Trisphosphate Receptor-mediated Internal Ca2+ Release from C

Vol. 268, No. 10, Issue of April 5, pp. 7290-7297,

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Bioehemistry and Molecular Biology. Inc.

1993 Printed in V.S.A.

The Involvement of Ankyrin in theRegulation of Inositol 1,4,5Trisphosphate Receptor-mediated Internal Ca2+Release from Ca2+ Storage Vesicles in Mouse T-lymphoma Cells* (Received for publication, October 27, 1992)

Lilly Y. W. BourguignonSQ,Hengtao JinS, Naoko IidaST, NeilR. Brandtll, and SheHui Zhang* From the $Departmentof Cell Biology and Anatomy, the 11Department of Molecular Pharmacology, School of Medicine, University of Miami, Miami, Florida 33101

Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, orlysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (1P3)to these internal vesicles reveals thepresence of a single, high affinity class of IP3 receptor with a dissociation constant (&) of 1.6 2 0.3 IIM. Using a panel of monoclonal and polyclonal antibodies against IPS receptor, we have established that the IPSreceptor (-260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysisof firststrand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IPS receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form)and not the long form (neuronal form)detected in rat braintissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and theIPSreceptor with a Kd of 0.2 nM. Most importantly, the bindingof ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+release. These findings suggest thatthe cytoskeleton plays a pivotal role in the regulation of IPS receptormediated internal Ca” release during lymphocyte activation.

Lymphocyte activationisinitiatedas aconsequence of ligand-receptor binding which often generates the onset of phospholipase C activity (1-3). Hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C results in the formation of two intracellular second messengers, diacylglycerol, and inositol 1,4,5-trisphosphate(IP3)’(4). Diacylglycerol is an essential cofactor in activating membersof the protein kinase Cfamily of serine/threoninekinases (5). IPS is a * This work was supported by National Institutes of Health Grant GM 36353 and American Heart Grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. § T o whom reprint requests should be addressed: Dept. of Cell Biology and Anatomy, School of Medicine, University of Miami, 1600 NW 10th Ave., Miami, FL 33101. Tel.: 305-547-6985; Fax: 305-5457166. 7 A postdoctoral fellow of the American Heart Association. The abbreviations used are: IP,, inositol 1,4,5-trisphosphate; IP,, inositol 1,3,4,5-tetrakisphosphate;IP,, inositol hexakisphosphate; MAPPing, message amplification phenotyping; DTT, dithiothreitol; - 2 -hydroxy- 1CHAPS, 3 - [ (3-cholamidopropyl)dimethylammonio] propanesulfonic acid PCR, polymerase chain reaction.

physiological ligand known to mediate internal Ca2+ release from intracellular Ca2+ storage sites by binding to a specific receptor on certain intracellular membranevesicles (4). The IP3 receptor hasbeen identified by a number of investigators based on the specific binding of [3H]IPs to internal vesicles in smooth muscle cells (6) and in several nonmuscle cell types such as liver (7), adrenal corticalcells (8, 9), brain cerebellum (lo), and cerebellum Purkinje fibers (11). The primaryintracellularstoragesites for internal Ca2+ were originally thought to be part of the endoplasmic reticulum (12-14). However, subcellular fractionation studies revealed that the distributionof Ca2+-pumping, Ips-responsive organelles does not correlate with markers for plasma membrane, endoplasmic reticulum, mitochondria, Golgi apparatus, nor any other known organelles (15). Consequently, the IP3-responsive vesicles appear to bea unique type which have been designated as “calciosomes” (16). Recently, an IPS receptor has been reported to exist on the plasma membrane of human T-lymphocytes (17). Since the structural and functional properties of this plasma membrane-associated IP3 receptorhave not been fully established, one cannotpreclude the possibility that this surface IPS receptor may be structurally or functionally related to several different plasma membrane receptors for inositol 1,3,4,5-tetrakisphosphate(IP,) or inositol hexakisphosphate (IPS) (18, 19). Consequently, the subcellular location of the IP3 receptor still remains be to determined. In mouse T-lymphoma cells, activation of phospholipase C by either a Gi,-like protein (3) or tyrosine kinase(s) (2) has also been shown to generate IP3. Although an Ips appears to be required for inducing internal Ca2+ release, the formation of receptor patching/capping (3,20) and general the activation of lymphocytes (1,2), very little isknown at the present time concerning the nature of the IP3 receptor and IP3-inducible internal storage sites in lymphocytes. In this study, we have isolated and partially characterized a mouse T-lymphoma IP3 receptor from a unique type of internal vesicle which bands at therelatively light density of 1.07 g/ml. This IPSreceptor, which shares immunological cross-reactivity with the brain IP, receptor, displays both high affinity IP, binding andCa2’ ionchannelproperties.Mostimportantly,theinteraction betweenmouse T-lymphoma IP3 receptor and ankyrin, an important membrane-associated cytoskeletal protein, significantly inhibits IP3 binding and IPS-mediated internal Ca2+ release. MATERIALS AND METHODS

Cell Culture The mouse T-lymphoma BW 5147 cell line (an AKR/J lymphoma line) weregrown at 37 “C in 5% C02/95% air using Dulbecco’s

