Store-operated Cation Entry Mediated by CD20 in Membrane Rafts

B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through store-operated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium. The activity of this SOC pathway was abolished by deletion of a cytoplasmic sequence in CD20 essential for its efficient raft localization. Strontium-permeable SOC channels were detected in B cells, and B cell receptor-stimulated influx was significantly reduced by downregulation of CD20 expression using short interfering RNA and also by cholesterol depletion. This is the first evidence that raft-associated CD20 constitutes a component of a SOC entry pathway activated by the B cell receptor. B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through store-operated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium. The activity of this SOC pathway was abolished by deletion of a cytoplasmic sequence in CD20 essential for its efficient raft localization. Strontium-permeable SOC channels were detected in B cells, and B cell receptor-stimulated influx was significantly reduced by downregulation of CD20 expression using short interfering RNA and also by cholesterol depletion. This is the first evidence that raft-associated CD20 constitutes a component of a SOC entry pathway activated by the B cell receptor. The function of CD20, a tetraspan transmembrane protein expressed in B lymphocytes, is not yet fully elucidated, although electrophysiological evidence of calcium channel activity has been reported. Increased Ca2+ conductance, detected by whole cell patch clamp recordings, was induced by membrane hyperpolarization in a variety of cell types expressing CD20 ectopically and was similar to the native conductance found in B cells (1Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (291) Google Scholar, 2Kanzaki M. Shibata H. Mogami H. Kojima I. J. Biol. Chem. 1995; 270: 13099-13104Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The type of calcium channel that is formed or regulated by CD20 and the conditions of its activation are not known. Intracellular calcium is an essential regulator of cell function (3Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2285) Google Scholar, 4Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4583) Google Scholar). Receptor-mediated activation of phospholipase C and consequent inositol 1,4,5-trisphosphate production leads to rapid release of Ca2+ from intracellular stores in the endoplasmic reticulum. Store depletion activates Ca2+ influx across the plasma membrane, replenishing empty stores and providing a sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]c). The Ca2+ influx has been termed capacitative or store-operated Ca2+ (SOC) 1The abbreviations used are: SOC, store-operated calcium; TRP, transient receptor potential; CRAC, Ca2+ release-activated Ca2+; CHO, Chinese hamster ovary; GMI, Galβ1,3GalNAcβ1,4(NeuAcα2,3)Gal-β1,4Glcβ1,1-ceramide; siRNA, short interfering RNA; BCR, B cell receptor; ANOVA, analysis of variance; Tg, thapsigargin; MBC, methyl-β-cyclodextrin; SOSr, store-operated Sr2+; 0Ca, Ca2+-free buffer containing 0.2 mm EGTA. entry and is essential for regulating diverse cellular responses to receptor-mediated stimuli, including enzyme activation, gene expression, secretion, and cell division (5Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1310) Google Scholar, 6Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1295) Google Scholar, 7Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Crossref PubMed Scopus (83) Google Scholar). Electrophysiological studies distinguish several classes of SOC channels, including the highly Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel described in mast cells and T lymphocytes and others with distinct characteristics (7Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Crossref PubMed Scopus (83) Google Scholar, 8Putney Jr., J.W. McKay R.R. BioEssays. 1999; 21: 38-46Crossref PubMed Scopus (359) Google Scholar). Despite intensive research efforts, the molecular identity of SOC channel proteins remains unclear. The mammalian homologues of the Drosophila transient receptor potential (TRP) gene products, now forming a TRP superfamily with more than 20 members in 3 subfamilies (9Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; : 229-231Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar), provide the only candidate SOC channel proteins currently known. All TRPs apparently form cation channels but with variable selectivity and activation properties (10Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (983) Google Scholar, 11Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar). In