title: An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes.
abstract: The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a beta-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa beta-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.
authors: Department of Chemistry, Purdue University, 1393 BRWN Building, West Lafayette, IN 47907, USA.
authors: Marko
authors: M
authors: Stefanovic
authors: Nicholas O
authors: NO
authors: Markham
authors: Erin M
authors: EM
authors: Parry
authors: Lisa J
authors: LJ
authors: Garrett-Beal
authors: Amanda P
authors: AP
authors: Cline
authors: Patrick G
authors: PG
authors: Gallagher
authors: Philip S
authors: PS
authors: Low
authors: David M
authors: DM
authors: Bodine
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017487:Ankyrins / Q000097:blood*; D001665:Binding Sites; D001789:Blood Group Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004907:Erythrocyte Deformability; D004910:Erythrocyte Membrane / Q000648:ultrastructure; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005091:Exons; D005998:Glycine; D051379:Mice; D008822:Mice, Transgenic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D001789:Blood Group Antigens; D011994:Recombinant Proteins; C523515:Slc4a1 protein, mouse; D005998:Glycine
references: Blood. 1993 Nov 15;82(10):2953-60
references: 8219186
references: Blood. 1991 Jan 1;77(1):165-73
references: 1702027
references: Cell. 1996 Sep 20;86(6):917-27
references: 8808627
references: Nat Genet. 1996 Oct;14(2):227-30
references: 8841202
references: Blood. 1996 Dec 1;88(11):4366-74
references: 8943874
references: Biochemistry. 1997 Aug 5;36(31):9596-604
references: 9236006
references: J Biol Chem. 1998 Jun 12;273(24):14819-26
references: 9614083
references: Biochemistry. 1998 Dec 22;37(51):17828-35
references: 9922149
references: J Clin Invest. 1999 Jun;103(11):1527-37
references: 10359562
references: Proc Natl Acad Sci U S A. 1999 Sep 14;96(19):10717-22
references: 10485892
references: Blood. 2005 Jan 15;105(2):627-34
references: 15358624
references: J Biol Chem. 2000 Apr 14;275(15):11383-8
references: 10753953
references: J Biol Chem. 2001 Dec 14;276(50):46968-74
references: 11595743
references: Blood. 2002 Sep 1;100(5):1878-85
references: 12176912
references: EMBO J. 2002 Dec 2;21(23):6387-96
references: 12456646
references: J Biol Chem. 2003 Feb 28;278(9):6879-84
references: 12482869
references: Blood. 2003 May 15;101(10):4180-8
references: 12531814
references: Nucleic Acids Res. 2003 May 15;31(10):e57
references: 12736323
references: J Biol Chem. 2003 Jul 11;278(28):25526-33
references: 12719424
references: Blood. 2004 Feb 1;103(3):1131-6
references: 14551146
references: Nature. 1979 Oct 11;281(5731):426-9
references: 573863
references: Methods Enzymol. 1983;96:313-24
references: 6228705
references: Biochim Biophys Acta. 1986 Sep 22;864(2):145-67
references: 2943319
references: Nature. 1987 Aug 6-12;328(6130):533-6
references: 3039371
references: N Engl J Med. 1988 Jan 28;318(4):230-4
references: 2961992
references: J Clin Invest. 1988 Mar;81(3):893-901
references: 2963832
references: J Biol Chem. 1989 Sep 25;264(27):15893-9
references: 2476434
references: Soc Gen Physiol Ser. 1988;43:91-100
references: 3077561
references: Nature. 1990 Jun 21;345(6277):736-9
references: 2141669
references: N Engl J Med. 1990 Oct 11;323(15):1046-50
references: 1977081
references: J Biol Chem. 1990 Sep 25;265(27):16166-72
references: 2144526
references: Cell. 1991 Jun 28;65(7):1153-63
references: 2065352
references: J Biol Chem. 1991 Aug 25;266(24):15726-37
references: 1874731
references: Br J Haematol. 1991 Aug;78(4):551-4
references: 1832935
references: Blood. 1992 Apr 1;79(7):1846-54
references: 1558976
references: J Biol Chem. 1993 Jun 5;268(16):11489-91
references: 8505285
references: J Biol Chem. 1993 Jul 15;268(20):14627-31
references: 8325839
references: Ann Hematol. 1993 Aug;67(2):89-93
references: 8347735
references: J Biol Chem. 1995 Sep 15;270(37):22050-7
references: 7665627