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. 2003 Sep 15;374(Pt 3):793–797. doi: 10.1042/BJ20030683

Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane.

Mohabir Ramjeesingh 1, Jackie F Kidd 1, Ling Jun Huan 1, Yanchun Wang 1, Christine E Bear 1
PMCID: PMC1223644  PMID: 12820897

Abstract

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700-10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

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Selected References

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  1. Angers S., Salahpour A., Joly E., Hilairet S., Chelsky D., Dennis M., Bouvier M. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3684–3689. doi: 10.1073/pnas.060590697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boucher R. C. An overview of the pathogenesis of cystic fibrosis lung disease. Adv Drug Deliv Rev. 2002 Dec 5;54(11):1359–1371. doi: 10.1016/s0169-409x(02)00144-8. [DOI] [PubMed] [Google Scholar]
  3. Chen J-H, Chang X-B, Aleksandrov A. A., Riordan J. R. CFTR is a monomer: biochemical and functional evidence. J Membr Biol. 2002 Jul 1;188(1):55–71. doi: 10.1007/s00232-001-0174-2. [DOI] [PubMed] [Google Scholar]
  4. Cheng S. H., Gregory R. J., Marshall J., Paul S., Souza D. W., White G. A., O'Riordan C. R., Smith A. E. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell. 1990 Nov 16;63(4):827–834. doi: 10.1016/0092-8674(90)90148-8. [DOI] [PubMed] [Google Scholar]
  5. Clarke L. L., Grubb B. R., Gabriel S. E., Smithies O., Koller B. H., Boucher R. C. Defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis. Science. 1992 Aug 21;257(5073):1125–1128. doi: 10.1126/science.257.5073.1125. [DOI] [PubMed] [Google Scholar]
  6. Denning G. M., Anderson M. P., Amara J. F., Marshall J., Smith A. E., Welsh M. J. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature. 1992 Aug 27;358(6389):761–764. doi: 10.1038/358761a0. [DOI] [PubMed] [Google Scholar]
  7. Doyle D. A., Morais Cabral J., Pfuetzner R. A., Kuo A., Gulbis J. M., Cohen S. L., Chait B. T., MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998 Apr 3;280(5360):69–77. doi: 10.1126/science.280.5360.69. [DOI] [PubMed] [Google Scholar]
  8. Dutzler Raimund, Campbell Ernest B., Cadene Martine, Chait Brian T., MacKinnon Roderick. X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature. 2002 Jan 17;415(6869):287–294. doi: 10.1038/415287a. [DOI] [PubMed] [Google Scholar]
  9. Dutzler Raimund, Campbell Ernest B., MacKinnon Roderick. Gating the selectivity filter in ClC chloride channels. Science. 2003 Mar 20;300(5616):108–112. doi: 10.1126/science.1082708. [DOI] [PubMed] [Google Scholar]
  10. Eskandari S., Wright E. M., Kreman M., Starace D. M., Zampighi G. A. Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11235–11240. doi: 10.1073/pnas.95.19.11235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fischer H., Machen T. E. CFTR displays voltage dependence and two gating modes during stimulation. J Gen Physiol. 1994 Sep;104(3):541–566. doi: 10.1085/jgp.104.3.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hall R. A., Hansen A., Andersen P. H., Soderling T. R. Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells. J Neurochem. 1997 Feb;68(2):625–630. doi: 10.1046/j.1471-4159.1997.68020625.x. [DOI] [PubMed] [Google Scholar]
  13. Jiang Y., MacKinnon R. The barium site in a potassium channel by x-ray crystallography. J Gen Physiol. 2000 Mar;115(3):269–272. doi: 10.1085/jgp.115.3.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kartner N., Augustinas O., Jensen T. J., Naismith A. L., Riordan J. R. Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland. Nat Genet. 1992 Aug;1(5):321–327. doi: 10.1038/ng0892-321. [DOI] [PubMed] [Google Scholar]