B lymphocytes, expression of specific genes requires sustained elevation in [Ca2+]c (12Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1580) Google Scholar). As in other cell types, B lymphocytes activate Ca2+ entry after store-depletion, but the molecular components and the mechanism of activation of the SOC channels are unclear. Recently, TRPC1, for which there is evidence of store-operated, diacylglycerol, or receptor-mediated channel activity in other cell types (10Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (983) Google Scholar), was genetically disrupted in chicken DT40 B cells and found to reduce but not ablate SOC entry (13Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 673-681Crossref PubMed Scopus (177) Google Scholar). This suggests that TRPC1 may form SOC channels in B cells and also indicates the existence of additional SOC entry pathways. In this report we demonstrate that CD20, when expressed in Chinese hamster ovary (CHO) cells, dramatically enhanced SOC entry through strontium-permeable ion channels, providing the first evidence that CD20 is a component of a SOC entry pathway. In B cells, short interfering RNA (siRNA) reduced both CD20 expression and B cell receptor (BCR)-stimulated strontium and calcium entry. Cholesterol depletion or deletion of a 7-residue cytoplasmic CD20 sequence that controls its localization to cholesterol-dependent membrane microdomains, i.e. lipid rafts, inhibited SOC entry, suggesting the involvement of rafts in regulating CD20 SOC activity. Cell Lines and Culture—Ramos, BJAB, and Molt-4 cells were maintained in RMPI, 10% fetal bovine serum, and CHO cells in α-minimum Eagle's medium, 10% fetal bovine serum. Antibiotic-antimycotic (Invitrogen) was added to all cell culture media. Molt-4 T cells expressing CD20 or CD20 deletion constructs were described previously (14Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar). CHO cells were transfected with CD20 cDNA or deletion mutants in BCMGSneo vector or vector alone as described (14Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar). Positive cells were selected by Geneticin (Invitrogen) and further sorted by flow cytometry. For RNA interference experiments, BJAB cells were transfected with 120 pmol of siRNAs in 400 μl of RPMI by electroporation at 300 V and 800 microfarads. Reagents and Antibodies—Fura-2/AM was purchased from Molecular Probes (Eugene, OR). Thapsigargin, SKF96365, ionomycin, and BAPTA/AM were purchased from Calbiochem. siRNAs against CD20 (CCACTCTTCAGGAGGATGT) and control (GCGCGCTTTGTAGGATTCG) were purchased from Dharmacon Research Inc. (Lafayette, CO). All other chemicals were from Sigma. Mouse IgG2a isotype control antibody and fluorescein isothiocyanate-conjugated goat anti-mouse antibody were purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL), and mouse B1 monoclonal antibody (IgG2a) against human CD20 was from Coulter Corp. (Hialeah, FL). For immunoblotting, anti-Gαi antibody was purchased from Oncogene Research Products (Boston, MA), and anti-CD45 and anti-caveolin antibodies were from Transduction Laboratories (Lexington, KY); rabbit antiserum generated against a CD20 C-terminal peptide was described previously (15Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (73) Google Scholar). Cholera toxin conjugated to horseradish peroxidase was purchased from Sigma. Calcium Solutions and Fura-2 Imaging—All buffers contained 125 mm NaCl, 5 mm KCl, 1 mm MgCl2, 20 mm HEPES, and the pH was adjusted to 7.4 with NaOH. CaCl2 and SrCl2 were added to the buffer at 1 mm final concentration as indicated. Ca2+-free buffer contained 0.2 mm EGTA. CHO cells were grown on glass cover slips in 0.05% fetal bovine serum for 2-3 days before the experiment. Ramos and BJAB cells were grown in normal culture medium and adhered to poly-lysine-coated cover slips by low speed centrifugation on the day of the experiment. The cells were loaded with fura-2 by incubation in 5 μm fura-2/AM in HEPES buffer with 1 mm Ca2+ for 40-50 min at room temperature followed by washing and a further 15-20-min incubation to ensure full de-esterification. Digital imaging of fura-2 fluorescence was performed using the ImageMaster system from Photon Technology International, Inc. (Lawrenceville, NJ), essentially as described previously (16Tsoi M. Rhee K.H. Bungard D. Li X.F. Lee S.L. Auer R.N. Lytton J. J. Biol. Chem. 1998; 273: 4155-4162Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The images were analyzed to give the averaged background-subtracted ratio of fluorescence excited at 340/380 nm (emission at 510 nm) for all the cells in one field (>20 CHO cells