Ryanodine Receptor-Ankyrin Interaction Regulates Internal Ca2+ Release in Mouse T-lymphoma Cells

In this study, we have identified and partially characterized a mouse T-lymphoma ryanodine receptor on a unique type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Analysis of the binding of 3Hryanodine to these internal vesicles reveals the presence of a single, low affinity binding site with a dissociation constant (Kd) of 200 nM. The second messenger, cyclic ADP-ribose, was found to increase the binding affinity of 3Hryanodine to its vesicle receptor at least 5-fold (Kd≈ 40 nM). In addition, cADP-ribose appears to be a potent activator of internal Ca2+ release in T-lymphoma cells and is capable of overriding ryanodine-mediated inhibition of internal Ca2+ release.Immunoblot analyses using a monoclonal mouse anti-ryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Double immunofluorescence staining and laser confocal microscopic analysis show that the ryanodine receptor is preferentially accumulated underneath surface receptor-capped structures. T-lymphoma ryanodine receptor was isolated (with an apparent sedimentation coefficient of 30 S) by extraction of the light density vesicles with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid (CHAPS) in 1 M NaCl followed by sucrose gradient centrifugation. Further analysis indicates that specific, high affinity binding occurs between ankyrin and this 30 S lymphoma ryanodine receptor (Kd = 0.075 nM). Most importantly, the binding of ankyrin to the light density vesicles significantly blocks ryanodine binding and ryanodine-mediated inhibition of internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of ryanodine receptor-mediated internal Ca2+ release during lymphocyte activation. In this study, we have identified and partially characterized a mouse T-lymphoma ryanodine receptor on a unique type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Analysis of the binding of 3Hryanodine to these internal vesicles reveals the presence of a single, low affinity binding site with a dissociation constant (Kd) of 200 nM. The second messenger, cyclic ADP-ribose, was found to increase the binding affinity of 3Hryanodine to its vesicle receptor at least 5-fold (Kd≈ 40 nM). In addition, cADP-ribose appears to be a potent activator of internal Ca2+ release in T-lymphoma cells and is capable of overriding ryanodine-mediated inhibition of internal Ca2+ release. Immunoblot analyses using a monoclonal mouse anti-ryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Double immunofluorescence staining and laser confocal microscopic analysis show that the ryanodine receptor is preferentially accumulated underneath surface receptor-capped structures. T-lymphoma ryanodine receptor was isolated (with an apparent sedimentation coefficient of 30 S) by extraction of the light density vesicles with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid (CHAPS) in 1 M NaCl followed by sucrose gradient centrifugation. Further analysis indicates that specific, high affinity binding occurs between ankyrin and this 30 S lymphoma ryanodine receptor (Kd = 0.075 nM). Most importantly, the binding of ankyrin to the light density vesicles significantly blocks ryanodine binding and ryanodine-mediated inhibition of internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of ryanodine receptor-mediated internal Ca2+ release during lymphocyte activation. Upon agonist stimulation, most cells release Ca2+ sequestered in internal storage sites through Ca2+ release channels, identified as the ryanodine receptor (RYR)1 1The abbreviations used are: RYRryanodine receptorIP3inositol 1,4,5-trisphosphatecADPRcADP-ribosePMSFphenylmethylsulfonyl fluorideConAconcanavalin APAGEpolyacrylamide gel electrophoresisMOPS4-morpholinepropanesulfonic acidCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. 1The abbreviations used are: RYRryanodine receptorIP3inositol 1,4,5-trisphosphatecADPRcADP-ribosePMSFphenylmethylsulfonyl fluorideConAconcanavalin APAGEpolyacrylamide gel electrophoresisMOPS4-morpholinepropanesulfonic acidCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid.(1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar) or the inositol 1,4,5-trisphosphate (IP3) receptor(2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6109) Google Scholar). These two Ca2+ channels are homotetrameric megaproteins (subunit molecular mass of 500 kDa for the ryanodine receptor and 270 kDa for the IP3 receptor) which share extensive homology in their C-terminal portion where the majority of the transmembrane segments are predicted to be located(2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6109) Google Scholar). ryanodine receptor inositol 1,4,5-trisphosphate cADP-ribose phenylmethylsulfonyl fluoride concanavalin A polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. ryanodine receptor inositol 1,4,5-trisphosphate cADP-ribose phenylmethylsulfonyl fluoride concanavalin A polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. Ryanodine receptors were originally found in the sarcoplasmic reticulum of skeletal muscle (type 1 receptor, RYR1) and cardiac muscle (type 2 receptor, RYR2)(3Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 4Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (224) Google Scholar). The two forms of the ryanodine receptors appear to be encoded by different genes(3Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 4Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (224) Google Scholar). Ca2+ release from the sarcoplasmic reticulum through these receptors plays a central role in regulating the contraction of skeletal and cardiac muscle fibers. In vivo, the Ca2+ release channel in skeletal muscle is activated by direct physical coupling to the voltage sensor in the transverse tubule (5Schneider M.F. Chandler W.K. Nature. 1973; 242: 244-246Crossref PubMed Scopus (630) Google Scholar, 6Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (628) Google Scholar) while the cardiac Ca2+ channel is activated by Ca2+ influx through plasma membrane-associated Ca2+ channels(7Fabiato A. Mol. Cell. Biochem. 1989; 89: 135-140Crossref PubMed Scopus (64) Google Scholar, 8Nabauer M. Callewaert G. Cleeman L. Morad M. Science. 1989; 244: 800-803Crossref PubMed Scopus (330) Google Scholar). Recently, a third ryanodine receptor gene (type 3 receptor, RYR3) has been detected in brain tissues(9Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (346) Google Scholar). Since this new isoform of RYR is also found in smooth muscle tissue and several types of non-muscle cells(4Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (224) Google Scholar, 9Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (346) Google Scholar, 10Giannini G. Clementi E. Ceci R. Marziali G. Sorrentino V. Science. 1992; 257: 91-94Crossref PubMed Scopus (211) Google Scholar), the concept that ryanodine receptors are muscle-specific Ca2+ channels must be changed. Furthermore, it has been suggested that activation of ryanodine receptors by a second messenger (e.g. cyclic ADP-ribose (cADPR)) plays a critical role in intracellular Ca2+ signaling occurring during agonist-induced cell activation in non-muscle cells(11Takasawa S. Nata K. Yonekura H. Okamoto H. Science. 1993; 259: 370-373Crossref PubMed Scopus (393) Google Scholar, 12States D.J. Walseth T.F. Lee H.C. Trends Biochem. Sci. 1992; 17: 495Abstract Full Text PDF PubMed Scopus (216) Google Scholar). Non-muscle cells, such as mouse T-lymphoma cells, may possess a specialized sarcoplasmic reticulum-like organelle required for regulating internal Ca2+ release. It has been shown that IP3 is involved in stimulating “light density vesicles” in mouse T-lymphoma cells to release Ca2+(13Bourguignon L.Y.W. Jin H. Iida N. Brandt N. Zhang S. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar). Recent cloning and cDNA sequence analysis show that the brain type of ryanodine receptor (RYR3) transcript is expressed in human Jurkat T-lymphocyte cells(14Hakamata Y. Nishimura S. Nakai J. Nakashima Y. Kita T. Imoto K. FEBS Lett. 1994; 352: 206-210Crossref PubMed Scopus (70) Google Scholar). However, direct biochemical evidence for the existence of a non-muscle ryanodine receptor (with a separate type of intracellular Ca2+-release channel) in lymphoid cells has not been reported. In this paper, we have isolated and partially characterized a mouse T-lymphoma ryanodine receptor from a type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Immunoblot analyses using a monoclonal mouse anti-ryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Most importantly, we have determined that ankyrin (a membrane-associated cytoskeletal protein) binds specifically to the mouse T-lymphoma ryanodine receptor, and that this binding significantly inhibits ryanodine binding to ryanodine receptor and blocks ryanodine-mediated inhibition of internal Ca2+ release. These results strongly suggest that the interaction between ryanodine receptor and ankyrin may play an important role in the regulation of internal Ca2+ release during lymphocyte activation. 