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modified Eagle's medium supplemented with 10% heat-inactivated horse serum (GIBCO), 1%penicilin, and 1%streptomycin. Cellular Fractionation The cells (suspended in 50 ml of ice-cold buffer consisting of 15 mM KCI, 1.5 mM Mg(OAc)z,1 mM dithiothreitol (DTT) and 10 mM HEPES (pH7.0)) weredisrupted by nitrogen cavitation inan Artisan homogenizer (Artisan Industries, Inc., Waltham, MA) held at 0 "C using a pressure of 60 p s i . for 15 min. After disruption, 0.10 volume of 700 mM KC1,40 m M Mg(OAc)z, 1mM DTT, and 400 mM HEPES (pH 7.0) was added, and nuclei were removed by centrifugation a t 500 X g,, for 4 min. The resulting supernatant was layered on a discontinuous sucrose gradient consisting of 0, 15, 25, 35, 40, and 50% sucrose (w/w) in a buffer containing 10 mM HEPES (pH 7.0), 50 mM KCl, 1 mM DTT,and 2 mM MgCl,. The gradient was centrifuged in a Beckman SW28 rotor at 25,000 rpm for 16 h. The membranous materials located in various sucrose layers were collected for further biochemical analyses including enzyme marker assays, IPB binding assays, Ca2+ flux measurement, and immunoblotting techniques as described below. Enzyme Marker Assays Na+/K+-ATPase activity was used as a specific enzyme marker for plasma membrane (21,22). NADPH-dependent cytochrome c reductase and sulfatase C activities were used as independent markers of endoplasmic reticulum (9, 23). Galactosyltransferase activity, which was used as a Golgi marker was assayed according to theprocedures described previously (24). P-N-Acetylglucosaminidase was used as a lysosomal marker (25, 26). Protein concentrations were determined using the Bio-Rad protein assay kit. Transmission Electron Microscopy Lymphoma light density vesicles collected from the 15-25% sucrose interface (according to the procedures described above), were fixed with 2% glutaraldehyde in phosphate-buffered saline (pH 7.3), postfixed with 1%OsO,, dehydrated through agraded ethanol series, and embedded in Spurrs embedding medium. Ultrathin sections were cut on a Sorvall MT2-B ultramicrotome, stained with uranyl acetate and lead citrate, and examined using a JEOL electron microscope at 80 kV. Immunoblotting Techniques Isolated lymphoma light density vesicles (collected from 15-25% sucrose interface) and brain IP, receptor (obtained from rat cerebellar membranes) (10) were either directly spotted on a sheet of nitrocellulose paper or analyzed by a 7.5% polyacrylamide gel electrophoresis followed by transferring to nitrocellulose sheets. Subsequently, the nitrocellulose sheets were incubated with the following various immunoreagents such as monoclonal mouse anti-IP3 receptor (IPR.1; it recognizes the C terminus cytoplasmic domain of IP3 receptor; full characterization of this monoclonal antibody willbe published elsewhere'), polyclonal rabbit anti-brain IP3 receptor (a gift from Dr. Thomas Sudhof, University of Texas Southestern Medical School, Dallas, TX), monoclonal mouse anti-calsequestrinor sheep anticalreticulin (kindly provided by Dr. D. H. MacLennan, university of Toronto,Canada). Nonimmune sheepserum, mouse serum, and rabbit serum were used as controls. Immunolabeled nitrocellulose sheets were subsequently incubated with biotinylated secondary antibodies and avidin H plus biotinylated horseradish peroxidase to form avidin biotin complex and to develop color reaction in the presence of diaminobenzidine with hydrogen peroxide. ImmunofluorescenceStaining Mouse T-lymphoma cells fixed in 2% paraformaldehyde were either surface-labeled with monoclonal mouse anti-IPa receptor antibody or rendered permeable by freezing (at -20 "C) and thawing (at room temperature) in the presence of 90% ethanol followed by staining with monoclonal mouse anti-IPS receptor antibody. These samples were then labeled with fluorescein-conjugated goat anti-mouse IgG. T o detect nonspecific antibody binding, cells were incubated with nonimmune mouse serum followed by fluorescein-conjugated goat anti-mouse IgG. No staining was observed in such control samples. H. Jin and L. Y. W. Bourguignon, submitted for publication.

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Purification of IPSReceptor Lymphoma light density vesicles, collected from the 15-25% sucrose interface (according to the procedures described above), were solubilized by adding Triton X-100 to a final concentrationof 1%(v/ v). Subsequently, the solubilized material was passed through a heparin-agarose column which was washed with 20 ml of a buffer (50 mM Tris-HC1 (pH 8.3), 1mM EDTA, 1 mM P-mercaptoethanol) plus 0.1% Triton X-100 and 0.25 M NaC1. The materials bound to the heparin-agarose were eluted with 3 ml of 50 mM Tris-HC1 (pH 7.71, 1 mM 0-mercaptoethanol, 0.1% Triton X-100,0.5 M NaC1; and then applied to an anti-IP3 receptor (IPR.l)-conjugated affinity column. The IP, receptor was eluted from the column with a solution containing 0.05 M diethylamine (pH ll.O), 10 mM EDTA, and 0.05% Triton X-100. Purity of the IPS receptor preparations was confirmed by SDS-polyacrylamide gel electrophoresis and silver staining. Purified IPS receptor was subsequently used for the incorporation into phospholipids (liposomes) and Ca2+flux measurements as described below.

