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. 1995 Aug 2;130(4):847–855. doi: 10.1083/jcb.130.4.847

Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function

PMCID: PMC2199966  PMID: 7642702

Abstract

A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca2+ buffering capacity of the intracellular Ca2+ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One- and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of approximately 3.5-fold and to maintain its Ca2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca2+ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca(2+)-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca2+ buffering within the IP3-sensitive, rapidly exchanging, Ca2+ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca2+ buffering within the IP3-sensitive stores accounts for approximately 45% of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca2+ binding proteins; (d) the free [Ca2+] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca2+ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+].

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Selected References

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  1. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  2. Bezprozvanny I., Watras J., Ehrlich B. E. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 1991 Jun 27;351(6329):751–754. doi: 10.1038/351751a0. [DOI] [PubMed] [Google Scholar]
  3. Brini M., Marsault R., Bastianutto C., Alvarez J., Pozzan T., Rizzuto R. Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. J Biol Chem. 1995 Apr 28;270(17):9896–9903. doi: 10.1074/jbc.270.17.9896. [DOI] [PubMed] [Google Scholar]
  4. Carafoli E., Chiesi M. Calcium pumps in the plasma and intracellular membranes. Curr Top Cell Regul. 1992;32:209–241. doi: 10.1016/b978-0-12-152832-4.50007-0. [DOI] [PubMed] [Google Scholar]
  5. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56:395–433. doi: 10.1146/annurev.bi.56.070187.002143. [DOI] [PubMed] [Google Scholar]
  6. Colyer J., Mata A. M., Lee A. G., East J. M. Effects on ATPase activity of monoclonal antibodies raised against (Ca2+ + Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum and their correlation with epitope location. Biochem J. 1989 Sep 1;262(2):439–447. doi: 10.1042/bj2620439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Damiani E., Heilmann C., Salvatori S., Margreth A. Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties. Biochem Biophys Res Commun. 1989 Dec 29;165(3):973–980. doi: 10.1016/0006-291x(89)92698-3. [DOI] [PubMed] [Google Scholar]
  8. Dedhar S. Novel functions for calreticulin: interaction with integrins and modulation of gene expression? Trends Biochem Sci. 1994 Jul;19(7):269–271. doi: 10.1016/0968-0004(94)90001-9. [DOI] [PubMed] [Google Scholar]
  9. Dedhar S., Rennie P. S., Shago M., Hagesteijn C. Y., Yang H., Filmus J., Hawley R. G., Bruchovsky N., Cheng H., Matusik R. J. Inhibition of nuclear hormone receptor activity by calreticulin. Nature. 1994 Feb 3;367(6462):480–483. doi: 10.1038/367480a0. [DOI] [PubMed] [Google Scholar]
  10. Diarra A., Sauvé R. Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations. Pflugers Arch. 1992 Oct;422(1):40–47. doi: 10.1007/BF00381511. [DOI] [PubMed] [Google Scholar]
  11. Fasolato C., Innocenti B., Pozzan T. Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends Pharmacol Sci. 1994 Mar;15(3):77–83. doi: 10.1016/0165-6147(94)90282-8. [DOI] [PubMed] [Google Scholar]
  12. Fasolato C., Zottini M., Clementi E., Zacchetti D., Meldolesi J., Pozzan T. Intracellular Ca2+ pools in PC12 cells. Three intracellular pools are distinguished by their turnover and mechanisms of Ca2+ accumulation, storage, and release. J Biol Chem. 1991 Oct 25;266(30):20159–20167. [PubMed] [Google Scholar]
