Hsia J A, Moss J, Hewlett E L, Vaughan M
J Biol Chem. 1984 Jan 25;259(2):1086-90.
Adenylate cyclase in NG108-15 (neuroblastoma X glioma hybrid) cells is responsive to both stimulatory and inhibitory ligands. Bordetella pertussis toxin (PT) catalyzes the ADP-ribosylation of a 41,000-Da peptide believed to be a subunit of the putative guanyl nucleotide-binding protein (Gi) involved in cyclase inhibition and abolishes inhibitory effects of opiate agonists. In studying the effects of PT on opiate receptors, we found that [3H]enkephalinamide binding was reduced by approximately 90% in membranes prepared from cells incubated with PT compared to control membranes. Agonist affinity, assessed by enkephalinamide competition for [3H]diprenorphine-binding sites, was markedly reduced in cells incubated with PT. Furthermore, inhibition by guanylylimidodiphosphate of ligand binding to opiate receptors was reduced following treatment with PT. The number of opiate receptors assessed by [3H]diprenorphine binding was unaltered by PT. These data are consistent with the hypothesis that PT-catalyzed ADP-ribosylation impairs the interaction of Gi with the inhibitory receptor-ligand complex, effectively uncoupling the inhibitory receptor from Gi and the cyclase catalytic unit.
NG108-15（神经母细胞瘤X胶质瘤杂交瘤）细胞中的腺苷酸环化酶对刺激性和抑制性配体均有反应。百日咳博德特氏菌毒素（PT）催化一种41,000道尔顿肽的ADP核糖基化，该肽被认为是参与环化酶抑制的假定鸟苷酸结合蛋白（Gi）的一个亚基，并消除阿片类激动剂的抑制作用。在研究PT对阿片受体的影响时，我们发现，与对照膜相比，在用PT孵育的细胞制备的膜中，[3H]脑啡肽酰胺结合减少了约90%。通过脑啡肽酰胺竞争[3H]二丙诺啡结合位点评估的激动剂亲和力，在用PT孵育的细胞中显著降低。此外，在用PT处理后，鸟苷酰亚胺二磷酸对配体与阿片受体结合的抑制作用降低。通过[3H]二丙诺啡结合评估的阿片受体数量不受PT影响。这些数据与以下假设一致，即PT催化的ADP核糖基化损害了Gi与抑制性受体-配体复合物的相互作用，有效地使抑制性受体与Gi和环化酶催化单元解偶联。
