Cellular and Molecular Life Sciences

, Volume 75, Issue 8, pp 1461–1482 | Cite as

Characterizations of PMCA2-interacting complex and its role as a calcium oxalate crystal-binding protein

  • Arada Vinaiphat
  • Visith Thongboonkerd
Original Article


Three isoforms of plasma membrane Ca2+-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca2+ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.


Crystal adhesion Crystal internalization Immuno-co-localization Interactomics IP-MS Kidney stone Renal calculi Renal tubular cells 



We thank Phornpimon Tipthara and Kedsarin Fong-ngern for their technical assistance. This study was supported by Mahidol University research grant, Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative, and the Thailand Research Fund (IRN60W0004 and IRG5980006). AV is supported by Siriraj Graduate Thesis Scholarship, whereas VT is supported by “Research Staff” Grant.

Author contributions

AV and VT designed research; AV performed experiments; AV and VT analyzed data; AVand VT wrote the manuscript; all authors reviewed the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

18_2017_2699_MOESM1_ESM.pdf (94 kb)
Supplementary material 1 (PDF 93 kb)


  1. 1.
    Strehler EE, Zacharias DA (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81:21–50CrossRefPubMedGoogle Scholar
  2. 2.
    Schuh K, Uldrijan S, Gambaryan S, Roethlein N, Neyses L (2003) Interaction of the plasma membrane Ca2+ pump 4b/CI with the Ca2+/calmodulin-dependent membrane-associated kinase CASK. J Biol Chem 278:9778–9783CrossRefPubMedGoogle Scholar
  3. 3.
    Carafoli E (1991) Calcium pump of the plasma membrane. Physiol Rev 71:129–153CrossRefPubMedGoogle Scholar
  4. 4.
    Oberleithner H, Westphale HJ, Gassner B (1991) Alkaline stress transforms Madin-Darby canine kidney cells. Pflug Arch 419:418–420CrossRefGoogle Scholar
  5. 5.
    Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35:919–922CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Strehler EE, Caride AJ, Filoteo AG, Xiong Y, Penniston JT, Enyedi A (2007) Plasma membrane Ca2+ ATPases as dynamic regulators of cellular calcium handling. Ann N Y Acad Sci 1099:226–236CrossRefPubMedGoogle Scholar
  7. 7.
    Domi T, Di Leva F, Fedrizzi L, Rimessi A, Brini M (2007) Functional specificity of PMCA isoforms? Ann N Y Acad Sci 1099:237–246CrossRefPubMedGoogle Scholar
  8. 8.
    Lee WJ, Roberts-Thomson SJ, Monteith GR (2005) Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines. Biochem Biophys Res Commun 337:779–783CrossRefPubMedGoogle Scholar
  9. 9.
    Spiden SL, Bortolozzi M, Di Leva F, de Angelis MH, Fuchs H, Lim D, Ortolano S, Ingham NJ, Brini M, Carafoli E, Mammano F, Steel KP (2008) The novel mouse mutation Oblivion inactivates the PMCA2 pump and causes progressive hearing loss. PLoS Genet 4:e1000238CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Schultz JM, Yang Y, Caride AJ, Filoteo AG, Penheiter AR, Lagziel A, Morell RJ, Mohiddin SA, Fananapazir L, Madeo AC, Penniston JT, Griffith AJ (2005) Modification of human hearing loss by plasma-membrane calcium pump PMCA2. N Engl J Med 352:1557–1564CrossRefPubMedGoogle Scholar
  11. 11.
    Kurnellas MP, Nicot A, Shull GE, Elkabes S (2005) Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury. FASEB J 19:298–300CrossRefPubMedGoogle Scholar
  12. 12.
    Xiong Y, Antalffy G, Enyedi A, Strehler EE (2009) Apical localization of PMCA2w/b is lipid raft-dependent. Biochem Biophys Res Commun 384:32–36CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kip SN, Strehler EE (2004) Vitamin D3 upregulates plasma membrane Ca2+-ATPase expression and potentiates apico-basal Ca2+ flux in MDCK cells. Am J Physiol Ren Physiol 286:F363–F369CrossRefGoogle Scholar
  14. 14.
    Friedman PA (2000) Mechanisms of renal calcium transport. Exp Nephrol 8:343–350CrossRefPubMedGoogle Scholar
  15. 15.
    Padanyi R, Xiong Y, Antalffy G, Lor K, Paszty K, Strehler EE, Enyedi A (2010) Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells. J Biol Chem 285:31704–31712CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kip SN, Strehler EE (2003) Characterization of PMCA isoforms and their contribution to transcellular Ca2+ flux in MDCK cells. Am J Physiol Ren Physiol 284:F122–F132CrossRefGoogle Scholar
  17. 17.
    Lopreiato R, Giacomello M, Carafoli E (2014) The plasma membrane calcium pump: new ways to look at an old enzyme. J Biol Chem 289:10261–10268CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Brini M (2009) Plasma membrane Ca(2+)-ATPase: from a housekeeping function to a versatile signaling role. Pflug Arch 457:657–664CrossRefGoogle Scholar
  19. 19.
    Strehler EE (2013) Plasma membrane calcium ATPases as novel candidates for therapeutic agent development. J Pharm Pharm Sci 16:190–206CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Gingras AC, Gstaiger M, Raught B, Aebersold R (2007) Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol 8:645–654CrossRefPubMedGoogle Scholar
  21. 21.
    Dukes JD, Whitley P, Chalmers AD (2011) The MDCK variety pack: choosing the right strain. BMC Cell Biol 12:43CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Sritippayawan S, Chiangjong W, Semangoen T, Aiyasanon N, Jaetanawanitch P, Sinchaikul S, Chen ST, Vasuvattakul S, Thongboonkerd V (2007) Proteomic analysis of peritoneal dialysate fluid in patients with different types of peritoneal membranes. J Proteome Res 6:4356–4362CrossRefPubMedGoogle Scholar
