Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains. 1. 4.1R in the Erythrocyte Membrane Skeleton The membrane skeleton, which underlies the erythrocyte plasma membrane, is made of a spectrin/actin lattice anchored to various transmembrane proteins via two specialized cytoskeletal proteins, 4.1R and red blood cell ankyrin, ankyrin-R [1]. 4.1R80 stabilizes horizontal interactions between spectrin heterodimers (α2-β2) and short actin (~14 molecules) filaments. Actin filaments interact with numerous accessory proteins, such as tropomyosin, myosin, tropomodulin, and adducin [1], which ensure reorganization of actin filaments. 4.1R80 interacts also with the transmembrane protein, glycophorin C (GPC) and with the membrane-associated guanylate kinase (MAGUK) protein p55, which also acts as an erythrocyte scaffolding protein (Figure 1). Figure 1: Structure of human erythrocyte membrane. Spectrin dimers underlying the membrane interact with transmembrane proteins band 3 through ankyrin and glycophorin C (GPC) through an actin complex and protein 4.1R 80 (4.1R 80). 4.1R 80 also forms a ternary complex with p55 and GPC. CaM binds to 4.1R 80 in a Ca 2+-independent manner. 1.1. GPC GPC is 32？kDa single transmembrane protein expressed at ~50,000？100,000 molecules/erythrocyte. The cytoplasmic domain consists of 47 amino acids residues (ID: P04921). The R82HK sequence has been identified as the 4.1R binding sequence [2–4]. This RHK motif is highly conserved in the
References
[1]
V. Bennett and A. J. Baines, “Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues,” Physiological Reviews, vol. 81, no. 3, pp. 1353–1392, 2001.
[2]
N. J. Hemming, D. J. Anstee, W. J. Mawby, M. E. Reid, and M. J. A. Tanner, “Localization of the protein 4.1-binding site on human erythrocyte glycophorins C and D,” Biochemical Journal, vol. 299, no. 1, pp. 191–196, 1994.
[3]
N. J. Hemming, D. J. Anstee, M. A. Staricoff, M. J. A. Tanner, and N. Mohandas, “Identification of the membrane attachment sites for protein 4.1 in the human erythrocyte,” Journal of Biological Chemistry, vol. 270, no. 10, pp. 5360–5366, 1995.
[4]
S. M. Marfatia, R. A. Lue, D. Branton, and A. H. Chishti, “Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein,” Journal of Biological Chemistry, vol. 270, no. 2, pp. 715–719, 1995.
[5]
M. Yageta, M. Kuramochi, M. Masuda et al., “Direct association of TSLC1 and DAL-1, two distinct tumor suppressor proteins in lung cancer,” Cancer Research, vol. 62, no. 18, pp. 5129–5133, 2002.
[6]
J. A. Girault, G. Labesse, J. P. Mornon, and I. Callebaut, “Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction,” Molecular Medicine, vol. 4, no. 12, pp. 751–769, 1998.
[7]
H. Kusunoki and T. Kohno, “Structural insight into the interaction between the p55 PDZ domain and glycophorin C,” Biochemical and Biophysical Research Communications, vol. 359, no. 4, pp. 972–978, 2007.
[8]
S. D. Dimitratos, D. F. Woods, D. G. Stathakis, and P. J. Bryant, “Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family,” BioEssays, vol. 21, no. 11, pp. 912–921, 1999.
[9]
B. J. Quinn, E. J. Welch, A. C. Kim, et al., “Erythrocyte scaffolding protein p55/MPP1 functions as an essential regulator of neutrophil polarity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 47, pp. 19842–19847, 2009.
[10]
P. Mburu, Y. Kikkawa, S. Townsend, R. Romero, H. Yonekawa, and S. D. M. Brown, “Whirlin complexes with p55 at the stereocilia tip during hair cell development,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 29, pp. 10973–10978, 2006.
[11]
P. Mburu, M. R. Romero, H. Hilton et al., “Gelsolin plays a role in the actin polymerization complex of hair cell stereocilia,” PLoS ONE, vol. 5, no. 7, article e11627, 2010.
[12]
W. Nunomura and Y. Takakuwa, “Regulation of protein 4.1R interactions with membrane proteins by Ca2+ and calmodulin,” Frontiers in Bioscience, vol. 11, no. 2, pp. 1522–1539, 2006.
[13]
P. Michaely and V. Bennett, “The ANK repeats of erythrocyte ankyrin form two distinct but cooperative binding sites for the erythrocyte anion exchanger,” Journal of Biological Chemistry, vol. 270, no. 37, pp. 22050–22057, 1995.
