forced expression of the above protein having molecular labels (histidine tag and the like) attached thereto, the protein can be extracted using the labels from a cell lysate. In some cases, other known purification methods (column chromatography and the like) may also be used.
Moreover, IRBIT and IP3PD are separately prepared, and then a complex thereof can be formed. Specifically, for example, GST-IP3BD expressed in Escherichia coli is purified with glutathione-Sepharose, and then a protein containing IRBIT or its IP3 receptor-binding region is expressed in Escherichia coli or Sf9 cells. Proteins containing the IRBIT or its IP3 receptor-binding region are allowed to contact with GST-IP3BD to form complexes, followed by glutathione-Sepharose pull-down, so that the complexes can be purified. Further alternatively, the above complex may also be prepared by pulling down IRBIT in the tissue with GST-IP3BD using glutathione-Sepharose from, for example, a high salt extract from the cerebellum.
A sample containing IP3 is allowed to contact with the thus prepared IRBIT-IP3 receptor/IP3BD complex (prepared, for example, at approximately 1 to 100 μM in 10 mM Hepes, 100 mM NaCl, 2 mM EDTA and 1 mM 2-ME (pH7.4)), followed by incubation on ice for approximately 5 to 30 minutes. Then, free IRBIT or the remaining IRBIT-IP3 receptor/IP3BD complex present in the solution is detected. The IRBIT-IP3 receptor/IP3BD complex to be used in reaction is previously bound to solid phase carriers, and then the solid phase carriers are separated from the liquid phase after reaction, so that free IRBIT in the liquid phase can be detected, or IRBIT in the IRBIT-IP3 receptor/IP3BD complex bound to the solid phase carriers can be detected. IRBIT may be detected with an immunological reaction system using an antibody that recognizes IRBIT. When labeled IRBIT is used, IRBIT can also be detected by detecting the label.
Furthermore, in an embodiment of the present invention, IP3 can also be quantified by a method using fluorescence resonance energy transfer, FRET.
Fluorescence resonance energy transfer (FRET) is a phenomenon wherein when two fluorephores are in close proximity and in the right orientation, excitation energy transfers from one fluorophore (energy donor) to the other fluorophore (energy acceptor) on the longer wavelength side. By labeling two molecules with certain 2 types of fluorescent molecules, changes in the interaction between the two molecules can be measured as changes in FRET efficiency. Further, changes in the concentration of another molecule that regulates the interaction between molecules can be measured.
Based on the FRET method, a method (72) for detecting changes in intracellular Ca2+ concentration, a method (71) for detecting changes in cAMP concentration, and the like have been developed.
IRBIT binds to the IP3BD of the IP3 receptor, and is dissociated from the IP3 receptor by IP3. Using this characteristic, IRBIT and IP3BD are labeled respectively with a combination of 2 types of fluorescent labels that are applicable to FRET. Thus, changes in in vitro or intracellular IP3 concentration can be detected. Specifically, for example, IRBIT is labeled with a yellow fluorescent protein (YFP) or with the improved protein thereof, Venus, and IP3BD is labeled with cyan fluorescent protein (CFP). In the absence of IP3, IRBIT binds to IP3BD and FRET occurs from CFP to Venus. However, in the presence of IP3, the two are dissociated from each other, and no FRET occurs. Further, when Venus-IRBIT and CFP-IP3BD are bound via a linker sequence (Venus-IRBIT-IP3BD-CFP), the molar ratios of the two will be the same, and this is efficient. When IP3 concentration is measured in vitro, Venus-IRBIT-IP3BD-CFP protein is added to a cell extract or the like, and then FRET is measured. Further, introduction of a Venus-IRBIT-IP3BD-CFP gene into a cell enables temporal and spatial analysis of changes in IP3 concentration within living cells. Measurement with the FRET method can be performed using a fluorescent microscope, fluorometer or the like.
Examples of IP3 detection methods using the FRET method will be described in more detail as follows.
