Bouaboula M, Perrachon S, Milligan L, Canat X, Rinaldi-Carmona M, Portier M, Barth F, Calandra B, Pecceu F, Lupker J, Maffrand J P, Le Fur G, Casellas P
Sanofi, 371 Rue du Pr. Joseph Blayac, 34184 Montpellier Cedex 04, France.
J Biol Chem. 1997 Aug 29;272(35):22330-9. doi: 10.1074/jbc.272.35.22330.
In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5'-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.
在本研究中，我们发现转染了人中枢大麻素受体（CB1）的中国仓鼠卵巢（CHO）细胞在丝裂原活化蛋白激酶（MAPK）和腺苷酸环化酶水平上均表现出高组成性活性。这些活性可被作为反向激动剂发挥作用的CB1选择性配体SR 141716A阻断。此外，结合研究表明，鸟嘌呤核苷酸降低了激动剂CP-55,940的结合，这是激动剂通常具有的效应，而它增强了反向激动剂SR 141716A的结合，这是反向激动剂的特性。出乎意料的是，我们发现SR 141716A的CB1介导效应包括抑制百日咳毒素敏感的受体酪氨酸激酶（如胰岛素或胰岛素样生长因子1受体）激活的MAPK，但不包括百日咳毒素不敏感的受体酪氨酸激酶（如成纤维细胞生长因子受体）激活的MAPK。当用直接激活Gi蛋白的马斯托帕兰类似物Mas-7刺激细胞时，我们也观察到了类似的结果。此外，SR 141716A抑制了CP-55,940或Mas-7在CHO-CB1细胞膜中诱导的鸟苷5'-O-(硫代三磷酸)摄取。这表明，除了抑制自身激活的CB1外，SR 141716A还可以传递一个生物信号，该信号阻断Gi蛋白，从而消除大部分Gi介导的反应。相比之下，SR 141716A对缺乏CB1受体的CHO细胞中胰岛素或IGF1激活的MAPK没有影响，排除了SR 141716A与Gi蛋白直接相互作用的可能性。这支持了Gi蛋白可能作为细胞内负性信号转导串扰分子的观点。基于这些极大地扩展了反向激动剂生物学特性的原始结果，我们提出了一种受体/配体相互作用的新模型。