[3H]IP3 Binding Assay Specific [3H]IP3binding was determined by the method described by Guillemette (8).Aliquots of each fraction (e.g. materials from the interface of 0-15, 15-25, 25-35, 35-40, and 40-50% sucrose layers) were incubated for 10 min at 4 "C in a 0.5 ml of a medium containing 25 mM NazHP04,100 mM KCI, 20 mM NaCI, 1 mM sodium EDTA, 1 mg/ml bovine serum albumin, and 0.05 pCi of [,H]IP3 (34.2 Ci/mmol, Amersham) at pH 7.4. In some cases, low density vesicleswere pretreated with either ankyrin (10 pg/ml) or monoclonal anti-IP, receptor antibody (IPR.l; 10 pg/ml) followedby [3H]IP3 binding. Binding was estimated in the presence of various concentrations of unlabeled 1P3 ranging from lo-" M to 1O"j M. The binding reaction was terminated by adding 2.5 ml of cold phosphate-buffered saline (pH 7.4) and filtrating through GF/B glass fiber filters that had been presoaked in phosphate-buffered saline containing 1%bovine serum albumin. The filter-associated radioactivity was analyzed by liquid scintillation counting. Ankyrin Binding Assay Humanerythrocyteankyrin was purified by the procedure of Bennett andStenbuck (27) and labeled with Nalz5Iusing IODO-GEN beads. '"1-Ankyrin(1-10 ng) was incubated with a nitrocellulose sheet coated with purified IP, receptor (obtained from anti-IP3receptor affinity column chromatography according to the procedures described above) in a binding solution containing 20 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 0.05% Triton X-100 and 0.1% bovine serum albumin for 30 min at room temperature. Following incubation, the nitrocellulose sheets were washed five times with the same binding solution and analyzed by dot assays followed by autoradiographic analysis or counted in a y counter (for Scatchard plot analysis). Background or nonspecific binding was determined by including a large excess of unlabeled ankyrin (at least 10-100-fold excess) in both dot assays and Schatchard plot analysis. The results were expressed as "specific binding" in which the background level of binding was subtracted. Ca'' Flux Measurement in Light Density Vesicles and IPSReceptor-

containing Phospholipid Vesicles (Liposomes) fluxes Ca" Flux Measurement in Light Density Ve~icle--'~Ca~+ were studied in a reaction mixture containing 120 mM KCl, 20 mM 10 mM phosphocreatine/ Tris-HEPES(pH 7.2). 0.3 mM MgC12, creatine kinase (10 units/ml) (Boehringer Mannheim), 3.75 FM ruthenium red, 1 mM Mg-ATP, 0.5 mM EGTA. CaClzwas added to this solution to generate a range of free Ca2+concentration between 100 and 300 nM. Subsequently, "Ca" (5-10 pCi/ml; 50 Ci/pg; Amersham) and light density vesicles (0.5 mg/ml) were added to the reaction mixture at 30 "C for 25 min. In Ca2+release experiments, IP, (10-100 nM) was added to these 45Ca2+-containingvesicles. The maximal amount of Ca2+release occurred 10 s after the addition of IP,. In some cases, low density vesicles were pretreated with either ankyrin (10 pglml) or monoclonal anti-IP, receptor antibody (IPR.l; 10 pg/ ml) followedby the addition of IPS (10-100nM) to the reaction mixture for ca2' flux measurements. The amount of Ca2+released from the light density vesicles wasdetermined hy a filtration method using Millipore filters (HAWP, 0.45 pm) and washing with a buffer containing 120 mMKC1 and 20 mM Tris-HEPES (pH7.2). Ca" Flux Measurement in IP3 Receptor-containing Phospholipid Vesicles (Liposomes)-Purified IPS receptor (obtained from the pro-

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IP3 Receptor andAnkyrin Interactionin T-lymphoma Cells