First, a Venus-IRBIT-IP3BD-CFP expression vector is prepared. Four cDNAs are amplified by PCR, each of which encodes a full-length IRBIT or a deletion mutant thereof containing a region sufficient for binding with IP3BD (e.g., a mutant comprising amino acid sequence 1-104 or amino acid sequence 1-277), a protein containing the IP3 binding region of IP3 receptor type I (a region comprising amino acid sequence 224-604 of SEQ ID NO: 7), Venus, and CFP. Then these 4 cDNAs are inserted into a mammalian cell expression vector (e.g., pcDNA3) so as to match the reading frames. The order for insertion is not specifically limited. For example, the order of Venus-IRBIT-IP3BD-CFP, wherein IRBIT and IP3BD are placed inside and Venus and CFP are placed outside, can be employed. For preparation of a recombinant protein, the cDNAs are inserted into an Escherichia coli expression vector (e.g., pET) or Sf9 expression vector (e.g., pFastBac). In this case, a tag (e.g., a His tag) can be linked to the N-terminus or the C-terminus for purification.
Venus-IRBIT-IP3BD-CFP is expressed in Escherichia coli or Sf9 cells, and is purified with ProBond resin (Invitrogen) or the like when a His tag is added. In some cases, it may be purified with an ion exchange column or a gel filtration column.
An IP3 solution having a known concentration is added to the purified Venus-IRBIT-IP3BD-CFP protein. The excitation wavelength (at around 440 nm) of CFP is applied to the solution, and then the CFP fluorescent wavelength (at around 480 nm, CFP fluorescence) and Venus fluorescent wavelength (at around 535 nm, FRET fluorescence) are measured. A calibration curve is then determined by plotting IP3 concentrations versus FRET fluorescence-to-CFP fluorescence ratios. Venus-IRBIT-IP3BD-CFP protein is added to a sample (e.g., a cell extract solution) with an unknown IP3 concentration to find a FRET fluorescence-to-CFP fluorescence ratio, and then the ratio is applied to the calibration curve, thereby quantifying IP3 concentration.
The IP3 concentration in a living cell can also be measured by the FRET method. First, a pcDNA-Venus-IRBIT-IP3BD-CFP gene is transfected into a culture cell (e.g., Cos-7 and HeLa) using a transfection reagent (e.g., TransIT (Mirus)). On 1-3 days after the transfection, cells on the glass bottom dish are observed using an inverted fluorescence microscope equipped with a cool CCD camera (e.g., IX71 (Olympus)). The CFP excitation wavelength (at around 440 nm) is applied, and then the CFP fluorescent wavelength (at around 480 nm, CFP fluorescence) and Venus fluorescent wavelength (at around 535 nm, FRET fluorescence) are measured. The FRET efficiency is quantified by taking the ratio of the two fluorescence intensities. An IP3-producing agonist (ATP in the case of Cos-7, histamine in the case of HeLa, and the like) is added to the glass-bottom dish, and then the FRET efficiency is measured with time. When IP3 is produced by the agonist, the FRET-to-CFP ratio is expected to be lowered.
The use of a combination of fluorescent labels, Venus and CFP, is as exemplified above. A combination of YFP and CFP or F1AsH and CFP can also be used, and use is not limited thereto. Further, various fluorescent molecules to be used for the FRET method are expected to be developed in the future, and they can also be used for implementing the present invention.
The present invention also encompasses the IP3 indicator of the present invention or a kit for IP3 detection to be used for the IP3 detection method of the present invention. The indicator or the kit contains a protein containing at least the IP3 receptor-binding region of IRBIT or a DNA or an RNA encoding the protein. In some cases, the indicator or the kit contains a protein containing at least the IP3-binding region of IP3 receptor, or a DNA or an RNA encoding the protein. Further, the indicator or the kit may also contain a labeling reagent (e.g., a fluorescent labeling compound) and/or an antibody that recognizes the above protein.
This specification includes part or all of the contents disclosed in specification and/or drawings of Japanese Patent Application No. 2002-299429, which are priority documents of present application.
The S-adenosylhomocysteine hydrolase activity in the hydrolytic direction of recombinant IRBIT-His (open circle), GST-IRBIT (triangle), GST (square), and S-adenosylhomocysteine hydrolase (closed circle) were measured according to the known method (43). Results are shown as the mean±standard deviation from three independent experimental results.
The present invention will be more particularly described with reference to the examples, but the present invention is not limited by these examples.
