Cussac D, Kordon C, Enjalbert A, Saltarelli D
ICNE UMR 9941 CNRS, Faculté de Médecine Secteur Nord, Marseille.
J Recept Signal Transduct Res. 1996 May-Jul;16(3-4):169-90. doi: 10.3109/10799899609039947.
Using antibodies raised against synthetic peptides of heterotrimeric GTP binding proteins, we demonstrate the presence of G alpha s, G alpha i1,2, G alpha i3, G alpha o2, and G beta subunits in pituitary cells. Pretreatment of pituitary cells with cholera toxin diminished the immunoreactivity of G alpha s and this decrease was kinetically coupled to the rate of G alpha s ADP-ribosylation. ADP-ribosylation by islet activating protein (IAP or Bordetella pertussis toxin) of G alpha i and G alpha o enhanced their immunoreactivities to antibodies raised against synthetic decapeptides that correspond to the G alpha carboxyl termini. Such enhancement was not observed when antibodies directed against the NH2-termini were used. These findings are consistent with the fact that ADP-ribosylation by IAP occurs on the cysteine located in the carboxyl terminal part of G alpha i and G alpha o. These observations mean that the kinetics and extent of Gi and Go ADP-ribosylation by IAP in whole pituitary cells and membrane preparations can be followed. It could be that ADP-ribosylation causes conformational changes in G alpha i and G alpha o. Indeed, we observed that ADP-ribosylated G alpha i was more sensitive to trypsin proteolysis and that the ADP-ribosylation rates of G alpha i and G alpha o in whole cells were comparable to the rate of loss of coupling between inhibitory neurohormone receptors and adenylyl cyclase.
利用针对异源三聚体GTP结合蛋白合成肽产生的抗体，我们证实了垂体细胞中存在Gαs、Gαi1,2、Gαi3、Gαo2和Gβ亚基。用霍乱毒素预处理垂体细胞可降低Gαs的免疫反应性，且这种降低在动力学上与Gαs的ADP核糖基化速率相关。胰岛激活蛋白（IAP或百日咳博德特氏菌毒素）对Gαi和Gαo的ADP核糖基化增强了它们对针对与Gα羧基末端对应的合成十肽产生的抗体的免疫反应性。当使用针对氨基末端的抗体时，未观察到这种增强。这些发现与IAP的ADP核糖基化发生在Gαi和Gαo羧基末端部分的半胱氨酸上这一事实一致。这些观察结果意味着可以追踪IAP在整个垂体细胞和膜制剂中对Gi和Go的ADP核糖基化的动力学和程度。可能是ADP核糖基化导致Gαi和Gαo发生构象变化。事实上，我们观察到ADP核糖基化的Gαi对胰蛋白酶消化更敏感，并且全细胞中Gαi和Gαo的ADP核糖基化速率与抑制性神经激素受体和腺苷酸环化酶之间偶联丧失的速率相当。