cedures described above) (100 pg/ml) was incorporated into phospha- gradient as described under “Materials and Methods.” Our tidylcholine/phosphatidyserine vesicles (a ratio of 1:1 IPS recep- results show the initial separation of various lymphomamemtor:phospholipids) in 1%CHAPS followed by dialysisagainst a buffer containing 20 mM Tris-HC1 (pH 7.4) at 25 “C, 100 mM NaCl, 100mM branes including soluble proteins (fraction A: 0-15% sucrose KCI, 2 mM 8-mercaptoethanol)for 72 h. These IPSreceptor-contain- interface) (Fig. la), “light density” membrane vesicles (fracing phospholipid vesicles (liposomes) were used to measure either IP, tion B: 15-25% sucrose interface) (Fig. la); Golgi membranes binding or IPS-induced Ca2+ release. The Ca2+flux measurement was (fraction C: 25-35% sucrose interface) (Fig. 1, a ande); initiated by adding 2 pCi of T a 2 +to the receptor-containing endoplasmic reticulum (Fig. 1, a and f ) and plasma memliposomes in the presence or absence of IP3 (10 nM) at 30 “C. The branes (fraction D: 35-45% sucrose interface) (Fig. 1, a and Ca2+flux measurement was terminated by adding a solution containing 0.5 mM CaCI2,5 mM MgSO,, and 100 pg/ml heparin and external g); and lysosomal membranes and otherlarge particles (mem45Ca2+ was removed by Dowex 50WX(Sigma) (28). The intravesicular brane-bound ribosomes) (fraction E: 40-50% sucrose layer) 46 (Fig. 1, a and h ) as determined by enzyme marker analyses Ca2+was counted by liquid scintillation counting. (Fig. 1, e-h). Message Amplification Phenotyping (MAPPing) Further analysis of the various sucrose gradient fractions First strand complementary DNA was synthesized at 42 “C for 1 h shows that the IP3 binding site is preferentially located in the in a final volumeof10plusing a reverse transcription system lightdensity vesicle fraction(fraction B, 15-25% sucrose (Promega).The reaction mixture contained1.0 pl of random primers interface) (Fig. Id) which represents approximately 10% of (0.5 mg/ml), 4.5 pl of RNA in H20, 1.0 pl of 10 X buffer (100 mM total membrane protein. Since there is alarge amount of Tris-HC1, pH 8.8, 500mM KCl, 1%Triton X-loo), 0.5 pl of RNAsin, protein in both fractions D and E, the specific activity of IP3 1.0 p1 of dNTP (dATP, dCTP, dGTP, dTTP mix (10 mM/nucleotide), is low compared with 2.0 p1 of 125 mM MgClz, and 1.0 p1 ofAMV (avian myeloblastosis binding detected in these fractions very virus) reverse transcriptase (25 units/ml). 86 pl of polymerase chain that in the B band (Fig. Id). Recently, a plasma membranereaction (PCR) mix was added to 4 pl of first strand cDNA. PCR mix associated IP3 receptor has been reported in Jurkat lymphocontains 59.5 p1 of sterile H20, 10 p1 of 10 X reaction buffer (500 mM cytes by Snyder and co-workers (17). It is possible that the KCI, 100 mM Tris-HC1 (pH 9.0), 15 mM MgCl2 and 1.0% Triton X- IP3 binding sites detected inC and D may represent a plasma loo), 16 pl of dNTP mix (1.25 mM/nucleotide) and 0.5 p1 (2.5 units) of the Thermus aquaticus thermostableDNA polymerase (Promega). membrane-associated species of IP3 receptor. T h e biochemical vesFive pl of each primerwas added to amplify a specific segmentof the similarities and/or differences between the “light density IPB receptor cDNA as described by Danoff et al. (29).The oligonucle- icle” IP3 receptor and the plasma membrane-associated IPS otide primers were 5’-CCG GAA TTC GTT TCA TCT GCAAGC receptor awaits furtheranalysis. A number of previous studies TAATAAAAC-3‘ and 5’-CCG GAA TTC AATGCT TTC ATG suggest that the intracellular IP3 binding sites are primarily GAA TAC TCG GTC-3’. The mixture was subjected to PCR ampli- located at the endoplasmic reticulum (12-14). The fact that fication withthe initial melting at 94 “C for 5 min, annealingat 50 “C for 2 min, and polymerizationat 72 “C for 2 min followed by 35 cycles the majority of IP3 binding is detected in the light density of denaturation at 94 “C for 30 s, annealing at 62 “C for 30 s, and vesicle fraction and not in the ERmay indicate that thereis polymerization at 72 “C for 1 min. Each samplewas analyzed by 2.0% a unique cellular location (possibly in a cell-specific manner) agarose gel electrophoresis and visualized by ethidium bromide stain- for IPBreceptors inlymphocytes. ing. In this paper, we have primarilyfocused on thehigh specific activity class of IPS binding sites detected in the light density SDS-Polyacrylamide Gel Electrophoresis and Autoradiographic vesicles (fraction B with a density of approximately 1.07 g/ Analyses ml) (Fig. 1, a and d ) . Morphological analysis using electron Electrophoresis was conducted using a 7.5 or 10% SDS-polyacrylmicroscopic techniques reveals that these light density vesiamide gelelectrophoresisslab gel and the discontinuous buffer system described byLaemmli (30). For autoradiographic analysis, all gels cles are smooth and heterogeneous in size; and occasionally were vacuum-dried and exposed to Kodak x-ray film (X-Omat XAR- they are present in tubular shapes (Fig. lb). These vesicles have negligible amounts of marker enzyme activities derived 5). from Golgi, endoplasmic reticulum, plasma membrane and RESULTS AND DISCUSSION lysosomes (Fig. 1, e-h). We have also determined that these low density vesicles are different from endosome structures Subcellular Localization of IP3 Receptor in Mouse Tusingmannose6-phosphatereceptorinternalization as a lymphoma Celk marker assay (25) (data not shown). Furthermore,using speInstriated muscle intracellularCa2+is released from a cific antibodies raised against calsequestrin (Fig. IC(i)) and light density specialized intracellular organelle, the sarcoplasmic reticulum calreticulin (Fig. IC(ii)), we have found that the (31). This structure contains an ATP-dependent Ca2+ pump vesicles (fraction B, 1 5 2 5 % sucrose interface) contain proand several Ca2+-binding proteins such as calsequestrin (32) tein molecules immunologically cross-reactive with these two and calreticulin (33). Ca2+ is released from the sarcoplasmic Ca*+-binding proteins (molecular masses of 63 and 55 kDa, respectively). We believe that this Western blot data are reticulum through a ryanodine-sensitive channel (homotetramer, molecular mass of each subunit is -565 kDa) which specific based on the facts that (a) the molecular masses of (55 kDa)in may be modulated by small molecules and calmodulinas well bothcalsequestrin (63 kDa)andcalreticulin as by muscle contraction (34-37). The observations of a lymphocyte light density vesicles are similar to those found membrane organelle with a Ca” pump, Ca2+-binding proteins in sarcoplasmic reticulumof striated muscle (32, 33);and ( b ) nonimmuneserum gives a negligible nonspecific reaction and a Ca2+ release channel led to the hypothesis that nonwe have tested whether IPB can muscle cells may possess a special sarcoplasmic reticulum- (data not shown). In addition, induce the release of Caz+ from the light density vesicles. like organelle generally referred to as the“calciosome” (16). that IP3causes a rapid release of internal In order to determine (i) whether mouse T-lymphoma cells Table I clearly shows actuallycontain “calciosome-like” structures, and (ii) how Ca2+from the light-density vesicles as early as 10-20 s after internal Ca” release is regulated in lymphocytes, we decided IPS addition. to establish a convenient and effective cellular fractionation Receptor from Characterization of MouseT-lymphoma procedure to isolate and identify those vesicles responsible for Light Density Vesicles internal CaZ+release. Mouse T-lymphoma cells were homogIP3 Binding Properties-The affinity of the light density enized and fractionatedby differential centrifugationfollowed by density gradient centrifugation ona discontinuous sucrose vesicles for [3H]IP3 was measured in binding inhibition ex-

