A new factor for cellular signal transduction-Studies of the structures and functions of soluble GTP binding proteins

GH_4C_1大鼠垂体细胞与生长抑素(SRIF)孵育8分钟后，粗膜G&lt；i2&gt；α亚基水平下降至对照的52%，而274,000×g上清液(胞浆部分)免疫印迹显示G&lt；i2&gt；α亚基。以百日咳毒素(PT)与β-淀粉复合体为底物，将溶解的胞浆部分进行ADP核糖化。PT可抑制SRIF对GH_4C_1细胞的作用。此外，在完整的GH_4C_1细胞中加入血管活性肠肽后，GH_4C_1细胞粗膜中G_S的α亚基(50 KDa)水平也明显降低，而胞浆中的G_Sα亚基的含量在前30分钟就明显下降。这些数据表明，在完整的GH_4C_1细胞中，PT敏感或霍乱毒素敏感的G蛋白确实在SRIF或VIP激活时解离为α亚基和β-淀粉复合体，并从膜向胞浆释放α亚基。

Incubation of the GH_4C_1 rat pituitary cells with somatostatin (SRIF) decreased the level of alpha subunit of G_<i2> to 52% of control values in the crude membrane during the first 8 min, whereas the 274,000 x g supernatant (cytosolic fraction) showed clearly the alpha subunit of G_<i2> by immunoblotting. The resolved cytosolic fraction was ADP-ribosylated by pertussis toxin (PT) with betagamma complex. The effects of SRIF were inhibited by the pretreatment of GH_4C_1 cells with PT. Furthermore, the addition of vasoactive intestinal peptide (VIP) to intact GH_4C_1 cells also decreased the level of alpha subunit (50 KDa) of G_s in crude membranes, whereas the cytosolic fraction revealed the alpha subunit of G_s the during first 30 min. These data indicate that PT-sensitive or cholera toxin-sensitive G proteins in intact GH_4C_1 cells do indeed dissociate into alpha subunit and betagamma complex upon SRIF or VIP activation and release alpha subunits from the membrane to cytosol.