Canals Meritxell, Milligan Graeme
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
J Biol Chem. 2008 Apr 25;283(17):11424-34. doi: 10.1074/jbc.M710300200. Epub 2008 Mar 4.
The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.
人μ阿片受体在Flp-In T-REx HEK293细胞中稳定表达。激动剂DAMGO（酪氨酸-d-丙氨酸-甘氨酸-N-甲基苯丙氨酸-甘醇）的占据导致ERK1/2丝裂原活化蛋白激酶磷酸化，该磷酸化被阿片拮抗剂纳洛酮阻断，但不被大麻素CB1受体反向激动剂SR141716A阻断。来自诱导型Flp-In T-REx位点的这些细胞中人大麻素CB1受体的表达并未改变μ阿片受体的表达水平。这使得大麻素CB1激动剂WIN55212-2能够刺激ERK1/2磷酸化，但导致DAMGO激活这些激酶的能力大幅降低。尽管对μ阿片受体缺乏亲和力，但同时添加SR141716A可使DAMGO的有效性恢复。相比之下，同时添加CB1受体中性拮抗剂O-2050则不能。CB1受体的诱导还导致基础[³⁵S]鸟苷5'-3-O-(硫代)三磷酸（GTPγS）结合增加，从而使DAMGO进一步刺激[³⁵S]GTPγS结合的能力大大降低。CB1反向激动剂减弱基础[³⁵S]GTPγS结合并恢复DAMGO刺激的能力。构建了Flp-In T-REx HEK293细胞，其组成性表达人μ阿片受体并携带缺乏组成性活性的修饰D163N大麻素CB1受体。修饰的大麻素CB1受体表达的诱导并不限制DAMGO介导的ERK1/2丝裂原活化蛋白激酶磷酸化，也不允许SR141716A增强DAMGO的功能。这些数据表明，是大麻素CB1受体固有的组成性活性降低了共表达的μ阿片受体的功能能力。
