Zuellig R A, Bornhauser B C, Knuesel I, Heller F, Fritschy J M, Schaub M C
Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland.
J Cell Biochem. 2000 Apr;77(3):418-31. 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 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.
已知肌营养不良蛋白和抗肌萎缩蛋白聚糖通过跨膜糖蛋白复合物将细胞内细胞骨架与细胞外基质连接起来。肌营养不良蛋白有四种短的C末端异构体（Dp71、Dp116、Dp140和Dp260），抗肌萎缩蛋白聚糖有三种（Up71、Up113和Up140）。我们在此首次描述了C6胶质瘤细胞中存在一种3.7-kb转录本和一种62-kDa蛋白，它们代表抗肌萎缩蛋白聚糖特有的短N末端异构体（N-抗肌萎缩蛋白聚糖）。从大鼠C6胶质瘤细胞cDNA文库中分离出20多个覆盖抗肌萎缩蛋白聚糖整个编码区的克隆。发现两个克隆编码一种含539个氨基酸的蛋白质。其序列与全长抗肌萎缩蛋白聚糖的序列相同，只是最后一个残基处半胱氨酸被缬氨酸取代。这种异构体包含肌动蛋白结合结构域（由两个钙调蛋白同源亚结构域组成），后面跟着两个血影蛋白样重复序列。对应于N-抗肌萎缩蛋白聚糖的重组片段在体外与F-肌动蛋白结合，平衡常数（亲和力）K为4.5×10⁵ M⁻¹，化学计量比为每五个肌动蛋白单体约结合一个片段。用针对抗肌萎缩蛋白聚糖不同区域的抗血清对C6胶质瘤细胞进行免疫细胞化学染色，共聚焦显微镜显示全长抗肌萎缩蛋白聚糖定位于膜下皮质肌动蛋白层。尽管预计N-抗肌萎缩蛋白聚糖定位于肌动蛋白应力纤维，但未检测到其独特的染色模式。推测它通过两个血影蛋白样重复序列与细胞膜上的全长抗肌萎缩蛋白聚糖共定位。