Method
Preparation of IP3R1 Affinity Column
The cDNA encoding the N-terminal region (amino acids 1-225) of mouse IP3R1 was inserted into glutathione S-transferase (GST) fusion vector pGEX-KG (40). The GST-IP3R1 (1-225) fragment was subcloned into the baculovirus transfer vector pBlueBac4.5 (Invitrogen). The region located downstream from Sma I site of the GST-IP3R1 (1-225) was replaced with the Sma I-EcoR I fragment of mouse IP3R1 (corresponding to amino acids 79-2217) to construct a GST-IP3R1 (1-2217) vector (termed GST-EL). A fragment encoding GST alone was subcloned into pBlueBac4.5 as a control. Sf9 cells were cultured in TNM-FH medium supplemented with 10% fetal bovine serum at 27° C. Recombinant baculoviruses carrying GST-EL or GST gene were generated with Bac-N-Blue™ Transfection Kit (Invitrogen) according to the instructions thereof. GST-EL and GST were expressed in 2×108 Sf9 cells by infecting recombinant baculoviruses at a multiplicity of infection of 5, and incubating for 48 hours. The cells were harvested and stored at −80° C. The cryopreserved cells were suspended in 10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol (2-ME), 0.1% Triton X-100, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μM leupeptin, 2 μM pepstatin A and 10 μM E-64), and were homogenized with a glass-Teflon homogenizer (1000 rpm, 10 strokes). The homogenate was centrifuged at 20,000×g for 30 minutes. The supernatant was incubated with 3 ml of glutathione-Sepharose 4B (Amersham Pharmacia Biotech) for 3 hours at 4° C. After washing 8 times with 40 ml of 10 mM Hepes (pH 7.4), 250 mM NaCl, 2 mM EDTA, 1 mM 2-ME, and 0.1% Triton X-100, GST-EL or GST coupled with glutathione-Sepharose was packed into columns and equilibrated with 10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, 1 mM 2-ME, and 0.1% Triton X-100. Approximately 5 mg of GST-EL was immobilized.
Purification and Partial Amino Acid Sequencing of IRBIT
Adult rat cerebella (approximately 5 g) were homogenized in 45 ml of a homogenize buffer (10 mM Hepes (pH 7.4), 20 mM Sucrose, 2 mM EDTA, 1 mM 2-ME and protease inhibitors) with a glass-Teflon homogenizer (950 rpm, 10 strokes). The homogenate was centrifuged at 1,000×g for 10 minutes, and then the supernatant (S1 fraction) was further centrifuged at 100,000×g for 60 minutes to obtain the cytosolic fraction (the supernatant) and the crude microsome (the pellet). The crude microsome was homogenized in 25 ml of a homogenize buffer containing 500 mM NaCl with a glass-Teflon homogenizer (1,200 rpm, 10 strokes), incubated on ice for 15 minutes, and then centrifuged at 100,000×g for 60 minutes to obtain the high salt extract (supernatant) and the stripped-crude microsome (the pellet). The high salt extract was diluted 5-fold with a dilution buffer (10 mM Hepes (pH 7.4), 2 mM EDTA, 1 mM 2-ME, 0.01% Brij 35 and protease inhibitors). The diluted high salt extract was pre-cleared with glutathione-Sepharose and loaded into GST-EL affinity column equilibrated with a binding buffer (10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA and 1 mM 2-ME). A GST column was used as a control. The columns were washed with the binding buffer in a quantity 20 times greater than the volume of the column and proteins bound to the column were eluted with the binding buffer containing 50 μM IP3 (Dojindo) and 0.05% Brij 35. The eluted material was concentrated, separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE), and stained with Coomassie Brilliant Blue (CBB). The approximate 60-kDa protein band was excised from the gel and digested with lysyl endopeptidase (Wako) according to the known method (41). The digested peptides were separated using a C-18 reversed-phase column (μRPC C2/C18 SC 2.1/10, Amersham Pharmacia Biotech) connected to a SMART system (Amersham Pharmacia Biotech). The amino acid sequence of each peptide was determined by 494 procise protein sequencer (Applied Biosystems). Two peptide sequences, N-YSFMATVTK-C (SEQ ID NO: 3) and N-QIQFADDMQEFTK-C (SEQ ID NO: 4) were obtained.
cDNA Cloning of IRBIT
BLAST searches of the two peptide sequences above, obtained from the 60-kDa protein above against the non-redundant database, revealed that these sequences matched a sequence (Accession number: CAC09285) in the database. Based on the databases of mouse-expressed sequence tags (Accession numbers: AW229870 and BE282170), primers (5′-ATGTCGATGCCTGACGCGATGC-3′ (SEQ ID NO: 5) and 5′-GCGTGGTTCATGTGGACTGGTC-3′ (SEQ ID NO: 6)) homologous to the cDNA were synthesized. The cDNA of IRBIT was amplified by polymerase chain reaction (PCR) using mouse cerebellum oligo dT-primed, first-strand cDNA as a template. The PCR product was cloned in pBluescript II KS(+) (Stratagene) and sequenced. Sequences of the three independent clones were confirmed.