  13. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fleischer S., Inui M. Biochemistry and biophysics of excitation-contraction coupling. Annu Rev Biophys Biophys Chem. 1989;18:333–364. doi: 10.1146/annurev.bb.18.060189.002001. [DOI] [PubMed] [Google Scholar]
  15. Furuichi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N., Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 1989 Nov 2;342(6245):32–38. doi: 10.1038/342032a0. [DOI] [PubMed] [Google Scholar]
  16. Grohovaz F., Zacchetti D., Clementi E., Lorenzon P., Meldolesi J., Fumagalli G. [Ca2+]i imaging in PC12 cells: multiple response patterns to receptor activation reveal new aspects of transmembrane signaling. J Cell Biol. 1991 Jun;113(6):1341–1350. doi: 10.1083/jcb.113.6.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  18. Hajnóczky G., Thomas A. P. The inositol trisphosphate calcium channel is inactivated by inositol trisphosphate. Nature. 1994 Aug 11;370(6489):474–477. doi: 10.1038/370474a0. [DOI] [PubMed] [Google Scholar]
  19. Hirose K., Iino M. Heterogeneity of channel density in inositol-1,4,5-trisphosphate-sensitive Ca2+ stores. Nature. 1994 Dec 22;372(6508):791–794. doi: 10.1038/372791a0. [DOI] [PubMed] [Google Scholar]
  20. Hoth M., Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. doi: 10.1038/355353a0. [DOI] [PubMed] [Google Scholar]
  21. Kao L. S., Cheung N. S. Mechanism of calcium transport across the plasma membrane of bovine chromaffin cells. J Neurochem. 1990 Jun;54(6):1972–1979. doi: 10.1111/j.1471-4159.1990.tb04900.x. [DOI] [PubMed] [Google Scholar]
  22. Lewis R. S., Cahalan M. D. Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells. Cell Regul. 1989 Nov;1(1):99–112. doi: 10.1091/mbc.1.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu N., Fine R. E., Simons E., Johnson R. J. Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells. J Biol Chem. 1994 Nov 18;269(46):28635–28639. [PubMed] [Google Scholar]
  24. Lucero H. A., Lebeche D., Kaminer B. ERcalcistorin/protein disulfide isomerase (PDI). Sequence determination and expression of a cDNA clone encoding a calcium storage protein with PDI activity from endoplasmic reticulum of the sea urchin egg. J Biol Chem. 1994 Sep 16;269(37):23112–23119. [PubMed] [Google Scholar]
  25. Lytton J., Nigam S. K. Intracellular calcium: molecules and pools. Curr Opin Cell Biol. 1992 Apr;4(2):220–226. doi: 10.1016/0955-0674(92)90036-c. [DOI] [PubMed] [Google Scholar]
  26. MacLennan D. H. Molecular tools to elucidate problems in excitation-contraction coupling. Biophys J. 1990 Dec;58(6):1355–1365. doi: 10.1016/S0006-3495(90)82482-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCauliffe D. P., Lux F. A., Lieu T. S., Sanz I., Hanke J., Newkirk M. M., Bachinski L. L., Itoh Y., Siciliano M. J., Reichlin M. Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen. J Clin Invest. 1990 May;85(5):1379–1391. doi: 10.1172/JCI114582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Menniti F. S., Bird G. S., Takemura H., Thastrup O., Potter B. V., Putney J. W., Jr Mobilization of calcium by inositol trisphosphates from permeabilized rat parotid acinar cells. Evidence for translocation of calcium from uptake to release sites within the inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pool. J Biol Chem. 1991 Jul 25;266(21):13646–13653. [PubMed] [Google Scholar]
  29. Michalak M., Milner R. E., Burns K., Opas M. Calreticulin. Biochem J. 1992 Aug 1;285(Pt 3):681–692. doi: 10.1042/bj2850681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mikoshiba K. Inositol 1,4,5-trisphosphate receptor. Trends Pharmacol Sci. 1993 Mar;14(3):86–89. doi: 10.1016/0165-6147(93)90069-v. [DOI] [PubMed] [Google Scholar]
