DANGER, a Novel Regulatory Protein of Inositol 1,4,5-Trisphosphate-Receptor Activity

Abstract

We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP3R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP3R. DANGER interacts with IP3R in vitro and co-immunoprecipitates with IP3R from cellular preparations. DANGER robustly enhances Ca2+-mediated inhibition of IP3 RCa2+ release without affecting IP3 binding in microsomal assays and inhibits gating in single-channel recordings of IP3R. DANGER appears to allosterically modulate the sensitivity of IP3 RtoCa2+ inhibition, which likely alters IP3R-mediated Ca2+ dynamics in cells where DANGER and IP3R are co-expressed. We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP3R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP3R. DANGER interacts with IP3R in vitro and co-immunoprecipitates with IP3R from cellular preparations. DANGER robustly enhances Ca2+-mediated inhibition of IP3 RCa2+ release without affecting IP3 binding in microsomal assays and inhibits gating in single-channel recordings of IP3R. DANGER appears to allosterically modulate the sensitivity of IP3 RtoCa2+ inhibition, which likely alters IP3R-mediated Ca2+ dynamics in cells where DANGER and IP3R are co-expressed. The inositol 1,4,5-trisphosphate receptor (IP3 R) 3The abbreviations used are: IP3 (and InsP3), inositol 1,4,5-trisphosphate; IP3R, IP3 receptor(s); ER, endoplasmic reticulum; aa, amino acids; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; siRNA, small interfering RNA; NGF, nerve growth factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; WT, wild type. 3The abbreviations used are: IP3 (and InsP3), inositol 1,4,5-trisphosphate; IP3R, IP3 receptor(s); ER, endoplasmic reticulum; aa, amino acids; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; siRNA, small interfering RNA; NGF, nerve growth factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; WT, wild type. is a large, endoplasmic reticulum (ER) resident protein, which is a key regulator of intracellular Ca2+ signaling (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4394) Google Scholar, 2Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (370) Google Scholar). Inositol 1,4,5-trisphosphate (IP3) is formed in response to the activation of G protein-coupled receptors or receptor tyrosine kinase receptors located in the plasma membrane (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4394) Google Scholar), which elicit IP3R-mediated Ca2+ release from ER stores. The IP3 recognition site of IP3R includes amino acids (aa) 225-578 in the N-terminal portion of the protein, while the Ca2+ channel domain comprises ∼300 aa in the extreme C terminus (2Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (370) Google Scholar). The IP3 binding site and the Ca2+ channel are separated by ∼2,000 aa, providing a large area for interactions with multiple regulatory proteins including calmodulin, chromogranins, glyceraldehyde-3-phosphate dehydrogenase, RACK1, and caldendrin (3Patterson R.L. Boehning D. Snyder S.H. Annu. Rev. Biochem. 2004; 73: 437-465Crossref PubMed Scopus (369) Google Scholar). While these proteins regulate IP3R function in diverse ways, only two regulators have been shown to influence Ca2+ sensitivity. Cytochrome c, which binds to the extreme C terminus of the IP3R, relieves the inhibitory actions of Ca2+ upon the channel (4Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (534) Google Scholar), and Bcl-XL binding to the C terminus also influences the Ca2+ dependence (5White C. Li C. Yang J. Petrenko N.B. Madesh M. Thompson C.B. Foskett J.K. Nat. Cell Biol. 2005; 7: 1021-1028Crossref PubMed Scopus (354) Google Scholar). We have identified a novel vertebrate protein, designated DANGER, which was isolated by yeast two-hybrid analysis with the regulatory region of the IP3R as bait. DANGER physiologically binds to IP3R and allosterically enhances the potency of Ca2+-inhibition of IP3R-mediated Ca2+ release without affecting ligand binding. Yeast Two-hybrid Analysis—The Matchmaker3 yeast-2-hybrid system from Clontech (Palo Alto, CA) was employed. AH109 β-galactosidase yeast was used. IP3R fragments were cloned into pGADT7 (β-galactosidase acceptor domain) vector and were screened against a rat brain and human fetal kidney library (Clontech) as per the manufacturer's specifications. Expression of these fragments was determined by Western blotting using antibodies from Clontech, corresponding to the expression vector. Positive clones grew on minimal SD agar (Clontech) lacking adenine, histidine, leucine, and tryptophan and had β-galactosidase activity. Calcium Release Measurements and Electrophysiology—Calcium release through recombinant type I (SII+)IP3R was measured exactly as described previously (13Boehning D. Mak D.O. Foskett J.K. Joseph S.K. J. Biol. Chem. 2001; 276: 13509-13512Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 14Joseph S.K. Bokkala S. Boehning D. Zeigler S. J. Biol. Chem. 2000; 275: 16084-16090Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Calcium concentrations in all solutions were calibrated with a calcium-selective minielectrode, and confirmed with fluorescent dyes. When the effects of DANGER were examined, it was added simultaneously with IP3. In the absence of IP3, SERCA activity was not affected by the addition of DANGER. At the concentrations used in this study, DANGER did not change the calcium concentration of the assay solution as determined by a calcium minielectrode. Spodoptera frugiperda (Sf9) cells (Invitrogen) were grown and maintained in SF-900II serum-free media (Invitrogen) as described previously (9Ionescu L. Cheung K.H. Vais H. Mak D.O. White C. Foskett J.K. J. Physiol. (Lond.). 2006; 573: 645-662Crossref Scopus (56) Google Scholar). An ice-cold nuclear isolation solution, containing (in mm): 140 KCl, 250 sucrose, 1.5 β-mercaptoethanol, 10 Tris-HCl (pH to 7.4), with complete protease inhibitor mixture (Roche Applied Science) and 0.05 mm phenylmethylsulfonyl fluoride, was added to the flask, and the cells were detached by gentle scraping. Homogenization of 1-2 ml of the mixture was performed using 2-4 strokes of the pestle in an ice-cold Dounce homogenizer. 20-30 μl of the homogenized mixture was added to 1 ml standard bath solution (in mm: 140 KCl, 10 HEPES, 0.5 BAPTA, pH 7.3, and free [Ca2+] adjusted to ∼300 nm) in an experimental chamber on the stage of an inverted microscope. Isolated nuclei are 5-10 μm in diameter and were distinguished from intact cells based on their unique morphology (9Ionescu L. Cheung K.H. Vais H. Mak D.O. White C. Foskett J.K. J. Physiol. (Lond.). 2006; 573: 645-662Crossref Scopus (56) Google Scholar). The standard pipette solution contained (in mm): 140 KCl, 0.5 Na2ATP, 10 HEPES, pH 7.3, 0.07 mm Ca2+, and 100 nm InsP3. All solutions were carefully buffered to desired free [Ca2+] (8Mak D.O. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (226) Google Scholar) confirmed by fluorometry. Data were acquired as described (8Mak D.O. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (226) Google Scholar). Segments of current traces exhibiting one or two InsP3R channels were used for open probability (Po) determinations (15Mak D.O. Foskett J.K. J. Gen. Physiol. 1997; 109: 571-587Crossref PubMed Scopus (142) Google Scholar). Single channel traces were used for dwell time analyses by QuB software (16Qin F. Auerbach A. Sachs F. Biophys. J. 2000; 79: 1915-1927Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The number of active channels in nuclear membrane patches (NA) and the mean channel activity duration Ta were determined as described (9Ionescu L. Cheung K.H. Vais H. Mak D.O. White C. Foskett J.K. J. Physiol. (Lond.). 