[14]
T. Jons and D. Drenckhahn, “Identification of the binding interface involved in linkage of cytoskeletal protein 4.1 to the erythrocyte anion exchanger,” EMBO Journal, vol. 11, no. 8, pp. 2863–2867, 1992.
[15]
D. Zhang, A. Kiyatkin, J. T. Bolin, and P. S. Low, “Crystallographic structure and functional interpretation of the cytoplasmic domain of erythrocyte membrane band 3,” Blood, vol. 96, no. 9, pp. 2925–2933, 2000.
[16]
X. L. An, Y. Takakuwa, W. Nunomura, S. Manno, and N. Mohandas, “Modulation of band 3-ankyrin interaction by protein 4.1: functional implications in regulation of erythrocyte membrane mechanical properties,” Journal of Biological Chemistry, vol. 271, no. 52, pp. 33187–33191, 1996.
[17]
W. Nunomura, Y. Takakuwa, R. Tokimitsu, S. W. Krauss, M. Kawashima, and N. Mohandas, “Regulation of CD44-protein 4.1 interaction by Ca2+ and calmodulin. Implications for modulation of CD44-ankyrin interaction,” Journal of Biological Chemistry, vol. 272, no. 48, pp. 30322–30328, 1997.
[18]
W. Nunomura, Y. Takakuwa, G. N. Cherr, and K. Murata, “Characterization of protein 4.1R in erythrocytes of zebrafish (Danio rerio): unique binding properties with transmembrane proteins and calmodulin,” Comparative Biochemistry and Physiology B, vol. 148, pp. 124–138, 2007.
[19]
K. Murata, W. Nunomura, Y. Takakuwa, and G. N. Cherr, “Two different unique cardiac isoforms of protein 4.1R in zebrafish, Danio rerio, and insights into their cardiac functions as related to their unique structures,” Development Growth and Differentiation, vol. 52, no. 7, pp. 591–602, 2010.
[20]
M. Salomao, X. Zhang, Y. Yang et al., “Protein 4.1R-dependent multiprotein complex: new insights into the structural organization of the red blood cell membrane,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 23, pp. 8026–8031, 2008.
[21]
E. Shafizadeh, B. H. Paw, H. Foott et al., “Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency,” Development, vol. 129, no. 18, pp. 4359–4370, 2002.
[22]
Y. Takakuwa, “Protein 4.1, a multifunctional protein of the erythrocyte membrane skeleton: structure and functions in erythrocytes and nonerythroid cells,” International Journal of Hematology, vol. 72, no. 3, pp. 298–309, 2000.
[23]
W. Diakowski, M. Grzybek, and A. F. Sikorski, “Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily,” Folia Histochemica et Cytobiologica, vol. 44, no. 4, pp. 231–248, 2006.
[24]
L. Y. Hung, C. J. C. Tang, and T. K. Tang, “Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the γ-tubulin complex,” Molecular and Cellular Biology, vol. 20, no. 20, pp. 7813–7825, 2000.
[25]
B. G. Han, W. Nunomura, Y. Takakuwa, N. Mohandas, and B. K. Jap, “Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization,” Nature Structural Biology, vol. 7, no. 10, pp. 871–875, 2000.
[26]
T. Ishida and K. Kinoshita, “PrDOS: prediction of disordered protein regions from amino acid sequence,” Nucleic Acids Research, vol. 35, pp. W460–W464, 2007.
[27]
G. M. Kelly, B. D. Zelus, and R. T. Moon, “Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1,” Journal of Biological Chemistry, vol. 266, no. 19, pp. 12469–12473, 1991.
[28]
E. Leclerc and S. Vetter, “Characterization of a calcium-dependent calmodulin-binding domain in the 135-kD human protein 4.1 isoform,” European Journal of Biochemistry, vol. 258, no. 2, pp. 567–571, 1998.
[29]
W. Nunomura, M. Parra, M. Hebiguchi, K. I. Sawada, N. Mohandas, and Y. Takakuwa, “Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation,” Biochemical Journal, vol. 417, no. 1, pp. 141–148, 2009.
[30]
D. Eliezer, “Biophysical characterization of intrinsically disordered proteins,” Current Opinion in Structural Biology, vol. 19, no. 1, pp. 23–30, 2009.
[31]
L. M. Espinoza-Fonseca, “Reconciling binding mechanisms of intrinsically disordered proteins,” Biochemical and Biophysical Research Communications, vol. 382, no. 3, pp. 479–482, 2009.
[32]
A. K. Dunker, M. S. Cortese, P. Romero, L. M. Iakoucheva, and V. N. Uversky, “Flexible nets: the roles of intrinsic disorder in protein interaction networks,” FEBS Journal, vol. 272, no. 20, pp. 5129–5148, 2005.