Preparation of Recombinant Proteins
The cDNAs encoding the full-length and N-terminal region (amino acids 1-104) of IRBIT were subcloned into the Escherichia coli hexahistidine (His) fusion vector pET-23a(+) (Novagen) to generate IRBIT-His and IRBIT(1-104)-His expression vectors, respectively. The same cDNAs were subcloned into the GST fusion vector pGEX-4T-1 (Amersham Pharmacia Biotech) to generate GST-IRBIT and GST-IRBIT(1-104) expression vectors, respectively. The cDNA fragments encoding the amino acids 1-225, 1-343, 341-923, 600-1248, 916-1581 and 1553-1943 of the amino acid sequence (SEQ ID NO: 7) of mouse IP3R1 were inserted into pGEX-KG to generate GST-Ia, GST-Iab, GST-IIab, GST-IIbIIIa, GST-IIIab and GST-IV expression vectors, respectively. The amino acids 1593-2217 of mouse IP3R1 were inserted into pGEX-4T-1 to obtain GST-IV-Va expression vector. These fusion proteins were expressed in Escherichia coli. GST-EL was expressed in Sf9 cells as described above. The expressed His-tagged fusion proteins were purified using ProBond resin (Invitrogen), and GST fusion proteins were purified using glutathione-Sepharose. GST-IbIIa (amino acids 224-604 of mouse IP3R1) and its site-directed mutants K508A and R441Q used herein were known previously (see Ref. 42; GST-IbIIa was referred to as G224 in the reference).
Assays for Enzyme Activity
To assay S-adenosylhomocysteine hydrolase activity in the hydrolytic direction, the color developed by the reaction between the product (Homocysteine) and 5,5′-dithiobis (2-nitrobenzoic acid) (Sigma) was measured spectroscopically using the purified IRBIT-His (3.0 μg), GST-IRBIT (4.3 μg), GST (1.3 μg) and rabbit S-adenosylhomocysteine hydrolase (Sigma) (2.4 μg), according to the known method (43). Absorbance at 412 nm was measured 0, 5, 20 and 60 minutes later using a DU 640 spectrophotometer (Beckman). Results are shown as the mean±standard deviation from three independent experiments (
Production of Affinity Purified Anti-IRBIT Antibody
A Japanese White rabbit was immunized with the purified IRBIT(1-104)-His by subcutaneous injection with the complete Freund's adjuvant at 14-day intervals. The anti-IRBIT antisera were passed through a GST-IRBIT(1-104) covalently coupled with cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech), and antibodies specifically bound to the column were eluted with 100 mM glycine-HCl (pH 2.5).
Subcellular Fractionation and Immunoblotting
The cerebrum, cerebellum, heart, lung, liver, kidney, thymus, spleen, testis and ovary were removed from adult mice and S1 fraction was obtained as described above. The cytosolic fraction, crude microsome, high salt extract, and stripped-crude microsome of mouse cerebellum were obtained in a manner similar to the above method. Proteins with the amount indicated in
Generation and Transfection of Mammalian Cell Expression Vectors
The cDNA encoding the full-length IRBIT was subcloned into the pcDNA3 (Invitrogen). The cDNA encoding the full-length IRBIT or its deletion mutants (amino acids 1-277, 1-104 and 105-530) were subcloned into the pEGFP-C1 (Clontech) to generate green fluorescent protein (GFP) fusion protein expression vectors. Mouse IP3R1 expression vector pBact-STneoB-C1 used herein was known previously (44). Cos-7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37° C. Transient transfections were performed using TransIT transfection reagents (Mirus) according to the instructions attached thereto. Two days after transfection, the transfected cells were used for immunoblotting, pulldown experiments, or immunostaining.
