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 fo r dystrophin and three for utrophin (Up71, Up113, and Up140). We describe h ere for the first time the existence of a 3.7-kb transcript and a 62-kDa pr otein in C6 glioma cells representing a short N-terminal isoform unique for utrophin (N-utrophin). More than 20 clones covering the entire coding regi on of utrophin were isolated from a rat C6 glioma cell cDNA library. Two cl ones were found to code for a protein with 53.9 amino acids. Its sequence i s identical to that of the hull-length utrophin, except for the last residu e where Cys is replaced by Val. This isoform contains the actin binding dom ain (consisting of two calponin homology subdomains), followed by two spect rin-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. I mmunocytochemical staining of C6 glioma cells with antisera specific for di fferent utrophin regions localised full-length utrophin in the submembraneo us cortical actin layer as revealed by confocal microscopy. A distinct stai ning 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 cel l membrane. J. Cell. Biochem. 77:418-431, 2000. (C) 2000 Wiley-Liss, Inc.