3HRyanodine (specific activity 54.7 Ci/mmol) was purchased from DuPont NEN. 45CaCl2 (specific activity 5-30 Ci/g) was obtained from ICN. cADPR was purchased from Amersham. The mouse T-lymphoma BW 5147 cell line (an AKR/J lymphoma line) was grown at 37°C in 5% CO2, 95% air using Dulbecco's modified Eagle's medium supplemented with 10% heatinactivated horse serum (Life Technologies, Inc.), 1% penicillin, and 1% streptomycin. The cells (suspended in 50 ml of ice-cold buffer consisting of 15 mM KCl, 1.5 mM Mg(OAc)2, 1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM HEPES (pH 7.0)) were disrupted by nitrogen cavitation in an Artisan homogenizer (Artisan Industries, Inc., Waltham, MA) held at 0°C using a pressure of 600 p.s.i. for 15 min. After disruption, one-tenth volume of 700 mM KCl, 40 mM Mg(OAc)2, 1 mM dithiothreitol, 400 mM HEPES (pH 7.0), 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF was added, and nuclei were removed by centrifugation at 500 × gav for 4 min. The resulting supernatant was layered on a discontinuous sucrose gradient consisting of 0%, 15%, 25%, 35%, 40%, 50% sucrose (w/w) in a buffer containing 10 mM HEPES (pH 7.0), 50 mM KCl, 1 mM dithiothreitol, 2 mM MgCl2, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. The gradient was centrifuged in a Beckman SW28 rotor at 25,000 rpm for 16 h as described previously(13Bourguignon L.Y.W. Jin H. Iida N. Brandt N. Zhang S. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar). The membranous materials located in various sucrose layers were collected for further biochemical analyses including enzyme marker assays, ryanodine binding assays, Ca2+ flux measurement, and immunoblotting techniques as described below. Na+/K+-ATPase activity was used as a specific enzyme marker for plasma membrane(15Monneron A. D'Alayer J.D. J. Cell Biol. 1978; 27: 211-231Crossref Scopus (51) Google Scholar, 16Bourguignon G.J. Bourguignon L.Y.W. Biochim. Biophys. Acta. 1981; 646: 109-118Crossref Scopus (8) Google Scholar). NADPH-dependent cytochrome c reductase and Sulfatase C activities were used as independent markers of endoplasmic reticulum(17Rossier M.F. Capponi A.M. Vallotton M.B. J. Biol. Chem. 1989; 264: 14078-14084Abstract Full Text PDF PubMed Google Scholar, 18Fleischer S. Fleischer B. Methods Enzymol. 1967; 10: 406-433Crossref Scopus (234) Google Scholar). Galactosyltransferase activity, which was used as a Golgi marker, was assayed to the described S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was used as a L.Y.W. K. M. J. Cell. Physiol. Scopus Google Scholar, A. J. Cell Biol. PubMed Scopus Google Scholar). were determined using the T-lymphoma cells were with Dulbecco's modified Eagle's medium and in the concanavalin A was to the cell at 0°C for 30 min. were with Dulbecco's modified Eagle's medium followed by at 37°C for 15 to or structures. were in containing M buffer (pH In cells were in with cells were by or followed by staining with an monoclonal mouse anti-ryanodine receptor antibody it was the C of the ryanodine These were with antibody cells were with mouse serum followed by staining was in such were with a confocal laser was using a 5% polyacrylamide gel and the discontinuous buffer described by Nature. PubMed Scopus Google Scholar). were and to ankyrin was by the of and V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). light density vesicles from the sucrose to the described were in a containing mM (pH mM KCl, 1 mM mM 1 mM of aprotinin, of leupeptin, and 10 3Hryanodine in the presence of various of ryanodine from 50 for 16 h at Ca2+ in the was by using a with a A and was used to the of to the Ca2+ In cyclic ADP-ribose or cytoskeletal (e.g. ankyrin or was also in the ryanodine binding The binding was by a containing mM mM (pH 1 mM MgCl2, and 1 mM and through a The was by The results were expressed as in which the binding as the with and the in the presence of was from the 3Hryanodine The of the 3Hryanodine binding (e.g. mouse lymphoma light density skeletal muscle sarcoplasmic cardiac muscle sarcoplasmic and brain were in a containing 1 M NaCl mM and a modified from a E. G. Nature. PubMed Scopus Google Scholar). were centrifuged on sucrose density in a Beckman