  15. Kartner N., Hanrahan J. W., Jensen T. J., Naismith A. L., Sun S. Z., Ackerley C. A., Reyes E. F., Tsui L. C., Rommens J. M., Bear C. E. Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. Cell. 1991 Feb 22;64(4):681–691. doi: 10.1016/0092-8674(91)90498-n. [DOI] [PubMed] [Google Scholar]
  16. Krouse M. E., Wine J. J. Evidence that CFTR channels can regulate the open duration of other CFTR channels: cooperativity. J Membr Biol. 2001 Aug 1;182(3):223–232. doi: 10.1007/s00232-001-0046-9. [DOI] [PubMed] [Google Scholar]
  17. Marshall J., Fang S., Ostedgaard L. S., O'Riordan C. R., Ferrara D., Amara J. F., Hoppe H., 4th, Scheule R. K., Welsh M. J., Smith A. E. Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro. J Biol Chem. 1994 Jan 28;269(4):2987–2995. [PubMed] [Google Scholar]
  18. Naren Anjaparavanda P., Cobb Bryan, Li Chunying, Roy Koushik, Nelson David, Heda Ghanshyam D., Liao Jie, Kirk Kevin L., Sorscher Eric J., Hanrahan John. A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci U S A. 2002 Dec 26;100(1):342–346. doi: 10.1073/pnas.0135434100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pasyk E. A., Foskett J. K. Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J Biol Chem. 1995 May 26;270(21):12347–12350. doi: 10.1074/jbc.270.21.12347. [DOI] [PubMed] [Google Scholar]
  20. Raghuram V., Mak D. O., Foskett J. K. Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. Proc Natl Acad Sci U S A. 2001 Jan 23;98(3):1300–1305. doi: 10.1073/pnas.031538898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ramjeesingh M., Huan L. J., Garami E., Bear C. E. Novel method for evaluation of the oligomeric structure of membrane proteins. Biochem J. 1999 Aug 15;342(Pt 1):119–123. [PMC free article] [PubMed] [Google Scholar]
  22. Ramjeesingh M., Li C., Kogan I., Wang Y., Huan L. J., Bear C. E. A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator. Biochemistry. 2001 Sep 4;40(35):10700–10706. doi: 10.1021/bi0108195. [DOI] [PubMed] [Google Scholar]
  23. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  24. Sheppard D. N., Welsh M. J. Structure and function of the CFTR chloride channel. Physiol Rev. 1999 Jan;79(1 Suppl):S23–S45. doi: 10.1152/physrev.1999.79.1.S23. [DOI] [PubMed] [Google Scholar]
  25. Tsui L. C., Durie P. Genotype and phenotype in cystic fibrosis. Hosp Pract (1995) 1997 Jun 15;32(6):115-8, 123-9, 134, passim. doi: 10.1080/21548331.1997.11443512. [DOI] [PubMed] [Google Scholar]
  26. Wang S., Yue H., Derin R. B., Guggino W. B., Li M. Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell. 2000 Sep 29;103(1):169–179. doi: 10.1016/s0092-8674(00)00096-9. [DOI] [PubMed] [Google Scholar]
  27. Zabner J., Smith J. J., Karp P. H., Widdicombe J. H., Welsh M. J. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol Cell. 1998 Sep;2(3):397–403. doi: 10.1016/s1097-2765(00)80284-1. [DOI] [PubMed] [Google Scholar]
  28. Zerhusen B., Zhao J., Xie J., Davis P. B., Ma J. A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules. J Biol Chem. 1999 Mar 19;274(12):7627–7630. doi: 10.1074/jbc.274.12.7627. [DOI] [PubMed] [Google Scholar]
  29. Zhang F., Kartner N., Lukacs G. L. Limited proteolysis as a probe for arrested conformational maturation of delta F508 CFTR. Nat Struct Biol. 1998 Mar;5(3):180–183. doi: 10.1038/nsb0398-180. [DOI] [PubMed] [Google Scholar]

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