Uphues I, Kolter T, Goud B, Eckel J
Laboratory of Molecular Cardiology, Diabetes Research Institute, Düsseldorf, Germany.
Biochem J. 1994 Jul 1;301 ( Pt 1)(Pt 1):177-82. doi: 10.1042/bj3010177.
Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.
从大鼠心室组织获得的亚细胞级分用于阐明小分子质量G蛋白与胰岛素反应性葡萄糖转运体GLUT4之间可能的功能关系。蛋白质通过SDS/PAGE分离并转移至硝酸纤维素膜。用[α-32P]GTP孵育显示，在质膜和微粒体膜中均存在两条主要的、明显不同的24 kDa和26 kDa GTP结合蛋白条带。利用微粒体膜与抗GLUT4抗体的免疫吸附作用来分离富含GLUT4的膜泡。发现该物质中小G蛋白的含量大幅降低，仅存在24 kDa的蛋白种类。体内胰岛素处理对微粒体膜中小GTP结合蛋白的含量没有影响，但使富含GLUT4囊泡中的24 kDa蛋白种类显著减少了36±5%（n = 3）。这与胰岛素处理动物后富含GLUT4囊泡回收率降低（降低30 - 40%）相关。用一组抗rab GTP结合蛋白抗血清对微粒体膜进行蛋白质印迹分析表明存在分子量为24 kDa的rab4A，而未观察到rab1A、rab2和rab6。在富含GLUT4的囊泡中几乎检测不到rab4A；然而，胰岛素使rab4A从胞质溶胶和微粒体级分大量转移至质膜，同时GLUT4平行增加。这些数据表明，一种小GTP结合蛋白与GLUT4共定位于胰岛素反应性细胞内区室，并强烈提示该蛋白参与心肌中GLUT4的胞吐作用。此外，观察到的rab4A易位与胰岛素诱导的内体再循环过程相符，可能包括葡萄糖转运体。