IPSReceptor and Ankyrin Interaction T-lymphoma in i.

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A

B C D E FRACTION

FIG. 1. Separation of various lymphoma membranes on a discontinuous sucrose gradient and enzyme marker analysis of various lymphoma membranes on a discontinuous sucrose gradient. a, fraction A represents soluble proteins (0-1596 sucrose interface); fraction B represents light density vesicle membrane (15-25% sucrose interface); fraction C represents Golgi membranes (25-35% sucrose interface); fraction D represents plasma membranes (35-40% sucrose interface); and fraction E represents lysosome membranes and other large particles (e.g. ribosomes) (40-5076 sucrose interface). b, an electron microscopic photograph of light density vesicle membranes collected from fraction C (15-25% sucrose interface) magnification X 80,000. (Arrowheads in b indicate the heterogenous vesicle population.) c, immunoblot of calsequestrin (c-i) and calreticulin (c-ii) using light density vesicles (fraction B) and specific antibodies against these two proteins (sheep anti-calreticulin (c-i) andmouse anti-calsequestrin (c-ii)) (Nonimmune sheep serumand nonimmune mouse serum were used as controls. No staining was observed in these control samples (data not shown).) d, [3H]IP3binding assay; e, galactosyltransferase assay (a Golgi maker); f, NADPH-dependentcytochrome c-reductase (an endoplasmic reticulum marker) (similar results were observed using sulphatase C assay as an independent marker of endoplasmic reticulum (data not shown)); g, Na+/K+-ATPaseassay (a plasma membrane marker); h, p-N-actyl-glucosaminidase assay (a lysosome marker). Fractions: A , 0-1575 sucrose interface; B, 15-25% sucrose interface; C, 25-35% sucrose interface; D,35-40’31 sucrose interface; and E, 40-50% sucrose interface. (Arrowheads in Fig. 1, cl-h indicate maximal level of enrichment.)

TABLE I Effects of monoclonal anti-IPS receptor on “Ca2+ releasefrom lymphoma light density vesicles The amount of Ca2+released for A23187-treated vesicle was designated as the maximal level of Ca2+release signal and experimental figures were compared to this maximal release. The incubation time for ‘%a2+ release was 10 s. The data shown are the averages of triplicate determinants. which varied bv less than 5%( n= 5). Internal ‘‘Ca*+ Treatments release % of m a x i m 0 1 rekase 1. A23187 100 2. Buffer (no treatment) 0 3. IPS (10-100 nM) 44 47 4. Monoclonal anti-IPS receptor antibody (10 pglml) 46 5. Monoclonal anti-IP3 receptor antibody (10 pg/ml) IPS (10-100 nM) 0 6. Nonimmune normal mouse IgG (10 d m l )

