Ignatova E G, Belcheva M M, Bohn L M, Neuman M C, Coscia C J
E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
J Neurosci. 1999 Jan 1;19(1):56-63. doi: 10.1523/JNEUROSCI.19-01-00056.1999.
Previously, we implicated the opioid receptor (OR), Gbetagamma subunits, and Ras in the opioid activation of extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family involved in mitogenic signaling. We now report that OR endocytosis also plays a role in the opioid stimulation of ERK activity. COS-7 and HEK-293 cells were cotransfected with the cDNA of delta-, mu;-, or kappa-OR, dynamin wild-type (DWT), or the dominant suppressor mutant dynamin K44A, which blocks receptor endocytosis. The activation of ERK by opioid agonists in the presence of DWT was detected. In contrast, parallel ectopic coexpression of the K44A mutant with OR, followed by agonist treatment, resulted in a time-dependent attenuation of ERK activation. Immunofluorescence confocal microscopy of delta-OR and DWT-cotransfected COS-7 cells revealed that agonist exposure for 10 min resulted in an ablation of cell surface delta-OR immunoreactivity (IR) and an intensification of cytoplasmic (presumably endosomal) staining as seen in the absence of overexpressed DWT. After 1 hr of delta-agonist exposure the cells displayed substantial internalization of delta-OR IR. If the cells were cotransfected with delta-OR and dynamin mutant K44A, OR IR was retained on the cell surface even after 1 hr of delta-agonist treatment. Parallel immunofluorescence confocal microscopy, using an anti-ERK antibody, showed that agonist-induced time-dependent ERK IR trafficking into perinuclear and nuclear loci was impaired in the internalization-defective cells. Thus, both biochemical and immunofluorescence confocal microscopic evidence supports the hypothesis that the opioid activation of ERK requires receptor internalization in transfected mammalian cells.
此前，我们发现阿片受体（OR）、Gβγ亚基和Ras参与了细胞外信号调节蛋白激酶（ERK）的阿片类激活过程，ERK是丝裂原活化蛋白（MAP）激酶家族的成员，参与有丝分裂信号传导。我们现在报告，OR内吞作用在阿片类物质刺激ERK活性中也起作用。将δ-、μ-或κ-OR的cDNA与野生型发动蛋白（DWT）或阻断受体内吞作用的显性抑制突变体发动蛋白K44A共转染到COS-7和HEK-293细胞中。在存在DWT的情况下检测到阿片类激动剂对ERK的激活。相比之下，K44A突变体与OR平行异位共表达，随后进行激动剂处理，导致ERK激活随时间衰减。对δ-OR和DWT共转染的COS-7细胞进行免疫荧光共聚焦显微镜检查发现，暴露于激动剂10分钟导致细胞表面δ-OR免疫反应性（IR）消失，细胞质（可能是内体）染色增强，这与未过表达DWT时的情况相同。暴露于δ-激动剂1小时后，细胞显示出δ-OR IR的大量内化。如果细胞与δ-OR和发动蛋白突变体K44A共转染，即使在δ-激动剂处理1小时后，OR IR仍保留在细胞表面。使用抗ERK抗体进行的平行免疫荧光共聚焦显微镜检查表明，在有内化缺陷的细胞中，激动剂诱导的ERK IR向核周和核位点的时间依赖性转运受损。因此，生化和免疫荧光共聚焦显微镜证据均支持以下假设：在转染的哺乳动物细胞中，ERK的阿片类激活需要受体内化。
