Mammalian Genome

, Volume 16, Issue 3, pp 137–151 | Cite as

Cardiac muscle cell cytoskeletal protein 4.1: Analysis of transcripts and subcellular location—relevance to membrane integrity, microstructure, and possible role in heart failure

  • Pamela M. Taylor-Harris
  • Lisa A. Keating
  • Alison M. Maggs
  • Gareth W. Phillips
  • Emma J. Birks
  • Rodney C.G. Franklin
  • Magdi H. Yacoub
  • Anthony J. Baines
  • Jennifer C. PinderEmail author
Original Contributions


The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies.


Intercalate Disc Ferm Domain Human Heart Tissue Sarcomeric Actin Human Dilate Cardiomyopathy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the British Heart Foundation (project grant numbers PG/99155 and PG/03/159/16422), the BBSRC, and the Medical Research Council. We thank Prof. John Conboy for considerable help in sharing unpublished results on 4.1 sequences, Dr. Elisabeth Ehler for rat cardiomyocytes, Dr. T. K. Tang for a full-length 4.1R cDNA clone, Dr. Philippe Gascard for anti-4.1R antibodies, and Dr. Leszek Kotula for an anti-αII-spectrin antibodies. Kate Kirwan’s help with the preparation of images and figures was much appreciated.


  1. Arber, S, Hunter, JJ, Ross, J,Jr, Hong, M, Sansig, G,  et al. 1997MLP dificient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failureCell88393403Google Scholar
  2. Auerbach, D, Bantle, S, Keller, S, Hinderling, V, Leu, M,  et al. 1999Different domains of the M-band protein myomesin are involved in myosin binding and M-band targetingMol Biol Cell1012971308Google Scholar
  3. Badorff, C, Lee, GH, Lamphear, BJ, Martone, ME, Campbell, KP,  et al. 1999Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathyNat Med5320326Google Scholar
  4. Baklouti, F, Huang, SC, Vulliamy, TJ, Delaunay, J, Benz, EJ,Jr 1997Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNAGenomics39289302Google Scholar
  5. Baumgartner, S, Littleton, JT, Broadie, K, Bhat, MA, Harbecke, R,  et al. 1996A Drosophila neurexin is required for septate junction and blood–nerve barrier formation and functionCell8710591068Google Scholar
  6. Bennett, V, Baines, AJ 2001Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissuesPhysiol Rev8113531392Google Scholar
  7. Bennett, PM, Baines, AJ, Lecomte, MC, Maggs, AM, Pinder, JC 2004Not just a plasma membrane protein: In cardiac muscle cells alpha-II spectrin also shows a close association with myofibrilsJ Muscle Res Cell Motil25119126Google Scholar
  8. Carver, T, Bleasby, A 2003The design of Jemboss: a graphical user interface to EMBOSSBioinformatics1918371843Google Scholar
  9. Chasis, JA, Coulombel, L, McGee, S, Lee, G, Tchernia, G,  et al. 1996Differential use of protein 4.1 translation initiation sites during erythropoiesis: implications for a mutation-induced stage-specific deficiency of protein 4.1 during erythroid developmentBlood8753245331Google Scholar
  10. Chishti, AH, Kim, AC, Marfatia, SM, Lutchman, M, Hanspal, M,  et al. 1998The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membraneTrends Biochem Sci23281282Google Scholar
  11. Cianci, CD, Zhang, Z, Pradhan, D, Morrow, JS 1999Brain and muscle express a unique alternative transcript of alphall spectrinBiochemistry381572115730Google Scholar
  12. Coleman, SK, Cai, C, Mottershead, DG, Haapalahti, JP, Keinanen, K 2003Surface expression of GluR-D AMPA receptor is dependent on an interaction between its C-terminal domain and a 4.1 proteinJ Neurosci23798806Google Scholar