  23. 23.
    Havanapan PO, Thongboonkerd V (2009) Are protease inhibitors required for gel-based proteomics of kidney and urine? J Proteome Res 8:3109–3117CrossRefPubMedGoogle Scholar
  24. 24.
    Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232CrossRefPubMedGoogle Scholar
  25. 25.
    Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723–C742CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Thongboonkerd V, Semangoen T, Chutipongtanate S (2006) Factors determining types and morphologies of calcium oxalate crystals: molar concentrations, buffering, pH, stirring and temperature. Clin Chim Acta 367:120–131CrossRefPubMedGoogle Scholar
  27. 27.
    Thongboonkerd V, Semangoen T, Sinchaikul S, Chen ST (2008) Proteomic analysis of calcium oxalate monohydrate crystal-induced cytotoxicity in distal renal tubular cells. J Proteome Res 7:4689–4700CrossRefPubMedGoogle Scholar
  28. 28.
    Chaiyarit S, Mungdee S, Thongboonkerd V (2010) Non-radioactive labelling of calcium oxalate crystals for investigations of crystal-cell interaction and internalization. Anal Methods 2:1536–1541CrossRefGoogle Scholar
  29. 29.
    Chaiyarit S, Singhto N, Thongboonkerd V (2016) Calcium oxalate monohydrate crystals internalized into renal tubular cells are degraded and dissolved by endolysosomes. Chem Biol Interact 246:30–35CrossRefPubMedGoogle Scholar
  30. 30.
    Fong-ngern K, Chiangjong W, Thongboonkerd V (2009) Peeling as a novel, simple, and effective method for isolation of apical membrane from intact polarized epithelial cells. Anal Biochem 395:25–32CrossRefPubMedGoogle Scholar
  31. 31.
    Fong-ngern K, Peerapen P, Sinchaikul S, Chen ST, Thongboonkerd V (2011) Large-scale identification of calcium oxalate monohydrate crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. J Proteome Res 10:4463–4477CrossRefPubMedGoogle Scholar
  32. 32.
    Holton M, Yang D, Wang W, Mohamed TM, Neyses L, Armesilla AL (2007) The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells. FEBS Lett 581:4115–4119CrossRefPubMedGoogle Scholar
  33. 33.
    Kosk-Kosicka D, Zylinska L (1997) Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells. Mol Cell Biochem 173:79–87CrossRefPubMedGoogle Scholar
  34. 34.
    Usachev YM, DeMarco SJ, Campbell C, Strehler EE, Thayer SA (2002) Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4. Neuron 33:113–122CrossRefPubMedGoogle Scholar
  35. 35.
    Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C (2015) STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:D447–D452CrossRefPubMedGoogle Scholar
  36. 36.
    DeMarco SJ, Chicka MC, Strehler EE (2002) Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes. J Biol Chem 277:10506–10511CrossRefPubMedGoogle Scholar
  37. 37.
    Perkins JR, Diboun I, Dessailly BH, Lees JG, Orengo C (2010) Transient protein–protein interactions: structural, functional, and network properties. Structure 18:1233–1243CrossRefPubMedGoogle Scholar
  38. 38.
    Boulon S, Ahmad Y, Trinkle-Mulcahy L, Verheggen C, Cobley A, Gregor P, Bertrand E, Whitehorn M, Lamond AI (2010) Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteom 9:861–879CrossRefGoogle Scholar
  39. 39.
    Bozulic LD, Malik MT, Powell DW, Nanez A, Link AJ, Ramos KS, Dean WL (2007) Plasma membrane Ca(2+)-ATPase associates with CLP36, alpha-actinin and actin in human platelets. Thromb Haemost 97:587–597PubMedGoogle Scholar
  40. 40.
    Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiol Rev 80:1483–1521CrossRefPubMedGoogle Scholar
  41. 41.
    von Mering C, Krause R, Snel B, Cornell M, Oliver SG, Fields S, Bork P (2002) Comparative assessment of large-scale data sets of protein–protein interactions. Nature 417:399–403CrossRefGoogle Scholar
  42. 42.
    Carafoli E, Santella L, Branca D, Brini M (2001) Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol 36:107–260CrossRefPubMedGoogle Scholar
  43. 43.
    Dardamanis M (2013) Pathomechanisms of nephrolithiasis. Hippokratia 17:100–107PubMedPubMedCentralGoogle Scholar
  44. 44.
    Vinaiphat A, Thongboonkerd V (2017) Prospects for proteomics in kidney stone disease. Expert Rev Proteom 14:185–187CrossRefGoogle Scholar
  45. 45.
    Lieske JC, Leonard R, Toback FG (1995) Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions. Am J Physiol 268:F604–F612CrossRefPubMedGoogle Scholar
  46. 46.
    Lieske JC, Leonard R, Swift H, Toback FG (1996) Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells. Am J Physiol 270:F192–F199CrossRefPubMedGoogle Scholar
  47. 47.
    Kanlaya R, Sintiprungrat K, Chaiyarit S, Thongboonkerd V (2013) Macropinocytosis is the major mechanism for endocytosis of calcium oxalate crystals into renal tubular cells. Cell Biochem Biophys 67:1171–1179CrossRefPubMedGoogle Scholar
  48. 48.
    Fong-ngern K, Sueksakit K, Thongboonkerd V (2016) Surface heat shock protein 90 serves as a potential receptor for calcium oxalate crystal on apical membrane of renal tubular epithelial cells. J Biol Inorg Chem 21:463–474CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand
  2. 2.Center for Research in Complex Systems ScienceMahidol UniversityBangkokThailand

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