Identification and characterisation of transcript and protein of a new short N-terminal utrophin isoform

• Published in: Journal of cellular biochemistry 2000-06, Vol.77 (3), p.418-431
• Main Authors: Zuellig, Richard A., Bornhauser, Beat C., Knuesel, Irene, Heller, Franziska, Fritschy, Jean-Marc, Schaub, Marcus C.
• Format: Article
• Language: English
• Subjects: actin binding; Actins - metabolism; Animals; Blotting, Northern; Blotting, Western; calponin homology domain; Cloning, Molecular; Cytoskeletal Proteins - biosynthesis; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - genetics; cytoskeleton; Diaphragm - metabolism; DNA, Complementary - metabolism; dystrophin; Dystrophin - metabolism; Gene Library; glioma cell line; Immunohistochemistry; Kidney - metabolism; Lung - metabolism; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Microscopy, Confocal; muscular dystrophy; Protein Binding; Protein Structure, Tertiary; Rats; Recombinant Proteins - metabolism; RNA, Messenger - metabolism; Tumor Cells, Cultured; Utrophin
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/(SICI)1097-4644(20000601)77:3<418::AID-JCB7>3.0.CO;2-Z

Dystrophin and utrophin are known to link the intracellular cytoskeleton to the extracellular matrix via a transmembraneous glycoprotein complex. Four short C‐terminal isoforms (Dp71, Dp116, Dp140, and Dp260) are described for dystrophin and three for utrophin (Up71, Up113, and Up140). We describe here for the first time the existence of a 3.7‐kb transcript and a 62‐kDa protein in C6 glioma cells representing a short N‐terminal isoform unique for utrophin (N‐utrophin). More than 20 clones covering the entire coding region of utrophin were isolated from a rat C6 glioma cell cDNA library. Two clones were found to code for a protein with 539 amino acids. Its sequence is identical to that of the full‐length utrophin, except for the last residue where Cys is replaced by Val. This isoform contains the actin binding domain (consisting of two calponin homology subdomains), followed by two spectrin‐like repeats. A recombinant fragment corresponding to N‐utrophin binds to F‐actin in vitro with an equilibrium constant (affinity) K of 4.5 × 105 M−1 and a stoichiometry of one fragment per around five actin monomers. Immunocytochemical staining of C6 glioma cells with antisera specific for different utrophin regions localised full‐length utrophin in the submembraneous cortical actin layer as revealed by confocal microscopy. A distinct staining pattern for the N‐utrophin was not detectable, although it was expected to localise at the actin stress fibers. It is assumed that it co‐localises via the two spectrin‐like repeats with the full‐length utrophin at the cell membrane. J. Cell. Biochem. 77:418–431, 2000. © 2000 Wiley‐Liss, Inc.