  13. Conboy, J 1999The role of alternative pre-mRNA splicing in regulating the structure and function of skeletal protein 4.1Proc Soc Exp Biol Med2207378Google Scholar
  14. Conboy, J, Kan, YW, Shohet, SB, Mohandas, N 1986Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeletonProc Natl Acad Sci USA8395129516Google Scholar
  15. Conboy, JG, Chan, JY, Chasis, JA, Kan, YW, Mohandas, N 1991Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1J Biol Chem26682738280Google Scholar
  16. Corpet, F 1988Multiple sequence alignment with hierarchical clusteringNucleic Acid Res161088110890Google Scholar
  17. Dan-Goor, M, Silberstein, L, Kessel, M, Muhlrad, A 1990Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosinJ Muscle Res Cell Motil11216226Google Scholar
  18. Denisenko–Nehrbass, N, Goutebroze, L, Galvez, T, Bonnon, C, Stankoff, B,  et al. 2003Association of Caspr/paranodin with tumour suppressor schwannomin/merlin and betal integrin in the central nervous systemJ Neurochem84209221Google Scholar
  19. Discher, DE, Winardi, R, Schischmanoff, PO, Parra, M, Conboy, JG,  et al. 1995Mechanochemistry of protein 4.1’s spectrrn-actui-binding domain: ternary complex interactions, membrane binding, network integration, structural strengtheningJ Cell Biol130897907Google Scholar
  20. Ehler, E, Horowits, R, Zuppinger, C, Price, RL, Perriard, E,  et al. 2001Alterations at the intercalated disk associated with the absence of muscle LIM proteinJ Cell Biol153763772Google Scholar
  21. Flick, MJ, Konieczny, SF 2000The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrinJ Cell Sci113(Pt 9)15531564Google Scholar
  22. Gascard, P, Mohandas, N 2000New insights into functions of erythroid proteins in nonerythroid cellsCurr Opin Hematol7123129Google Scholar
  23. Gimm, JA, An, X, Nunomura, W, Mohandas, N 2002Functional characterization of spectrin-actin-binding domains in 4.1 family of proteinsBiochemistry4172757282Google Scholar
  24. Graham, RM, Owens, WA 1999Pathogenesis of inherited forms of dilated cardiomyopathyN Engl J Med34117591762Google Scholar
  25. Hayes, NV, Scott, C, Heerkens, E, Ohanian, V, Maggs, AM,  et al. 2000Identification of a novel C-terminal variant of beta II spectrin: two isoforms of beta II spectrin have distinct intracellular locations and activitiesJ Cell Sci11320232034Google Scholar
  26. Hoover, KB, Bryant, PJ 2000The genetics of the protein 4.1 family: organizers of the membrane and cytoskeletonCurr Opin Cell Biol12229234Google Scholar
  27. Hu, Z, Frith, M, Niu, T, Weng, Z 2003SeqVISTA: a graphical tool for sequence feature visualization and comparisonBMC Bioinformatics41Google Scholar
  28. Huang, J, Tang, C, Kou, G, Marchesi, V, Benz, E,Jr,  et al. 1993Genomic structure of the locus encoding protein 4.1. Structural basis for complex combinational patterns of tissue-specific alternative RNA splicingJ Biol Chem26837583766Google Scholar
  29. Isayama, T, Goodman, SR, Zagon, IS 1993Localization of spectrin isoforms in the adult mouse heartCell Tissue Res274127133Google Scholar
  30. Jons, T, Drenckhahn, DT 1992Identification of the binding interface involved in linkage of cytoskeletal protein 4.1 to the erythrocyte anion exchangerEmbo1128632867Google Scholar
  31. Kittimyom, K, Mastronardi, M, Roemer, M, Wells, WA, Greenberg, ER,  et al. 2004Allele-specific loss of heterozygosity at the DAL-1/4.1B (EPB41L3) tumor-suppressor gene locus in the absence of mutationGenes Chromosomes Cancer41190203Google Scholar