+

periments using increasing concentrations of unlabeled IP3. The displacement curve reveals the presence of a single high affinity IP3 receptor with a dissociation constant (Kd) of 1.6

k 0.3 nM (mean k S.D., n = 4) (Fig. 2). The binding affinity of the light densityvesicles for [3H]IP3 is comparable to those reported for internal vesicles isolated from other cell types (6). Immunological Analyses of Mouse T-lymphoma IPS Receptor-The IP3 receptor in nonmuscle cells has been shown to be a homotetramer (2500 amino acid subunits, molecular mass -260 kDa) with limited homology to the ryanodine receptor in striated muscle (38, 39). Using a polyclonal rabbit anti-IPS receptor antibody, we have determined that theT-lymphoma light densityvesicles (Fig. 3, A and B ) contain an IPS receptor analogous to brain IP3 receptor (Fig. 3, D and E ) with a molecular mass of -260 kDa. In addition, amonoclonal mouse anti-IP3 receptor antibody (IPR.l) which does not interfere IP3 binding (Table 11) and recognizes a short sequence at the C terminus cytoplasmic side of IP3 receptor was also used. Similar immunocross-reactivitywas observed using this newly developed IP3 receptor antibody (Fig. 3, C and F ) . Using immunofluorescence staining, we have found that the IP3 receptor is preferentially associated with numerous vesicular structures located in the cytoplasm (Fig. 4B).Althoughthe resolution of light microscopy is limited, no

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in T-lymphoma Cells

100

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FIG.2. Binding of ['HIIPS to light density vesicles. Light density vesicle membranes (collected from 15-25% sucrose interface) were incubated with[3H] IP3 in the presence of various concentration of unlabeled IP3binding in buffer as described under "Materialsand Methods."

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TABLEI1 Effects of ankyrin and monoclonal anti-IPS receptor on [3HlIP~ binding in low density vesicles Low density vesicles (either pretreated with ankyrin (10 pg/ml) or monoclonal anti-IPS receptor antibody (10 pg/ml) or without any treatment) were incubated with [3H]IP3 as described under "Materials and Methods." Specific [3H]IP3 Samples

1

binding

116.4

85

No treatment (control) Ankyrin-treated Monoclonal anti-IPS receptor-treated

CPM x I V2/mg protein 20.5 f 0.8 2.4 f 0.2

19.8 0.4

cate that no cell surface label is detected using our newly developed monoclonal anti-IP3 antibody (IPR.l) (Fig. 4A). Most importantly,binding of this monoclonal anti-IPSreceptor antibody to 260-kDa protein-containing low density vesiFIG.3. SDS-polyacrylamide gel electrophoresis analysis of cles induces asignificant amount of internal ca'+release lymphoma light density vesicle membrane proteins and rat analogous to IP3-mediated Ca2+ stimulation (Table I). We brain cerebellum membrane proteins. A , total lymphoma light believe this anti-IP3 receptor antibody-induced Ca2+ release density vesicle membrane proteins. B, immunoblot of lymphoma light mouse IgG shows no density vesicle membrane proteins with polyclonal rabbit anti-rat is specific, sincenonimmunenormal brain IP3 receptor antibody. (As a control, nonimmune rabbit serum stimulation on Ca2+ release activity (Table I). There is no additive effect on the stimulation of internal Ca2+release if was used. No staining was detected onthese samples (datanot shown).) C, silver staining of purified lymphoma IPSreceptor obtained both a monoclonal anti-IP3receptor antibody (IPR.l) and IPS from monoclonal mouse anti-IPS receptor-conjugated affinity column. are added togetherto thelow density vesicles (Table I). These (As a control, nonimmune mouse serum-conjugated column was used. results suggest that the epitope which this monoclonal antiNo protein was detected on these columns(data notshown).) D, total rat brain cerebellum membrane proteins. E , immunoblot of rat brain IP3 antibody (IPR.l) recognizes must be very close to the cerebellum membrane proteins with polyclonal rabbit anti-rat brain proposed Ca2+ channel region in the C terminus of the IP3 IPS receptor antibody. F, silver staining of purified rat brain IPS receptor protein (40). Also, the binding of this antibody to IPS receptor obtained from monoclonal mouse anti-IPR receptor-conju- receptor possibly induces aconformationalchange of the gated affinity column. receptor which mimics the effect of IP3 binding to the receptor. Together, these findings clearly indicate that the lymphoma 260-kDa protein is an IP3receptor-like molecule (Fig. obvious endoplasmic reticulum-like network structureis 3, C and F ) . observed. In order to verify that our immunofluorescence Demonstration of IP3-induced Ca" Flux in Lipid Vesicles staining is specific for the intracellular IP3 receptor and not Reconstituted with the Putative 260-kDa ZP3 Receptor-The surface-bound IP3 receptor, we also carried out this immu- first direct demonstration that IP3 induces the openings of nolabeling procedure using intact cells (i.e. without any per- Ca'+ channels was obtained in planarlipid bilayers into which meabilization procedures) as a control. Our data clearly indi- vesicles made from aortic smooth muscle sarcoplasmic retic-

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FIG.4. Immunofluorescence staining of intracellular IPS receptors in mouse T-lymphoma cells using fluorescein-conjugated monoclonal anti-IP3 receptor. A, staining of surface exposed IPS receptor (note that only a background level of label was detected); B, staining of intracellular IPS receptor (note thata significant amount of label was detected invesicular structures).