2006; 573: 645-662Crossref Scopus (56) Google Scholar). All data are averages of at least six experiments performed at the same [InsP3] and [Ca2+]i. Error bars indicate S.E. Cell Lines and Expression Protocols—Human HEK cells were obtained from ATCC and cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 1% penicillin-streptomycin. PC12 cells were cultured in Dulbecco's minimal essential medium supplemented with 10% horse serum, 5% fetal bovine serum, 2 mm l-glutamine, and 1% penicillin-streptomycin. Transfection of PC12 and HEK with either 100 nm small interfering RNA (siRNA) duplex and 1 μg YFP, or 1 μg of DANGER and 1 μg of YFP, was performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's specifications. Expression Constructs—The type 1 IP3R SII+ splice variant and SERCA-2b in pcDNA3.1 have been described elsewhere (17Boehning D. Joseph S.K. J. Biol. Chem. 2000; 275: 21492-21499Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and were kindly provided by Dr. Suresh K. Joseph (Thomas Jefferson University, Philadelphia, PA). DANGER was cloned from human, rat, and mouse cDNA libraries generated from brain using the Roche reverse transcriptase. PCR was performed using 5′ and 3′ 21-mer primers corresponding to the following sequences human (GI: 29789287), Mouse (GI: 39645725), and Rat (GI:30466774), which have 9-mer overhangs on each end to engineer the appropriate restriction sites. Two siRNA sequences were used for DANGER deletion, each with similar efficacy: 5′-aagaatgccccagcgctcatt-3′ (human) and 5′-aatacgagtttgaccttgctt-3′ (rat). Antibody Generation—Rabbit polyclonal antiserum against DANGER was generated by injecting New Zealand White male rabbits with peptide RAEPLNLFRPFVLQR SLYR coupled to keyhole limpet hemocyanin (Pierce). Initial injection was with Complete Freund's adjuvant, and boost injections at days 14, 21, and 49 were with Incomplete Freund's Adjuvant. Rabbit injections, bleeds, and housing were performed by Cocalico Biologicals (Reamstown, PA). Monoclonal type 1 IP3R antibody m18A10 was a kind gift form K. Mikoshiba. Anti-Myc antibody was from Sigma. Subcellular Fractionation—After inducing differentiation by NGF, cells were harvested by gently scraping plates with a cell scraper, and were washed once with cold PBS. The washed pellet was subjected to one freeze-thaw cycle in liquid nitrogen. Pellets were resuspended in 1 ml of Buffer A (250 mm sucrose, 10 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, one protease inhibitor pill). Cells were then homogenized on ice using a 1-ml glass Dounce homogenizer with a tight fitting pestle until ∼95% of cells were disrupted as judged by trypan blue staining. Crude lysates were centrifuged at 1,000 × g for 15 min at 4 °C to remove nuclei and unbroken cells. The supernatant was collected and the pellet (P1) discarded. The low speed supernatant was then subjected to 10,000 × g centrifugation for 15 min, which yielded the 10,000 × g pellet (P2). The supernatant from the P2 pellet was centrifuged at 15,000 × g to completely rid the supernatant of any remaining mitochondria. Finally the 15,000 × g supernatant was separated into cytosol (S3) and light membrane (P3) fractions by centrifugation at 100,000 × g for 1 h. The 100,000 × g supernatant was collected as the S3 fraction, and the pellet was resuspended in 40-70 μl of Buffer A. P2 was washed twice by resuspending cells in 100 μl of Buffer A and pelleting (10,000 × g for 15 min). After the final wash, P2 was resuspended in 50-100 μl of Buffer A. All fractionations were repeated a minimum of four times with essentially identical results. Subcellular fractions were characterized using nucleoporin (P1, nuclear), cytochrome c oxidase (P2, mitochondria, heavy ER), heme oxygenase 2 (P3, light ER), and lactate dehydrogenase (S3, cytosol). Co-immunoprecipitation—Cell lysis buffer (150 mm NaCl, 50 mm Tris, pH 7.8, 1% Triton, 1 mm EDTA) was added to 100 μg of cell lysate to bring samples to a total volume of 500 μl. Samples were cleared of insoluble debris by centrifugation at 10,000 × g. Anti-IP3R-1, pre-immune sera, mouse IgG, or anti-DANGER antibodies, and protein A-Sepharose beads were added and