  32. Kontrogianni–Konstantopoulos, A, Huang, SC, Benz, EJ,Jr 2000A nonerythroid isoform of protein 4.1R interacts with components of the contractile apparatus in skeletal myofibersMol Biol Cell3138053817Google Scholar
  33. Kostin, S, Scholz, D, Shimada, T, Maeno, Y, Molnau, H,  et al. 1998The internal and external protein scaffold of the T-tubular system in cardiomyocytesCell Tissue Res94449460Google Scholar
  34. Laemmli, UK 1970Cleavage of structural proteins during the assembly of the head of bacteriophage T4Nature227680685Google Scholar
  35. Leonoudakis, D, Conti, LR, Anderson, S, Radeke, CM, McGuire, LM,  et al. 2004aProtein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteinsJ Biol Chem2792233122346Google Scholar
  36. Leonoudakis, D, Conti, LR, Radeke, CM, McGuire, LM, Vandenberg, CA 2004bA multiprotein trafficking complex composed of SAP97, CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium channelsJ Biol Chem2791905119063Google Scholar
  37. Linn, SC, Askew, GR, Menon, AG, Shull, GE 1995Conservation of anAE3 ClHCO 3 exchanger cardiac-specific exon and promoter region and AE3 mRNA expression patterns in murine and human heartsCirc Res76584591Google Scholar
  38. Lu, D, Yan, H, Othman, T, Turner, CP, Woolf, T,  et al. 2004Cytoskeletal protein 4.1G binds to the third intracellular loop of the A1 adenosine receptor and inhibits receptor actionBiochem J3775159Google Scholar
  39. Maggs, AM, Taylor–Harris, P, Peckham, M, Hughes, Sm 2000Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle developmentJ Muscle Res Cell Motil21101113Google Scholar
  40. Maximov, A, Tang, TS, Bezprozvanny, I 2003Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neuronsMol Cell Neurosci22271283Google Scholar
  41. Minamisawa, S, Hojima, M, Chu, G, Ward, CA, Frank, K,  et al. 1999Chronic phospholamban–sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathyCell99313322Google Scholar
  42. Mohler, PJ, Schott, JJ, Gramolini, AO, Dilly, KW, Guatimosim, S,  et al. 2003Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac deathNature421634639Google Scholar
  43. Moura Lima, PR, Salles, TS, Costa, FF, Saad, ST 2003Alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoformJ Cell Biochem8912151221Google Scholar
  44. Parra, M, Gascard, P, Walensky, LD, Snyder, SH, Mohandas, N,  et al. 1998Cloning and characterization of 4.1G (EPB41L2), a new member of the skeletal protein 4.1 (EPB41) gene familyGenomics49298306Google Scholar
  45. Parra, M, Gascard, P, Walensky, LD, Gimm, JA, Blackshaw, S,  et al. 2000Molecular and functional characterization of protein 4.1B, a novel member of the protein 4.1 family with high level, focal expression in brainJ Biol Chem27532473255Google Scholar
  46. Parra, M, Gee, S, Chan, N, Ryaboy, D, Dubchak, I,  et al. 2004Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitate expression of diverse tissue-specific isoformsGenomics84637646Google Scholar
  47. Pastenack, GR, Racusen, RH 1989Erythrocyte protein 4.1 binds and regulates myosinProc Natl Acad Sci USA8697129716Google Scholar
  48. Ramez, M, Blot–Chabaud, M, Cluzeaud, F, Chanan, S, Patterson, M,  et al. 2003Distinct distribution of specific members of protein 4.1 gene family in the mouse nephronKidney Int6313211337Google Scholar
  49. Richards, SM, Jaconi, ME, Vassort, G, Puceat, M 1999A spliced variant of AE1 gene encodes a truncated form of Band 3 in heart: the predominant anion exchanger in ventricular myocytesJ Cell Sci11215191528Google Scholar