for 16 h at × were by with 3Hryanodine which an of various gradient in a containing mM (pH mM KCl, 1 mM mM 1 mM of aprotinin, of leupeptin, and 10 3Hryanodine in the presence of various of ryanodine from 50 for 16 h at 3Hryanodine the was a in a containing 1 M mM and The was collected by low centrifugation × and the was by was with using was with a with ryanodine receptors S obtained from lymphoma light density skeletal muscle sarcoplasmic cardiac muscle sarcoplasmic or brain vesicles to the described in the presence of various of ankyrin from in a binding containing mM (pH mM and serum for 16 h at the were with the binding and in a The results were expressed as specific binding in which the of binding was a we removed ryanodine receptors by the 30 S with anti-ryanodine receptor specific ankyrin binding was detected in these ryanodine were in a containing mM (pH mM KCl, mM 1 mM of and of leupeptin, 1 in the presence or of various of ryanodine In cyclic ADP-ribose or ankyrin was also in the medium in which the Ca2+ was to The Ca2+ was by the of mM The of Ca2+ from the light density vesicles was determined by a using and with a buffer containing mM and mM (pH The was by including lymphoma 30 S lymphoma light density vesicles from sucrose lymphoma skeletal muscle cardiac muscle and brain vesicles were by a 5% polyacrylamide gel electrophoresis followed by to Ryanodine were detected by an using a of an monoclonal mouse anti-ryanodine receptor antibody and a of followed by to mouse serum was used as a It is that Ca2+ plays an important role in the regulation of a of activities including lymphocyte receptor L.Y.W. 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Iida N. Brandt N. Zhang S. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar) indicate that the organelle (e.g. Golgi endoplasmic and (e.g. are located at the sucrose the sucrose and the sucrose T-lymphoma ryanodine binding are preferentially located in the light density vesicle at the sucrose In this we have on the ryanodine binding sites detected in the light density vesicles with a density of 1.07 The that the mouse T-lymphoma IP3 receptor is also found in this light density vesicle L.Y.W. Jin H. Iida N. Brandt N. Zhang S. J. Biol. 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In this study, we have found that cyclic ADP-ribose the binding affinity of 3Hryanodine to its receptor at least 5-fold (Kd≈ 40 It is that low affinity ryanodine binding sites are expressed in the lymphoid These low affinity ryanodine binding sites may be affinity ryanodine binding sites a second messenger, such as is during lymphocyte activation by It has been shown that cADPR and its ADP-ribose, are capable of the Ca2+ release R. 1994; PubMed Google Scholar). In this we the that cADPR ADP-ribose are for the increase of the of 3Hryanodine have also found that the light density vesicles Ca2+ release activities which be by These findings suggest that the mouse T-lymphoma light density vesicles contain a Ca2+ channel and the cADPR is a potent activator of internal Ca2+ and activation is not by ryanodine the of of cADPR in regulating Ca2+ channel activity is not It is that cADPR on and Ca2+ channel of by ryanodine may be by the extensive cADPR on is in It is that a similar overriding of ryanodine inhibition of internal Ca2+ release by cADPR has also been to in cardiac muscle sarcoplasmic reticulum and brain L. J. A. Nature. 1993; PubMed Scopus Google Scholar). also indicate that cADPR not as a direct activator of Ca2+ release activity with the skeletal or cardiac muscle ryanodine R. 1994; PubMed Google Scholar, FEBS Lett. 1994; 352: PubMed Scopus (51) Google Scholar). the skeletal muscle ryanodine receptor and the cardiac ryanodine receptor are different from the brain and lymphoma ryanodine receptor It is cADPR high affinity binding with has a low binding affinity with and or the binding affinity for cADPR to may be that in and The interaction between cADPR and not may Ca2+ release channels in and A analysis of cADPR binding to the types of ryanodine receptors and RYR3) is to this of the light density vesicles with in 1 M followed by sucrose gradient centrifugation and with a with a sedimentation coefficient of 30 similar to the skeletal muscle ryanodine receptor analysis followed by staining of the 30 S reveals a polypeptide mass of immunoblotting we have found that the monoclonal anti-ryanodine receptor antibody a polypeptide (with a molecular mass of lymphoma and antibody also binds the ryanodine receptor isolated from skeletal muscle cardiac muscle brain and the lymphoma 30 S These findings indicate that mouse T-lymphoma cells in contain ryanodine In we have shown that the cells were with by anti-ryanodine receptor-mediated staining be detected that ryanodine receptor is not located on the surface of mouse T-lymphoma cells the of were with followed by and anti-ryanodine receptor-mediated intracellular ryanodine receptors are found to be the of the cell and the of the cells were with an such as concanavalin A the intracellular ryanodine receptors and preferentially with surface and structures. Since is to be involved in lymphocyte activation and L.Y.W. Bourguignon G.J. J. Cell. Biochem. PubMed Scopus Google Scholar), these results suggest that ryanodine receptor be important for internal Ca2+ release at the of receptor and lymphocyte activation. are in the of which and with internal vesicle It is that the of a direct between the cytoskeleton and is of the to during L.Y.W. of Scholar). In a of cytoskeletal which are of the (e.g. (a and are found to be involved in during cell activation by V. Physiol. Rev. 1990; PubMed Scopus Google Scholar). is a cytoskeletal to be to such as 3 V. Physiol. Rev. 1990; PubMed Scopus Google Scholar) and lymphocyte Bourguignon L.Y.W. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, L.Y.W. J. Bourguignon G.J. Mol. Cell. Biol. 1992; PubMed Scopus Google Scholar, Bourguignon L.Y.W. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, L.Y.W. J. 1993; Google Scholar, N. Bourguignon L.Y.W. J. Cell Biol. 1994; PubMed Scopus Google Scholar), and to In addition, ankyrin has been found to to the IP3 receptor in brain tissue S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and lymphoma L.Y.W. Jin H. Iida N. Brandt N. Zhang S. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar, L.Y.W. Jin H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the binding of ankyrin to the IP3 receptor IP3 binding and internal Ca2+ L.Y.W. Jin H. Iida N. Brandt N. Zhang S. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar, L.Y.W. Jin H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to the binding interaction between lymphoma ryanodine receptor and the cytoskeletal such as we have lymphoma 30 S ryanodine with in the presence of various of ankyrin indicate that specific, high affinity binding occurs between ankyrin and this lymphoma 30 S ryanodine receptor (Kd = 0.075 In addition, we have shown that the binding of to various 30 S ryanodine receptors isolated from skeletal muscle cardiac muscle and brain tissue is also specific ankyrin with binding to these ryanodine receptors and These suggest that the acid sequence of the ankyrin binding on various ryanodine receptors must be different cell types and tissues. Recently, we have shown that an from sequence between two ankyrin binding and IP3 receptor) the binding of ankyrin to mouse lymphoma IP3 L.Y.W. Jin H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). indicate that this acid the ankyrin binding also blocks ankyrin binding to lymphoma 30 S ryanodine receptor with an apparent inhibition constant of nM. Most importantly, the binding of ankyrin not to the ryanodine receptor in the light density vesicles also significantly inhibits ryanodine binding and blocks the of ryanodine on internal Ca2+ release it is that ankyrin binding or with ryanodine binding may overriding on ryanodine-mediated inhibition of internal Ca2+ release in these vesicles ankyrin binding may in the of ryanodine receptor resulting in a in ryanodine binding as as a in the of ryanodine inhibition of internal Ca2+ release. The that ankyrin also IP3 binding and Ca2+ release in mouse T-lymphoma cells L.Y.W. Jin H. Iida N. Brandt N. Zhang S. J. Biol. Chem. 1993; 268: 7290-7297Abstract Full Text PDF PubMed Google Scholar, L.Y.W. Jin H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) strongly that ankyrin is of the important regulating Ca2+ we are using in and techniques to further the ankyrin binding on the mouse T-lymphoma ryanodine In findings the that mouse T-lymphoma cells contain ryanodine receptors which as a second intracellular Ca2+ release channel with the IP3 Most importantly, we that cADPR and the cytoskeletal play a pivotal role in the regulation of internal Ca2+ release during lymphocyte activation. the of J. Bourguignon in the of this