In Vitro Binding Experiments
Mouse cerebellar cytosolic fraction was diluted 2-fold with 10 mM Hepes (pH 7.4), 200 mM NaCl, 2 mM EDTA, 1 mM 2-ME and 0.02% Triton X-100. The high salt extract was diluted 5-fold with 10 mM Hepes (pH 7.4), 2 mM EDTA, 1 mM 2-ME and 0.01% Triton X-100. The diluted fractions (the final NaCl concentration of both fractions was 100 mM) were incubated with 20 μg of GST-EL or GST for 2 hours at 4° C. After adding 10 μl of glutathione-Sepharose and another 2 hours of incubation, the resins were washed 5 times with a wash buffer (10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, 1 mM 2-ME, and 0.01% Triton X-100), and bound proteins were eluted with 20 mM glutathione.
The eluted proteins were analyzed by Western blotting with anti-IRBIT antibody. For dephosphorylation, the diluted high salt extract was incubated in the presence of or the absence of Escherichia coli alkaline phosphatase (Toyobo) with 2 mM MgCl2 for 30 minutes at 37° C. 5 mM EDTA was added, and then the resultant was subjected to pulldown assay as described above.
For dissociation experiments, IRBIT in the diluted high salt extract was pulled down with GST-EL for precipitation and washed as described above. To resins, 100 μl of a wash buffer containing IP3, inositol 4,5-bisphosphate (IP2) (Dojindo), inositol 1,3,4,5-tetrakisphosphate (IP4) (Calbiochem), inositol 1,2,3,4,5,6-hexakisphosphate (IP6) (Calbiochem) or ATP (Amersham Pharmacia Biotech) (0.1, 0.3, 1, 3 or 10 μM) were added. After incubation on ice for 10 minutes, samples were centrifuged at 10,000 rpm for 1 minute, and the supernatant was subjected to immunoblot analysis with anti-IRBIT antibody or goat anti-GST antibody (Amersham Pharmacia Biotech). For quantification, Alexa 680-conjugated goat anti-rabbit IgG (Molecular Probes) was used as a secondary antibody. The fluorescence intensity of the immunoreactive bands of IRBIT was measured using the Odyssey infrared imaging system (Aloka). Quantitative data (the mean±SD from at least three independent experiments) is expressed as a percentage of the amount of IRBIT in 10 μM IP3 eluate.
For the determination of the IRBIT binding region and the critical amino acid residue of IP3R1, the diluted high salt extract was subjected to pulldown assay as described above with 100 pmol of GST, GST-EL, GST-Ia, GST-Iab, GST-IbIIa, GST-IIab, GST-IIbIIIa, GST-IIIab, GST-IV, GST-IV-Va, K508A or R441Q, and analyzed by Western blotting with anti-IRBIT antibody.
For the determination of the IP3R1-interacting region of IRBIT, Cos-7 cells expressing GFP-tagged full-length IRBIT or its truncated mutants were lysed in a lysis buffer (10 mM Hepes (pH7.4), 100 mM NaCl, 2 mM EDTA, 1 mM 2-ME, 0.5% Nonidet P-40, and protease inhibitor) for 30 minutes at 4° C., followed by centrifugation (100,000×g, 30 minutes). The supernatants were subjected to pulldown assay with GST-EL or GST as described above, and bound proteins were subjected to immunoblot analysis with anti-GFP antibody (Medical & Biological Laboratories).
Indirect Immunofluorescence and Confocal Microscopy
Transfected Cos-7 cells cultured on glass cover slips, washed once in phosphate-buffered saline (PBS), fixed in 4% formaldehyde-containing PBS for 15 minutes, permeabilized in 0.1% Triton X-100-containing PBS for 5 minutes, and then blocked in PBS containing 2% goat serum for 60 minutes at room temperature. For washing out cytosolic proteins, the cell membranes of transfected cells were punctured in an ice-cold permeabilization buffer (80 mM PIPES (pH 7.2), 1 mM MgCl2, 1 mM EGTA and 4% polyethylene glycol) containing 0.1% saponin, and washed twice with the ice-cold permeabilization buffer, followed by fixation. The cells were allowed to react with rabbit anti-IRBIT antibody (1 μg/ml, room temperature, 60 minutes) and rat anti-IP3R1 antibody 18A10 (45) (overnight at 4° C.). After four times of 5 minutes of washing with PBS, Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-rat IgG (Molecular Probes) were applied to reaction for 45 minutes at 37° C. After 4 instances of washing (5 minutes each) with PBS, the cover slips were mounted with Vectashield (Vector Laboratories) and observed via IX-70 confocal fluorescence microscopy (Olympus) with a 60× objective lens.