  31. Muller S. R., Sullivan P. D., Clegg D. O., Feinstein S. C. Efficient transfection and expression of heterologous genes in PC12 cells. DNA Cell Biol. 1990 Apr;9(3):221–229. doi: 10.1089/dna.1990.9.221. [DOI] [PubMed] [Google Scholar]
  32. Putney J. W., Jr A model for receptor-regulated calcium entry. Cell Calcium. 1986 Feb;7(1):1–12. doi: 10.1016/0143-4160(86)90026-6. [DOI] [PubMed] [Google Scholar]
  33. Racchetti G., Papazafiri P., Volpe P., Meldolesi J. Calstorin, a new Ca2+ binding protein of the microsome lumen which is abundant in the rat brain. Biochem Biophys Res Commun. 1994 Sep 15;203(2):828–833. doi: 10.1006/bbrc.1994.2257. [DOI] [PubMed] [Google Scholar]
  34. Raichman M., Panzeri M. C., Clementi E., Papazafiri P., Eckley M., Clegg D. O., Villa A., Meldolesi J. Differential localization and functional role of calsequestrin in growing and differentiated myoblasts. J Cell Biol. 1995 Feb;128(3):341–354. doi: 10.1083/jcb.128.3.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rizzuto R., Bastianutto C., Brini M., Murgia M., Pozzan T. Mitochondrial Ca2+ homeostasis in intact cells. J Cell Biol. 1994 Sep;126(5):1183–1194. doi: 10.1083/jcb.126.5.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rizzuto R., Brini M., Murgia M., Pozzan T. Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science. 1993 Oct 29;262(5134):744–747. doi: 10.1126/science.8235595. [DOI] [PubMed] [Google Scholar]
  37. Rizzuto R., Brini M., Pozzan T. Targeting recombinant aequorin to specific intracellular organelles. Methods Cell Biol. 1994;40:339–358. doi: 10.1016/s0091-679x(08)61121-8. [DOI] [PubMed] [Google Scholar]
  38. Rojiani M. V., Finlay B. B., Gray V., Dedhar S. In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits. Biochemistry. 1991 Oct 15;30(41):9859–9866. doi: 10.1021/bi00105a008. [DOI] [PubMed] [Google Scholar]
  39. Takeshima H., Nishimura S., Matsumoto T., Ishida H., Kangawa K., Minamino N., Matsuo H., Ueda M., Hanaoka M., Hirose T. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature. 1989 Jun 8;339(6224):439–445. doi: 10.1038/339439a0. [DOI] [PubMed] [Google Scholar]
  40. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Treves S., De Mattei M., Landfredi M., Villa A., Green N. M., MacLennan D. H., Meldolesi J., Pozzan T. Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain. Biochem J. 1990 Oct 15;271(2):473–480. doi: 10.1042/bj2710473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van P. N., Peter F., Söling H. D. Four intracisternal calcium-binding glycoproteins from rat liver microsomes with high affinity for calcium. No indication for calsequestrin-like proteins in inositol 1,4,5-trisphosphate-sensitive calcium sequestering rat liver vesicles. J Biol Chem. 1989 Oct 15;264(29):17494–17501. [PubMed] [Google Scholar]
  43. Villa A., Podini P., Panzeri M. C., Söling H. D., Volpe P., Meldolesi J. The endoplasmic-sarcoplasmic reticulum of smooth muscle: immunocytochemistry of vas deferens fibers reveals specialized subcompartments differently equipped for the control of Ca2+ homeostasis. J Cell Biol. 1993 Jun;121(5):1041–1051. doi: 10.1083/jcb.121.5.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Villa A., Sharp A. H., Racchetti G., Podini P., Bole D. G., Dunn W. A., Pozzan T., Snyder S. H., Meldolesi J. The endoplasmic reticulum of Purkinje neuron body and dendrites: molecular identity and specializations for Ca2+ transport. Neuroscience. 1992 Jul;49(2):467–477. doi: 10.1016/0306-4522(92)90111-e. [DOI] [PubMed] [Google Scholar]
  45. Zacchetti D., Clementi E., Fasolato C., Lorenzon P., Zottini M., Grohovaz F., Fumagalli G., Pozzan T., Meldolesi J. Intracellular Ca2+ pools in PC12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-trisphosphate and caffeine-ryanodine. J Biol Chem. 1991 Oct 25;266(30):20152–20158. [PubMed] [Google Scholar]

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