or >100 B cells). To quantitatively describe the Ca2+ or Sr2+ entry, the peak ratio value of the Ca2+ or Sr2+ entry was subtracted by the base-line fura-2 ratio value, and this gives Ca′ or Sr′. The Ca′ or Sr′ was then divided by ATP′, Tg′, or IgM′ (ATP′, Tg′, or IgM′ are the peak ratio values of Ca2+ release after the stimulation subtracted by the base-line fura-2 ratio value). Statistical significance was tested using Student's t test or ANOVA as indicated. Cell Stimulation and Pretreatment Conditions—ATP and thapsigargin (Tg) were used at 100 μm and 1 μm final concentration, respectively. Tg was delivered to the chamber by pipetting in a 1-ml volume and was maintained for about 10-20 min. ATP was added to the flow solution and maintained after addition. SKF96365 was added to the EGTA buffer at 50 μm final concentration and maintained for about 9 min. Methyl-β-cyclodextrin (MBC) and cholesterol/MBC (11:181 weight ratio) were used to pretreat cells at 2% (w/v) for 1 h at room temperature. Antibody was used at 1 μg/106 cells. Flow Cytometry—Transfected BJAB or CHO cells were incubated with B1 anti-CD20 monoclonal antibody followed by fluorescein isothiocyanate-conjugated anti-mouse IgG. The data were acquired using a and analyzed using the and for was described previously J.P. M.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). BJAB, or cells or 5 CHO cells were in was added to a final concentration of and with 5 of and 5 of were for h at using a with a were from to and the was in buffer of were loaded on and at were to for immunoblotting, and horseradish antibodies were detected by For of were and with The images were acquired and analyzed using a and SOC in CHO cells expressing CD20 or control CHO cells transfected with vector alone were loaded with the and the fluorescence values in a field of cells were the of Intracellular stores were by receptor-mediated activation of phospholipase 1,4,5-trisphosphate using ATP V. G. Rev. 1998; Google or with Tg, a specific of the endoplasmic S. A. 1990; PubMed Scopus Google Scholar). then tested both Sr2+ and Ca2+ entry after depletion Sr2+ entry distinguish SOC channels from expressed channels in Birnbaumer L. K. 2000; PubMed Scopus Google Scholar). As in in base-line fluorescence was detected the control and CHO cells. ATP [Ca2+]c in the of Ca2+ in both cell as [Ca2+]c to to at the plasma membrane and by M.D. L. P. M. M.J. Lipp P. Cell Biol. 2001; PubMed Scopus Google Scholar). of Sr2+ induced a increase in the fluorescence ratio in cells, suggesting that influx of Sr2+ in these cells in to depletion. cells not significantly to that SOC channels are to Sr2+ the conditions The store-operated Sr2+ entry after ATP stimulation in CHO cells was about than that in CHO cells first was Sr2+ entry before depletion that CD20 expression not to Sr2+ in CHO cells. The were averaged from a of the of cells as in which the fura-2 fluorescence at the as and in store-operated cation entry in CHO in a Tg was then used to intracellular stores As after receptor-mediated Sr2+ CHO cells more than control cells after depletion and Sr2+ was and fluorescence values to Ca2+ was introduced the of Ca2+ cells that these cells maintained a normal Ca2+ entry pathway after Tg Calcium influx cells was that in cells and Sr2+ entry cells was reduced about by a channel used to Ca2+ entry R. A. J. 1990; PubMed Scopus Google Scholar), the that the enhanced Sr2+ entry in cells is through SOC ion channels. CD20 SOC in B that CD20 has the to form a SOC entry pathway with to CD20 is expressed in B lymphocytes, we then tested B cells are permeable to Sr2+ after depletion. used Ramos cells, an cell from The SOC channels were activated with to the or with Tg Sr2+ entry after stimulation with and was inhibited by SKF96365 and first and Ca2+ entry was also inhibited by SKF96365 using similar as in and Ramos B cells SOC entry channels that are permeable to Sr2+ and to cation entry in B cells reduced by SKF96365 and CD20 in a Recently, we that stimulation the lipid rafts and with CD20 (15Petrie R.J. Deans J.P. J. Immunol. 2002; 169: 2886-2891Crossref PubMed Scopus (73) Google Scholar). with of anti-CD20 monoclonal antibodies on responses T.F. P. Immunol. Full Text PDF PubMed Scopus Google a CD20 and the To the of CD20 to SOC entry in B cells we used the RNA interference Nat. Rev. 2002; PubMed Scopus Google to CD20 expression in the human B cell interfering RNA was to the of CD20 that is of the days after CD20 expression was and reduced by as with cells transfected with control of the was entry was significantly inhibited after of CD20 expression with anti-CD20 siRNA, control and of CD20 expression and entry were both of anti-CD20 of CD20 expression also inhibited Ca2+ entry after stimulation using the as in but Sr2+ with Ca2+ CD20 with we that CD20 is in but after antibody and on with the that with lipid rafts J.P. M.