Identification and characterisation of transcript and protein of a new short N-terminal utrophin isoform.
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 x 10(5) 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.
Zuellig
R A
RA
Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland.
Bornhauser
B C
BC
Knuesel
I
I
Heller
F
F
Fritschy
J M
JM
Schaub
M C
MC
D000199:Actins / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D003964:Diaphragm / Q000378:metabolism; D016189:Dystrophin / Q000378:metabolism; D015723:Gene Library; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018613:Microscopy, Confocal; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D049411:Utrophin
D000199:Actins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D016189:Dystrophin; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049411:Utrophin