Kitamura Y, Imai S, Matsueda R, Nomura Y
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Mol Pharmacol. 1990 Aug;38(2):184-91.
To gain insight into the coupling mechanism of inhibitory receptors, 5-hydroxytryptamine1A receptors and alpha 2-adrenoceptors, with GTP-binding proteins (G proteins) in the central nervous system, we examined the effects of two 3-nitro-2-pyridinesulfenyl compounds, S-(3-nitro-2-pyridinesulfenyl)-L-cysteine [Cys(Npys)] and N-t-butoxy-carbonyl-S-(3-nitro-2-pyridinesulfenyl)-L-cysteine [Boc-Cys(Npys)], on 1) specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (5-hydroxytryptamine1A agonist) and [3H]clonidine (alpha 2-agonist) to rat brain membranes, 2) [35S]guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) binding, and 3) pertussis toxin (islet-activating protein) (IAP)-catalyzed ADP-ribosylation of purified Go (an IAP-sensitive G protein present in abundance in the mammalian brain). Treatment with Cys(Npys) led to decreased [3H]8-OH-DPAT and [3H]clonidine binding, similar to the inhibitory effects of IAP and N-ethylmaleimide (NEM) on such binding. However, further treatment of Cys(Npys)-pretreated membranes with dithiothreitol completely abolished the inhibitory effect of Cys(Npys) on the binding of both ligands. On the other hand, treatment with Boc-Cys(Npys) inhibited the effect of several GTP analogs (GTP gamma S, guanylyl-imidodiphosphate, guanylyl)-(beta, gamma-methylene)-diphosphate, and GTP) on [3H]8-OH-DPAT and [3H]clonidine binding. Dithiothreitol and mercaptoethanol treatment of Boc-Cys(Npys)-pretreated membranes did not lead to a recovery of the effect of GTP analogs on agonist binding. Regardless of the presence or absence of GTP gamma S, agonist binding to Boc-Cys(Npys)-pretreated membranes was decreased by further addition of NEM or Cys(Npys). Cys(Npys) blocked [35S]GTP gamma S binding as well as IAP-catalyzed ADP-ribosylation in purified Go. In contrast, Boc-Cys(Npys) partially inhibited ADP-ribosylation and did not affect [35S]GTP gamma S binding. These results suggested that Cys(Npys) modifies the receptor-coupling domain in G proteins, followed by the uncoupling of inhibitory receptors from G proteins, similar to the effects of NEM and IAP. Boc-Cys(Npys), however, seems to stabilize the coupling state between the receptors and G proteins, thus abolishing the GTP gamma S effect.
为深入了解中枢神经系统中抑制性受体、5-羟色胺1A受体和α2-肾上腺素能受体与GTP结合蛋白（G蛋白）的偶联机制，我们研究了两种3-硝基-2-吡啶硫基化合物，即S-(3-硝基-2-吡啶硫基)-L-半胱氨酸[Cys(Npys)]和N-叔丁氧羰基-S-(3-硝基-2-吡啶硫基)-L-半胱氨酸[Boc-Cys(Npys)]对以下方面的影响：1）[3H]8-羟基-2-(二正丙基氨基)四氢萘（8-OH-DPAT，5-羟色胺1A激动剂）和[3H]可乐定（α2激动剂）与大鼠脑膜的特异性结合；2）[35S]鸟苷5'-O-(3-硫代)三磷酸（GTPγS）结合；3）百日咳毒素（胰岛激活蛋白）（IAP）催化的纯化Go（哺乳动物脑中大量存在的一种对IAP敏感的G蛋白）的ADP-核糖基化。用Cys(Npys)处理导致[3H]8-OH-DPAT和[3H]可乐定结合减少，类似于IAP和N-乙基马来酰亚胺（NEM）对这种结合的抑制作用。然而，用二硫苏糖醇对经Cys(Npys)预处理的膜进行进一步处理，完全消除了Cys(Npys)对两种配体结合的抑制作用。另一方面，用Boc-Cys(Npys)处理抑制了几种GTP类似物（GTPγS、鸟苷酰亚胺二磷酸、鸟苷酰-(β,γ-亚甲基)-二磷酸和GTP）对[3H]8-OH-DPAT和[3H]可乐定结合的影响。用二硫苏糖醇和巯基乙醇处理经Boc-Cys(Npys)预处理的膜，并未导致GTP类似物对激动剂结合作用的恢复。无论是否存在GTPγS，进一步添加NEM或Cys(Npys)都会使激动剂与经Boc-Cys(Npys)预处理的膜的结合减少。Cys(Npys)阻断了纯化Go中的[35S]GTPγS结合以及IAP催化的ADP-核糖基化。相反，Boc-Cys(Npys)部分抑制了ADP-核糖基化，且不影响[35S]GTPγS结合。这些结果表明，Cys(Npys)修饰G蛋白中的受体偶联结构域，随后使抑制性受体与G蛋白解偶联，类似于NEM和IAP的作用。然而，Boc-Cys(Npys)似乎稳定了受体与G蛋白之间的偶联状态，从而消除了GTPγS的作用。