[33]
Y. Minezaki, K. Homma, and K. Nishikawa, “Intrinsically disordered regions of human plasma membrane proteins preferentially occur in the cytoplasmic segment,” Journal of Molecular Biology, vol. 368, no. 3, pp. 902–913, 2007.
[34]
A. B. Sigalov, “Membrane binding of intrinsically disordered proteins: critical importance of an appropriate membrane model,” Self/Nonself—Immune Recognition and Signaling, vol. 1, no. 2, pp. 129–132, 2010.
[35]
A. N？rholm, R. Hendus-Altenburger, G. Bjerre, M. Kjaergaard, S. F. Pedersen, and B. B. Kragelund, “The intracellular distal tail of the Na+/H+ exchanger NHE1 is intrinsically disordered: implications for NHE1 trafficking,” Biochemistry, vol. 50, pp. 3469–3480, 2011.
[36]
M. K. Parra, S. Gee, N. Mohandas, and J. G. Conboy, “Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice,” The Journal of Biological Chemistry, vol. 286, pp. 6033–6039, 2011.
[37]
M. K. Parra, J. S. Tan, N. Mohandas, and J. G. Conboy, “Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene,” EMBO Journal, vol. 27, no. 1, pp. 122–131, 2008.
[38]
W. Nunomura, Y. Takakuwa, M. Parra, J. G. Conboy, and N. Mohandas, “Ca2+-dependent and Ca2+-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins,” Journal of Biological Chemistry, vol. 275, no. 9, pp. 6360–6367, 2000.
[39]
R. V. Yelamarty, B. A. Miller, R. C. Scaduto Jr., F. T. S. Yu, D. L. Tillotson, and J. Y. Cheung, “Three-dimensional intracellular gradients in single human brust-forming units-erythroid-derived erythroblasts induced by erythropoietin,” The Journal of Clinical Investigation, vol. 85, pp. 1799–1809, 1990.
[40]
B. A. Miller, R. C. Scaduto Jr., D. L. Tillotson, J. Botti, and J. Y. Cheung, “Erythropoietin stimulates a rise in intracellular free calcium concentartion in single early human erythroid precursors,” The Journal of Clinical Investigation, vol. 825, pp. 309–315, 1988.
[41]
W. Nunomura, K. Kinoshita, M. Parra et al., “Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells,” Biochemical Journal, vol. 432, no. 2, pp. 407–416, 2010.
[42]
M. Parra, P. Gascard, L. D. Walensky, S. H. Snyder, N. Mohandas, and J. G. Conboy, “Cloning and characterization of 4.1G (EPB41l2), a new member of the skeletal protein 4.1 (EPB41) gene family,” Genomics, vol. 49, no. 2, pp. 298–306, 1998.
[43]
P. Gascard, G. Lee, L. Coulombel et al., “Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation,” Blood, vol. 92, no. 11, pp. 4404–4414, 1998.
[44]
W. Nunomura, Y. Takakuwa, M. Parra, J. Conboy, and N. Mohandas, “Regulation of protein 4.1R, p55, and Glycophorin C ternary complex in human erythrocyte membrane,” Journal of Biological Chemistry, vol. 275, no. 32, pp. 24540–24546, 2000.
[45]
S. C. Huang, E. S. Liu, S. H. Chan et al., “Mitotic regulation of protein 4.1R involves phosphorylation by cdc2 kinase,” Molecular Biology of the Cell, vol. 16, no. 1, pp. 117–127, 2005.
[46]
A. Kontrogianni-Konstantopoulos, C. S. Frye, E. J. Benz Jr., and S. C. Huang, “The prototypical 4.1R-10-kDa domain and the 4.1G-10-kDa paralog mediate fodrin-actin complex formation,” Journal of Biological Chemistry, vol. 276, no. 23, pp. 20679–20687, 2001.
[47]
J. A. Gimm, X. An, W. Nunomura, and N. Mohandas, “Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins,” Biochemistry, vol. 41, no. 23, pp. 7275–7282, 2002.
[48]
D. Lu, H. Yan, T. Othman, C. P. Turner, T. Woolf, and S. A. Rivkees, “Cytoskeletal protein 4.1G binds to the third intracellular loop of the A1 adenosine receptor and inhibits receptor action,” Biochemical Journal, vol. 377, no. 1, pp. 51–59, 2004.
[49]
M. Saito, M. Sugai, Y. Katsushima, T. Yanagisawa, J. Sukegawa, and N. Nakahata, “Increase in cell-surface localization of parathyroid hormone receptor by cytoskeletal protein 4.1G,” Biochemical Journal, vol. 392, no. 1, pp. 75–81, 2005.