FIG. 5. Time course of IP3-induced %‘a2+flux in phospholipid vesicles (liposomes) reconstituted with thepurified lymphoma IP3 receptor (260-kDa protein). PurifiedIPS receptor (100 pg/ml) was incorporated into phosphatidylcholine/phosphatidyserine vesicles(liposomes) as described under“Materialsand Methods.” These IPS receptor-containing phospholipid vesicles (liposomes) were used to measure IPS-induced Ca2+release. The Ca2+ flux measurement was initiated by adding 2 pCi of “Ca” to the IPB receptor-containing liposomes in the presence ofIPn (-10 nM) as described under “Materials andMethods.”

ulum were incorporated (28,40). Subsequently, purified brain IP3 receptor hasbeen shown to mediateCa2+ fluxin reconstituted lipid vesicles (28). In this studywe have used anti-IP3 receptor-affinity chromatography to obtain a highly purified -615 260-kDa protein(IP, receptor-likemolecule) fraction (Fig. 369 3C). Subsequently, this purified 260-kDa protein was incorporated into synthetic phospholipid vesicles (liposomes). Our -123 data indicate thatCa2+ fluxactivity in these260-kDa proteinin uiuo containing lipid vesicles responds toIPS at the normal concentrations (-10nM) (Fig. 5). This functional evidence FIG.6. The MAPPing products of the regional IPS receptor further supports the contention that the lymphoma 260-kDa cDNA from mouse T-lymphoma cells and rat brain cerebelprotein contains both the IP3 binding site and the Ca2+ ion lum. Lane I , mouse T-lymphoma cells; lane 2, rat brain cerebellum; and lune 3, mouse T-lymphoma cells with GAPDH primers. (Markers channel property. Detection of the IP3 Receptor Transcripts by MAPPing for the molecular size (in base pairs) areshown on the right.) Techniques-Since two different IP3 receptor transcripts derived by alternative splicing have been identified and shown of the short form of the transcript (Fig. 6, lane 1); whereas cerebellum appear to be expressed in a tissue-specific manner (neuronal uersus the IP3 receptor transcripts in the rat brain non-neuronal tissues) (29), total RNA materials isolated from to display the long form (Fig. 6, lane 2 ) . These PCR products Corp.) mouse T-lymphoma cells and rat braincerebellum were ana- were further cloned using aTA cloning kit (Invitrogen lyzed for the presenceof such specific transcripts by MAPPing and sequenced. The nucleotide sequence data confirms that the PCR-amplified fragments represent the segment of the techniques.MAPPing wasdeveloped toanalyzeRNAsin small numbers of cells. This technique utilizes reverse tran- IPS receptorcDNA reported previously (29). As a control, the scription of total cellular RNA to synthesize complementary house keeping gene GAPDH primers were used to verify the DNA, followed by the polymerase chain reaction to specifi- specificity and sensitivity of MAPPing techniques (Fig. 6, lane 3 ) . This finding suggests that only the non-neuronal cally amplify DNA fragments of interest. in mouse T-lymphoma Previously, MAPPing techniques from various rat tissues form of the IP3 receptor is synthesized cells. have revealed two distinct IP3 receptor transcripts (29). A long form (also considered to be neuronal-specific) contains Interaction of IP3 Receptor and the Cytoskeleton a 120-nucleotide insert between the two CAMP-dependent protein kinase phosphorylation consensus sequences, and is Cytoskeleton proteins such as ankyrinhave been shown to predominantly detected in adult brain tissues. A short form be involved in regulating a number of cellular activities in(also considered to be a non-neuronal type) lacks the insert, cluding receptor patching and capping (41-46), cell adhesion and is primarily found in fetal brain and peripheral tissues (47-49), organelle movement, cell motility, protein secretion, (29). Specific oligonucleotide primers were utilized in the PCR and cell division (50, 51). Putney et al. (7) have reported that reactions to differentiate the long neuronal type from the IP3receptor-containing vesicles may be attachedtothe short non-neuronal type of the IP3 receptor transcripts. Our plasma membrane through cytoskeletal elements such asacresults with mouse T-lymphoma cells indicate the existence tin. Van Bennett andco-workers (52) have also reported that

1 2 3

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IPSReceptor and Ankyrin Interaction T-lymphoma in

FIG.7. Scatchard plot analysis and dot assays of ankyrin binding to purified lymphoma IPS receptor. ‘”1-Ankyrin (1-10 ng) was incubated with a nitrocellulose sheet coated with purified IPS receptor (obtained from monoclonal mouse anti-IPS receptor (IPR.l) affinity column chromatography) according to the procedures described under “Materials and Methods.” The results were expressed as “specific binding” in which the background level of binding was subtracted. A, Scatchard plot analysis of ankyrin binding to purified lymphoma IPS receptor. B, dot assays of lZ5I-ankyrin(5 ng) binding to purifiedlymphoma IPS receptorin the absence (i) and the presence of a 10-fold (ii) or 100-fold (iii) excess amount of unlabeled ankyrin.

