Li J G, Benovic J L, Liu-Chen L Y
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Mol Pharmacol. 2000 Oct;58(4):795-801.
Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.
此前，我们发现稳定表达于中国仓鼠卵巢（CHO）细胞中的人κ-阿片受体（hkor）在长时间接受U50,488H处理后会发生下调。在本研究中，我们确定了这一过程背后的机制。U50,488H导致hkor显著下调，而埃托啡则不会。U50,488H和埃托啡均未导致大鼠κ-阿片受体下调。因此，与内化作用类似，κ-阿片受体下调存在激动剂和物种差异。显性负性突变体抑制蛋白-2（319-418）或发动蛋白I-K44A的表达显著降低了U50,488H诱导的hkor下调。共表达GRK2或GRK2与抑制蛋白-2可使埃托啡诱导hkor下调，尽管单独表达抑制蛋白-2或发动蛋白I则不会。显性负性突变体rab5A-N133I或rab7-N125I的表达减弱了U50,488H诱导的下调。用溶酶体酶抑制剂[（2S,3S）反式环氧琥珀酰-L-亮氨酰胺基-3-甲基丁烷乙酯或氯喹]或蛋白酶体抑制剂（蛋白酶体抑制剂I、MG-132或乳胞素）预处理可降低U50,488H诱导的下调程度。氯喹和蛋白酶体抑制剂I联合使用可消除U50,488H诱导的下调。这些结果表明，U50,488H诱导的hkor下调涉及GRK、抑制蛋白-2、发动蛋白、rab5和rab7依赖性机制，且受体似乎被转运至溶酶体和蛋白酶体进行降解。因此，U50,488H诱导的hkor内化和下调具有共同的初始机制。据我们所知，这些结果首次报道了rab5和rab7参与激动剂诱导的G蛋白偶联受体下调。此外，本研究也是首次表明蛋白酶体参与激动剂诱导的G蛋白偶联受体下调的研究之一。
