Fels Institute for Cancer Research and Molecular Biology, Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2011 Jun 10;286(23):20354-65. doi: 10.1074/jbc.M110.177303. Epub 2011 Mar 18.
We have previously shown that the μ-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKCζ. Inhibition of PKCζ by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKCζ is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKCζ. The phosphorylation of PKCζ can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKCζ and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKCζ-YFP and PDK1-CFP. In addition, cells expressing PKCζ kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKCζ through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.
我们之前已经表明，μ 阿片受体（MOR）能够介导几种趋化因子受体（包括 CCR5）的交叉脱敏，但其生化机制尚未完全阐明。我们进行了一系列功能和生化研究，发现 MOR 诱导的 CCR5 交叉脱敏的机制涉及 PKCζ 的激活。PKCζ 的伪底物抑制剂、siRNA 或显性负突变体的抑制作用抑制了 CCR5 的交叉脱敏。我们的结果进一步表明，PKCζ 的激活是通过涉及磷酸肌醇依赖性激酶-1（PDK1）的途径介导的。此外，MOR 的激活增加了 PKCζ 的磷酸化水平和激酶活性。PKCζ 的磷酸化可以通过 PDK1 的显性负突变体抑制。我们观察到，在 MOR 激活后，根据表达 PKCζ-YFP 和 PDK1-CFP 的细胞中荧光共振能量转移的分析，PKCζ 和 PDK1 之间的相互作用立即增加。此外，表达 PKCζ 激酶结构域突变体（Lys-281、Thr-410、Thr-560）的细胞未能表现出完全由 MOR 诱导的 CCR5 活性脱敏。总之，我们提出，在 DAMGO 处理后，MOR 通过 PDK1 依赖性信号通路激活 PKCζ，从而诱导 CCR5 磷酸化和脱敏。由于 CCR5 是一种高度促炎受体，也是 HIV-1 的关键核心受体，这些结果可能为开发治疗某些炎症性疾病或 AIDS 的特定治疗剂提供一种新方法。