Immunoprecipitation
Detergent-solubilized extract from the crude microsome was prepared with 1% Nonidet P-40-containing 50 mM Hepes (pH 7.4), 1 mM MgCl2, and protease inhibitors for 30 minutes at 4° C., and centrifuged at 20,000×g for 30 minutes. The supernatant was diluted 10-fold in an immunoprecipitation buffer (10 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM MgCl2, and 1% Nonidet P-40) and incubated with rabbit anti-IRBIT antibody (3 μg) or control rabbit IgG (3 μg) for 2 hours at 4° C. Protein G Sepharose 4 fast flow (Amersham Pharmacia Biotech) was added for reaction with immune complexes for 2 hours at 4° C. Resins were washed three times with an immunoprecipitation buffer and subjected to Western blotting with anti-IRBIT antibody or mouse anti-IP3R1 antibody KM1112 (47).
Results
Purification and cDNA Cloning of a Novel IP3R-binding Protein
To identify IP3 receptor-interacting molecules, the N-terminal region of 2217 amino acid residues encoding the greater part of the cytoplasmic portion of mouse IP3R1 containing the IP3 binding domain and regulatory domain (10, 11) was used as a fusion protein (GST-EL) with GST. GST-EL and GST were expressed by the baculovirus/Sf9 cell system and conjugated to glutathione-Sepharose. The fraction extracted with a high salt buffer (containing 500 mM NaCl) from the crude microsome of cerebella was thought to be enriched with membrane-bound proteins. The fraction was loaded into a glutathione-Sepharose affinity column in which GST-EL or GST was immobilized. To detect proteins which were dissociated from the IP3 receptor in the presence of IP3, the proteins bound to the affinity columns were eluted using 50 μM IP3. A protein with a molecular mass of approximately 60 kDa was eluted from the GST-EL column (
The C-terminal region (amino acids 105-530) of IRBIT was shown to be homologous (51% identical and 74% similar) to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (EC3.3.1.1.) (48) (
Because IRBIT had homology with S-adenosylhomocysteine hydrolase, which catalyzes the reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine, whether IRBIT had the same enzyme activity was investigated using recombinant IRBIT expressed in Escherichia coli. C-terminally His-tagged IRBIT (IRBIT-His) and N-terminally GST-tagged IRBIT (GST-IRBIT) were purified, and their enzyme activities were measured in hydrolysis direction. As shown in
Tissue Distribution and Subcellular Localization of IRBIT
An affinity-purified antibody against the N-terminal region of IRBIT (
Next, subcellular localization of IRBIT was examined by fractionation of mouse cerebellum. IRBIT was present in both cytosolic and crude microsome fractions (
IRBIT in High Salt Extract Interacted with IP3R1 and the N-terminal Region of IRBIT was Essential for the Interaction.
IRBIT was present in both cytosolic and peripherally membrane-bound fractions of mouse cerebellum (
To examine the effect of phosphorylation, the high salt extract was treated with alkaline phosphatase, a nonspecific phosphatase, and then incubated with GST-EL or GST. As shown in
Based on the fact that IRBIT has 17 potential phosphorylation sites, seven of which are concentrated on the above N-terminal region, which is necessary for the interaction with the IP3 receptor, it is predicted that phosphorylation may be involved in the interaction with the IP3 receptor. It was hypothesized based on these findings that the dephosphorylated form of IRBIT is free in cytosol, whereas the phosphorylated form of IRBIT is membrane-bound via the interaction with the IP3 receptor. Although it could not be demonstrated yet, the interaction between IRBIT and the IP3 receptor may be dualistically regulated by IP3, and by either direct or indirect phosphorylation.
IRBIT co-localized with IP3R1 on Endoplasmic Reticulum in Cos-7 Cells
To investigate the subcellular localization of IRBIT, Cos-7 cells over-expressing IRBIT and IP3 receptor were analyzed by confocal immunofluorescence microscopy. IRBIT was diffusely distributed in cytoplasm, with no immunoreactivity in nucleus (
IRBIT Interacted with IP3R1 In Vivo
To confirm an association between IRBIT and IP3R1 in tissue, co-immunoprecipitation was performed using a mouse cerebellum. Detergent extracts of the crude microsome of the mouse cerebellum were immunoprecipitated with anti-IRBIT antibody and the immunoprecipitates were analyzed by immunoblotting with anti-IP3R1 antibody. IP3R1 was co-immunoprecipitated with anti-IRBIT antibody, but not with a control antibody (
Physiological Concentration of IP3 Selectively Dissociated IRBIT from IP3R1
IRBIT was originally identified in the eluate from the GST-EL column with 50 μM IP3 (
IRBIT in the high salt extract from the mouse cerebellar crude microsome was trapped with GST-EL. Then elution was attempted with 0.1 to 10 μM IP3, and other inositolpolyphosphates, IP2, IP4, IP6 or ATP. As shown in
The dissociation efficiency of IP3 was approximately 50 times greater than those of other inositol polyphosphates. ATP did not dissociate IRBIT even at a concentration of 10 μM. These results indicate that IRBIT was dissociated from IP3R1 selectively with physiological concentrations of IP3.