  50. Robb, VA, Li, W, Gascard, P, Perry, A, Mohandas, N,  et al. 2003Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesisNeurobiol Dis13191202Google Scholar
  51. Rumbaugh, G, Sia, GM, Garner, CC, Huganir, RL 2003Synapse-associated protein-97 isoform-specific regulation of surface AMPA receptors and synaptic function in cultured neuronsJ Neurosci2345674576Google Scholar
  52. Schischmanoff, PO, Yaswen, P, Parra, MK, Lee, G, Chasis, JA,  et al. 1997Cell shape-dependent regulation of protein 4.1 alternative pre-mRNA splicing in mammary epithelial cellsJ Biol Chem2721025410259Google Scholar
  53. Scott, C, Keating, L, Bellamy, M, Baines, AJ 2001aProtein 4.1 in forebrain postsynaptic density preparations: enrichment of 4.1 gene products and detection of 4.1R binding proteinsEur J Biochem26810841094Google Scholar
  54. Scott, C, Phillips, GW, Baines, AJ 2001bProperties of the C-terminal domain of 4.1 proteinsEur J Biochem26837093717Google Scholar
  55. Shen, L, Liang, F, Walensky, LD, Huganir, RL 2000Regulation of AMPA receptor GluR1 subunit surface expression by a 4.1 N-linked actin cytoskeletal associationJ Neurosci2079327940Google Scholar
  56. Sun, CX, Robb, VA, Gutmann, DH 2002Protein 4.1 tumor suppressors: getting a PERM grip on growth regulationJ Cell Sci11539914000Google Scholar
  57. Takakuwa, Y 2000Protein 4.1, a multifunctional protein of the erythrocyte membrane skeleton: structure and functions in erythrocytes and nonerythroid cellsInt J Hematol72298309Google Scholar
  58. Tang, TK, Leto, TL, Marchesi, VT, Benz, EJ,Jr 1988Expression of specific isoforms of protein 4.1 in erythroid and non-erythroid tissuesAdv Exp Med Biol2418195Google Scholar
  59. Tang, TK, Qin, Z, Leto, T, Marchesi, VT, Benz, EJ,Jr 1990Heterogeneity of mRNA and protein products arising from the protein 4.1 gene in erythroid and nonerythroid tissuesJ Cell Biol110617624Google Scholar
  60. Tang, Y, Katuri, V, Dillner, A, Mishra, B, Deng, CX,  et al. 2003Disruption of transforming growth factor-beta signaling in ELF beta-spectrin-deficient miceScience299574577Google Scholar
  61. Walensky, LD, Blackshaw, S, Liao, D, Watkins, CC, Weier, HU,  et al. 1999A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1J Neurosci1964576467Google Scholar
  62. Xu, J, Ziemnicka, D, Scalia, J, Kotula, L 2001Monoclonal antibodies to alphal spectrin Src homology 3 domain associate with macropinocytic vesicles in nonerythroid cellsBrain Res898171177Google Scholar
  63. Zhang, S, Mizutani, A, Hisatsune, C, Higo, T, Bannai, H,  et al. 2003Protem 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin–Darby canine kidney cellsJ Biol Chem27840484056Google Scholar
  64. Zolk, O, Caroni, P, Bohm, M 2000Decreased expression of the cardiac LIM domain protein MLP in chronic human heart failureCirculation10126742677Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Pamela M. Taylor-Harris
    • 1
  • Lisa A. Keating
    • 2
  • Alison M. Maggs
    • 1
  • Gareth W. Phillips
    • 1
  • Emma J. Birks
    • 3
  • Rodney C.G. Franklin
    • 3
  • Magdi H. Yacoub
    • 3
  • Anthony J. Baines
    • 2
  • Jennifer C. Pinder
    • 1
    Email author
  1. 1.Randall Division of Cell and Molecular BiophysicsKing’s College London, Guy’s CampusUK
  2. 2.Department of BiosciencesUniversity of KentCanterburyUK
  3. 3.Heart Science CentreImperial College LondonHarefieldUK

Personalised recommendations