Molecular Characterization of ADP-ribosyl Cyclase Coupled with Receptors

我们先前报道，刺激β-肾上腺素能和血管紧张素II受体可以调节cADP-核糖合成酶--腺苷二磷酸核糖环酶的活性，并表明cADP-核糖是交感神经增强心脏收缩、出生后心脏生长和神经元-神经胶质细胞相互作用的关键分子。在本研究中，我们使用相同的策略来探索mADP-核糖是mGluRs下游的第二信使的想法。我们首先测定了在谷氨酸和/或GTP存在或不存在的情况下，不同大鼠和小鼠神经组织的粗膜中ADP-核糖环化酶的活性。其次，在NG108-15神经母细胞瘤x神经胶质瘤杂交细胞中检测mGluRs对ADP-核糖环化酶的偶联偏好。谷氨酸盐通过III组mGIuRs刺激大鼠或小鼠视网膜粗膜或通过I组mGluRs刺激颈上神经节的ADP-核糖环化酶活性。MGluR6缺陷小鼠的视网膜对谷氨酸没有表现出ADP-核糖环化酶水平的增加。GTP可提高基础环化酶和谷氨酸突变型环化酶活性的起始速率。GTP-γ-S也能刺激基础活动。为了确定mGluR与ADP-核糖环化酶的偶联方式是否是单个克隆mGIuRs的共同特征，我们在NG108-15神经母细胞瘤x胶质瘤杂交细胞中表达了每一种mGluR亚型。谷氨酸诱导的环化酶刺激优先发生在高表达mGluRs1、3、5和6的NG108-15细胞中。表达mGluR2或mGluRs4和7的细胞分别表现出抑制或无偶联。经霍乱或百日咳毒素预处理后，谷氨酸对环化酶活性的激活或抑制作用分别被消除。因此，mGIuRs亚型特异性地通过G蛋白偶联到ADP-核糖环化酶表明，一些谷氨酸引起的神经元功能是由cADP-核糖介导的。

We previously reported that stimulation of β-adrenergic and angiotensin II receptors modulates activity of ADP-ribosyl cyclase, a synthetic enzyme of cADP-ribose, and showed that cADP-ribose is a critical molecule in sympathetic potentiation of heart contraction, post-natal heart growth, and neuron-glia interaction. In the present study, we used the same strategy to explore the idea mat cADP-ribose is a second messenger downstream of the mGluRs. We first measured ADP-ribosyl cyclase activity in crude membranes from various rat and mouse nervous tissues in the presence or absence of glutamate and/or GTP. Second, the coupling preference of mGluRs to ADP-ribosyl cyclase was examined in NG108-15 neuroblastoma x glioma hybrid cells over-expressing each subtype.Glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGIuRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6 deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimutated cyclase activity. GTP-γ-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyciase is a feature common to individual cloned mGIuRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pretreatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGIuRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal function is mediated by cADP-ribose.