Cells

0.8

1251- Ankyrin Bound

( f mollg)

the inhibitory effect on IP, function in thesevesicles (Fig. 8, A and B ) . Alternatively, ankyrin binding may cause some conformational changes at the regulatory domain(s) of IPa receptor resulting in a decrease in IP3 binding as well as a reduction in the potency of IP3 in releasing Ca2+ as shown previously by protein kinase A-mediated phosphorylation in cerebellar membranes (53). Currently, we are using in uitro mutagenesis and deletion mutation techniques to define further the ankyrin-binding domain(s) on mouse T-lymphoma IP3 receptor. Ankyrin is well known to bind a number of plasma membrane-associated proteins including band 3 (54), two other members of the anion exchange gene family (55, 56), Na+/ K’-ATPase (57-59), amiloride-sensitive Na’ channel (60), FIG.8. Effects of ankyrin on ‘%a2+ release in lymphoma the voltage-dependent Na’ channel (61,62) and GP85(CD44) light density vesicles. In these experiments, the amount of Ca2+ (43-48). The fact that ankyrin also binds to intracellular released for IPS-treated vesicle was designated as the maximal level IP, receptor on light density vesicles (or 100%) of Ca2+release signal and experimental figures were com- proteins such as the pared to this maximal release. The incubation time for ‘‘Ca2+ flux suggests that ankyrin may be involved in multiple functions was approximately 20 s. A, IP3 (100 nM) alone; B, ankyrin (10 pg/ml) during cellular regulation. In lymphocytes, for example, an+ IP3 (100 nM); C, monoclonal anti-IP3receptor antibody (10 gg/ml); kyrin mayplaya pivotal role inlinking surfaceadhesion D, ankyrin (10 pg/ml) + monoclonal anti-IPS receptor antibody (10 molecule (e.g. GP85(CD44)) and intracellular Ca2’ storage pdml). organelle membraneproteins (e.g. IP, receptor).Thisankyrin-based linkage between plasma membrane proteins and a complex consisting of IP3 receptor, ankyrin, and GP180 can organelle Ca2+channel molecules may be criticallyimportant be isolated from brain tissue. In this study, we have used a for receptor-mediated signal transduction duringlymphocyte newly developed in uitro assay whichinvolves the use of activation. nitrocellulose paperscoatedwithpurifiedlymphoma IP3 Acknowledgment-We gratefully acknowledge the assistance of Dr. receptor (obtained from anti-IP3antibody(IPR.l)affinity column chromatography as shown in Fig. 3C) to determine Gerard J. Bourguignon in the preparation of this manuscript. IP3 receptor’s binding to ‘251-ankyrin. Our data clearly indiREFERENCES cate that the lymphoma IP3 receptor binds to ankyrin (Fig. 1. Weiss, A. (1989) in Fundamental Immunology (Paul, W. E., ed) pp. 359384, Raven Press, New York 7). T o further establish the specificity and affinity of IP3 A., Koretzky, G., Schatzman, R., and Kadlecek, T. (1991) Proc. receptor’s binding to ankyrin,we have incubated lZ5I-ankyrin 2. Weiss, Natl. Acad. Sct. U.S. A. 88,5484-5488 3. Boureuienon. L.Y. W.. Walker.. G... and Huanp. H. S. (1990) J. Immunol. with IP3 receptor-coated nitrocellulose sheets in the absence 142,324212252 ’ (Fig. 7B (i)) and presence of various concentrations of unla4. Berridge, M. J. (1987) Annu. Reu. Biochem. 56,159-193 beled ankyrin (Fig, 7B (ii) and (iii)). Scatchard plot analysis 5. Nishizuka, Y. (1988) Nature 334,661-665 6. Chadwick, C. C., Saito, A., and Fleischer, S. (1990) Proc. Natl. Acad. Sei. reveals the presence of a single high affinity class of ankyrin U. S. A. 8 7 , 2132-2136 a dissociation constant( K d ) binding sites on IPS receptor with 7. Rossier. M. F... Bird,. G. S. J., and Putney, J., Jr. (1991) Biochem. J. 274. 643-650 of 0.2 nM (Fig. 7A). 8. Guillemette, G., Balla, T., Baukal, A. J., Spat, A., and Catt, K. J. (1987) J. Finallyandmostimportantly, we havefound that the Biol. Chem. 262, 1010-1015 9. Rossier, M. F., Capponi, A. M., and Vallotton, M. B. (1989) J. Biol. Chem. binding of ankyrin to the IPS receptor in light density vesicles 264,14078-14084 significantly inhibits IP3 binding (Table 11) and IP3-stimu- 10. Supattapone, S., Worley, P. F.,Baraban, J. M., and Snyder, S. H. (1988) J . Biol. Chem. 263,1530-1534 lated internal Ca2+ release (Fig. 8, A and B ) . This ankyrin- 11. Walton, P. D., Airey, J. A., Sutko, J. L., Beck, C. F., Mignery,G. A., Sudhof, T. C., Deerinck, T. J., and Ellisman, M. H. (1991) J. Cell Biol. mediated inhibitory effect on IPS-inducible Ca2+ release ap1 1 3 , 1145-1157 pears to be very selective, since ankyrin fails toblock mono- 12. Mignery, G. A., Sudhof, T. C., Takei, K., and Camilli, P. D. (1989) Nature clonal anti-IP3 receptor-stimulated internalCa2+ release(Fig. 342,192-195 A. H., Snyde, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 8, C and D).I t is possible that ankyrin binding competes or 13. Sharp, 7444-7449 overlaps with IPS binding sites (Table11). This may explain 14. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Science 252,443-446 ~~

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