IRBIT Interacted with the IP3-binding Region of IP3R1 and Lys-508 of IP3R1 was Essential for the Interaction with both IRBIT and IP3
To examine which region of the IP3-binding region or the regulatory region of IP3R1 was essential for the interaction with IRBIT, 8 types of IP3R1 deletion mutants constructed as GST fusion proteins based on the domain structure of IP3R1 (51) were used (
It is concluded based on the above results that IRBIT is normally associated with IP3R1, and is dissociated from IP3R1 when IP3 concentration is elevated by extracellular stimulation. IRBIT is shown to be a sole IP3 receptor-binding protein, whose interaction with IP3 receptor can be regulated by P3.
IRBIT Lowered the Affinity of IP1R to IP3 in a Phosphorylation Dependent Manner
The effect of IRBIT on binding of IP3 to IP3R was examined. GST-EL (0.2 μg) was incubated with 0.1, 1, or 10 μg of purified His-tagged IRBIT expressed in Sf9 cells or E. coli in a solution containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM 2-mercaptoethanol, for 30 min on ice. Then 8.7 nM [3H]IP3 (PerkinElmer Life Sciences) was added to the samples and incubated for 10 min on ice (total volume was 50 μl). The samples were mixed with 2 μl of 50 mg/ml γ-globulin and 50 μl of 30% PEG6000, 50 mM Tris-HCl (pH 8.0), and incubated for 5 min on ice. Non-specific binding was measured in the presence of 10 μM cold IP3. After centrifugation at 20,000×g for 5 min, the precipitate was dissolved in SOLVABLE™ (Packard) and the radioactivity was measured with a liquid scintillation counter (Beckman Coulter). The result is shown in
IP3-Induced Calcium Release of HeLa Cells was Increased when the Expression of IRBIT was Suppressed with RNA Interference
RNA interference experiment showed that IP3-induced calcium release of HeLa cells was increased when the expression of IRBIT was suppressed with RNA interference (
IRBIT Interacted with type II Phosphatidylinositol Phosphate Kinase.
IRBIT was transfected into Cos-7 cells with Myc-tagged type II phosphatidylinositol phosphate kinase α, β or γ (PIPKII α, β or γ). After two days, cells were lysed in lysis buffer, followed by centrifugation (100,000×g, 30 min). The supernatants were incubated with 3 μg of anti-IRBIT antibody, rabbit IgG, mouse anti-Myc antibody, or mouse IgG for 1 h at 4° C. After adding 5 μl of Protein G beads and another 1-h incubation, the beads were washed five times with lysis buffer and analyzed by Western blotting with anti-IRBIT antibody or HRP conjugated anti-Myc antibody.
Immunoprecipitation of Myc-PIPKII α, β or γ with anti-Myc antibody co-precipitated IRBIT (
IRBIT Interacted with Sodium Bicarbonate Cotransporter.
Mouse cerebellar cytosol fraction or detergent extract of microsome fraction was immunoprecipitated with 50 μg of anti-IRBIT antibody or rabbit IgG. Immunoprecipitates were separated by 10% SDS-PAGE gel, and stained with Coomassie Brilliant Blue (
In summery, the IRBIT protein that we have found herein interacts with the IP3 receptor and the interaction is disrupted by IP3 in an IP3 concentration-dependent manner. Thus, the IRBIT was shown to be a useful protein molecule as an indicator for detecting and quantifying IP3 both intracellularly and in a cell-free system. Further, since the sequence from amino acid residues 1 to 104 of the amino acid sequence of IRBIT is a region essential for binding to IP3, it is considered that in addition to IRBIT, a protein containing at least the amino acid sequence (1-104) is also useful as an indicator for IP3.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2002-299429 | Oct 2002 | JP | national |