incubated on a rotator for 30 min at 4 °C. The protein A-Sepharose beads were washed three times with lysis buffer and quenched with 20 μl of SDS sample buffer. Co-immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot analysis. Immunofluorescence and Immunohistochemistry—PC12 cells were grown on coverslips for 56 h then fixed with 0.4% paraformaldehyde in 2× PBS for 30 min and washed three times in 2× PBS for 15 min. Coverslips were quenched in 50 mm NH4Cl for 15 min and washed three times in 2× PBS for 15 min. Cells were permeabilized in saponin solution (2× PBS, 1% bovine serum albumin, 1% goat serum, and 0.075% w/v saponin) for 1 h. Coverslips were washed three times in 2× PBS for 15 min and subsequently inverted on 250 μl of anti-DANGER and IP3R in a wet chamber overnight at 4 °C. Coverslips were washed three times in 2× PBS for 15 min and inverted on 250 μl of secondary antibody solution 1:300 for 1 h at room temperature. Finally, coverslips were washed three times in 2× PBS for 15 min and mounted onto slides with Slo-fade. Fluorescence signals were detected with a Zeiss LSM410 confocal laser scanning microscope, using a 63×/NA 1.4 objective. Formalin-fixed, paraffin-embedded 5 μm sections were dewaxed, rehydrated, and subjected to antigen retrieval by incubation in 10 mm sodium citrate, pH 6.0, at 98 °C for 20 min. Sections were then immunostained using the Vectastain™ ABC system according to the manufacturer instructions using affinity purified anti-DANGER antibody described above at a dilution of 1/50. Antibodies and Reagents—Plasmids were from the following sources: EYFP, Matchmaker©, and Myc-CMV vector cDNA from CLONETECH and human M5 muscarinic receptor cDNA from L. Birnbaumer (National Institutes of Health). Human Northern blot from Clontech (Palo Alto, CA). Carbachol, protein A-agarose, GST-agarose, Sigma. Fura-2/AM was from Molecular Probes (Eugene, OR). UTP was from Invitrogen. siRNA duplex was from Qiagen (Valencia, CA). Ca45 was from Amersham Biosciences. We conducted a yeast two-hybrid analysis using multiple fragments of IP3R as described previously (6Patterson R.L. van Rossum D.B. Barrow R.K. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2328-2332Crossref PubMed Scopus (82) Google Scholar) (Fig. 1A). A novel interacting sequence, corresponding to a previously uncharacterized expressed sequence tag designated KIAA1754 (7Nagase T. Kikuno R. Ohara O. DNA Res. 2001; 8: 319-327Crossref PubMed Scopus (48) Google Scholar), bound to aa 923-1581 fragment of IP3R. This expressed sequence tag, which we now denote as DANGER, maps to a human genomic sequence on chromosome 10q25.1 comprising a single coding exon (4.3 kb). The open reading frame codes for a protein of 547 aa and is predicted to contain several coiled-coil regions, an N-terminal signal peptide for ER/plasma-membrane retention, and a partial MAB-21 domain (Fig. 1A, schematic). Direct binding of DANGER to full-length IP3R was detected in vitro by co-immunoprecipitation with both overexpressed and endogenous DANGER proteins. We observe robust interactions between a GST-DANGER fusion protein and IP3R (type 1 and type 3) from cell lysates as well as purified type 1 IP3R from rat cerebellum (Fig. 1B). Binding to type 2 IP3R could not be confirmed as the type2 IP3R antibodies we employed failed to demonstrate immunoreactivity to the samples assayed. To study endogenous DANGER, we a polyclonal antibody against aa of DANGER (Fig. a DANGER expression in PC12 the polyclonal antibody both endogenous and DANGER at the same (Fig. as shown by of overexpressed DANGER. Western analysis of PC12 cell lysates with polyclonal anti-DANGER a single corresponding to the full-length protein as well as two of and To the of the we employed siRNA to endogenous DANGER from PC12 cells (Fig. The siRNA DANGER immunoreactivity the three by antibody from the same we the of endogenous DANGER and IP3R in intact cells. We co-immunoprecipitation of DANGER with IP3R