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). a than raft that may of than with we found that when B cells were in CD20 with the low that may CD20 on the with and a of proteins that is to rafts is from rafts and was not detected in the of with other J.K. J. Cell Biol. PubMed Scopus Google Scholar, R.N. J. Exp. Med. 1999; PubMed Scopus Google Scholar, Immunol. 2000; PubMed Scopus Google and that the membrane in not an from BJAB cells are in data were using Ramos cells raft are in which to to K. D. Nat. Rev. Mol. Cell Biol. 2000; 1: PubMed Scopus Google Scholar). Pretreatment of BJAB cells with the reduced the of CD20 in the the cholesterol of we a short cytoplasmic sequence that was for CD20 in (14Polyak M.J. Tailor S.H. Deans J.P. J. Immunol. 1998; 161: 3242-3248PubMed Google Scholar). To this sequence was a of raft of CD20, lipid rafts were from Molt-4 T cells transfected with CD20 cDNA or the deletion The that deletion of the of CD20 with the of in Ramos of cholesterol by cellular including receptor-mediated K. D. Nat. Rev. Mol. Cell Biol. 2000; 1: PubMed Scopus Google Scholar), and specific of on SOC entry been in other J. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (359) Google Scholar, G. M.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). CD20 was found to the of on entry in Ramos B cells was with were to to the with report that on or on calcium after stimulation R.J. M. Deans J.P. J. Immunol. 2000; PubMed Scopus Google Scholar), Ramos cells normal Ca2+ release Sr2+ entry, was significantly reduced in cells as with cells and but was normal in cells with B and a of on the entry pathway. in CHO by of were found to for efficient of CD20 with lipid rafts, we tested these store-operated cation influx in transfected CHO cells. and for were expressed in CHO cells. The expression of these constructs were similar to one but were significantly than the expression of native CD20 used for the in a of CHO cells was that expressed CD20 at a similar to the deletion mutants entry was in the CHO cell expressing of CD20 and was found to significantly in cells with reduced CD20 expression of the cytoplasmic of CD20 on Sr2+ entry when with cells expressing a similar of CD20, the deletion abolished Sr2+ entry after depletion B and raft of ectopically expressed CD20 in CHO cells was of significantly reduced of CD20 on the the deletion and localization in the raft as a control was not by the expression of of the CD20 This is the first report of an membrane protein of the TRP as a component of a SOC entry pathway. a of calcium channel CD20 is a with transmembrane and cytoplasmic containing activity or protein or The as and after of proteins (1Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (291) Google and is containing CD20 as the component M.J. Deans J.P. 2002; PubMed Scopus Google Scholar). As entry transfected CHO cells with the of CD20 expression and was abolished by of these a for CD20 in ion channel SOC entry in cells to by channels, as described in mast cells and T cells A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1295) Google Scholar, 7Lewis R.S. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 279-307Crossref PubMed Scopus (83) Google Scholar). SOC channels distinct from are in other cell types L. J. Physiol. PubMed Google Scholar, M. Y. R.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google but not been described in cells of the and the has been (1Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (291) Google both are and with similar electrophysiological is highly permeable to Ca2+ and has selectivity for with Ca2+ Sr2+ A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1295) Google Scholar). has not been described in mammalian B lymphocytes but was in B cells M. Lewis R.S. J. Physiol. 2001; PubMed Scopus Google Scholar, K. N. Kurosaki T. S. J. 2001; PubMed Scopus Google Scholar). analysis of the ion selectivity of the has not been although a report a well by CD20 (1Bubien J.K. Zhou L.J. Bell P.D. Frizzell R.A. Tedder T.F. J. Cell Biol. 1993; 121: 1121-1132Crossref PubMed Scopus (291) Google Scholar). that the CD20 SOC was permeable to The fura-2 imaging data that Sr2+ the cells more than Ca2+; this may selectivity of the channel for Ca2+ than for Sr2+ or may to Sr2+ and Ca2+ with to fura-2 or L. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). 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