using either polyclonal antibody to DANGER or a antibody to type 1 IP3R (Fig. Subcellular of PC12 cells (Fig. the of DANGER in fractions P2 and which contain ER with in the nuclear The DANGER isolated from the yeast two-hybrid comprises aa which contain a partial MAB-21 We to the site in DANGER binds IP3R using yeast two-hybrid and fusion proteins of DANGER not we are to binding to IP3R with any fragment of DANGER the one isolated from the yeast two-hybrid the of the region is for binding. To carefully the intracellular of DANGER, we performed using DANGER antibody and type 1 IP3R antibodies in PC12 cells with for 2 We observe a membrane for endogenous DANGER, which a of with type 1 IP3R in 50 cells (Fig. We also observe a small of DANGER in the with the from the of DANGER with type 1 IP3R the plasma membrane DANGER and type also in to a similar as in the cell (Fig. DANGER is in which contain the of the brain (Fig. immunoreactivity for to DANGER is also in cells. DANGER is also expressed the in cell including and not To a of DANGER on IP3R we release of Ca2+ from microsomal of cells with type 1 IP3R and (Fig. as described previously (4Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (534) Google Scholar). with incubation with purified DANGER nm) on Ca2+ release in the of nm Ca2+ a wide of IP3 concentrations (Fig. In or (siRNA) of DANGER not IP3 in response to muscarinic and receptor not At low Ca2+ nm) Ca2+ enhances IP3R channel while concentrations of Ca2+ IP3R (8Mak D.O. McBride S. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15821-15825Crossref PubMed Scopus (226) Google Scholar). DANGER nm) on the actions of Ca2+ enhances the inhibitory effects of Ca2+ (Fig. in the absence of DANGER, Ca2+ and not influence IP3R to 100 nm in the of DANGER release of Ca2+ is at these Ca2+ effects are with 100 nm DANGER not on IP3R-mediated Ca2+ release not to from in binding affinity of IP3 for IP3R as DANGER not IP3 binding affinity (Fig. We the effects of DANGER on activity of IP3R by single IP3R channel recordings with We endogenous the membrane of isolated nuclei (5White C. Li C. Yang J. Petrenko N.B. Madesh M. Thompson C.B. Foskett J.K. Nat. Cell Biol. 2005; 7: 1021-1028Crossref PubMed Scopus (354) Google Scholar, L. Cheung K.H. Vais H. Mak D.O. White C. Foskett J.K. J. Physiol. (Lond.). 2006; 573: 645-662Crossref Scopus (56) Google Scholar). The of the channel is to the pipette solution containing μm Ca2+ and 100 nm InsP3. In this the channel open probability (Fig. as previously (9Ionescu L. Cheung K.H. Vais H. Mak D.O. White C. Foskett J.K. J. Physiol. (Lond.). 2006; 573: 645-662Crossref Scopus (56) Google Scholar). of 1 μm DANGER in the pipette solution channel by is as a of an of the mean time DANGER the channel In the of channel by the channel activity duration Ta and the mean number of channels in each is by DANGER (Fig. In DANGER is a novel membrane-associated IP3R binding protein enhances the sensitivity of IP3R to inhibition by cytochrome c and which also modulate Ca2+ sensitivity of IP3R, the inhibitory actions of Ca2+ (4Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (534) Google Scholar, C. Li C. Yang J. Petrenko N.B. Madesh M. Thompson C.B. Foskett J.K. Nat. Cell Biol. 2005; 7: 1021-1028Crossref PubMed Scopus (354) Google Scholar). be the of of Ca2+ inhibition of IP3R channel function is In microsomal membranes with DANGER, IP3R channel function by as [Ca2+] from to a This in Ca2+ is a regulator of channel DANGER in not in cells is unique neuronal IP3R in cells are to IP3 K. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K. D. J. Biophys. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). This sensitivity well with the influences we have of DANGER on IP3R channel which of the Ca2+ inhibition of the proteins which have been to to the IP3R have not been for their to IP3R activity a large of Ca2+ it be of IP3R Ca2+ dependence IP3R binding be a function been described We R. J. and M. for