Cellular Pharmacology of Kappa Opiod Receptor

描述(由申请人提供)：？阿片受体(KOPR)是阿片受体的三种主要类型之一。然后呢？)阿片受体在体内介导阿片类药物的作用。激活KOPR可产生止痛、烦躁不安、利水、体温过低和调节免疫反应。KOPR拮抗剂可能对抑制可卡因的渴望以及作为抗抑郁剂和抗焦虑剂有潜在的帮助。KOPR是7TMR家族的成员，通过百日咳毒素敏感的G蛋白与多种效应器偶联。对于7TMR，激动剂调节下游信号分子的能力取决于细胞表面受体的可用性。细胞表面7TMR的数量反映了生物合成和内吞途径之间的平衡。激活后的内吞事件已经被很好地记录下来；然而，对生物合成途径的调控却知之甚少。这项赠款申请的重点是描述沿生物合成途径对KOPR贩运的调节，特别是通过我们发现与KOPR相互作用并参与KOPR出口贩运的GEC1、Sortilin和14-3-3蛋白。核心假设是，出口贩运是由与KOPR相互作用的分子调控的。具体目标如下。(1)描述GEC1促进的KOPR表达和贩运的潜在机制。GEC1通过提高NSF的ATPase活性，而不是通过脂质结合而不是通过膜结合来增强KOPR表达的假设将得到验证。(2)探讨14-3-3在KOPR转运和细胞药理中的作用。假设14-3-3蛋白与KOPR C末端结构域和/或I3环结合，从而屏蔽COPI结合，使KOPR能够分选到质膜上。(3)研究KOPR与山梨素的相互作用及其功能后果。需要检验的假设是，山梨素与KOPR结合，将受体分选到内小体和溶酶体，这代表了一种新的ER后质量控制机制。我们还将研究KOPR与这些蛋白质的相互作用是否影响信号和调控。拟议的研究将有助于更好地了解KOPR的细胞生物学，并为KOPR的出口监管提供机械性的见解。此外，这种理解将对其他膜结合受体具有重要意义，因为这些出口调节机制很可能适用于其他一些蛋白质。与公共健康相关：拟议的研究将更好地了解细胞表面受体数量是如何调节的，这些受体在应对外部刺激(包括药物和神经递质)时发挥着重要作用。

DESCRIPTION (provided by applicant): The ? opioid receptor (KOPR) is one of the three major types (?, ? and ?) of opioid receptors that mediate effects of opioids in vivo. Activation of KOPR produces analgesia, dysphoria, water diuresis, hypothermia and modulation of immune responses. KOPR antagonists may be potentially useful for curbing cocaine craving and as anti-depressants and anti-anxiety agents. KOPR, a member of the 7TMR family, is coupled through pertussis toxin-sensitive G proteins to a variety of effectors. For 7TMRs the capacity of agonists to modulate downstream signaling molecules depends on the availability of the receptors on cell surface. The number of cell surface 7TMRs reflects a balance between biosynthesis and endocytosis pathways. The post-activation endocytic events have been well-documented; however, regulation along the biosynthesis pathway is much less understood. The focus of this grant application is to characterize the regulation of the KOPR trafficking along the biosynthesis pathway, in particular by the proteins GEC1, sortilin and 14-3-3, which we found to interact with the KOPR and to be involved in KOPR export trafficking. The central hypothesis is that the export trafficking is regulated by molecules interacting with the KOPR. The specific aims are as follows. (1) To delineate mechanisms underlying GEC1-promoted expression and trafficking of the KOPR. The hypothesis that GEC1 enhances KOPR expression by enhancing ATPase activity of NSF, but not by membrane association by lipid conjugation will be tested. (2) To investigate the role of 14-3-3 in the trafficking and cellular pharmacology of the KOPR. The hypothesis is that 14-3-3 proteins bind to KOPR C-terminal domain and/or i3 loop, which masks COPI binding, allowing the KOPR to sort to plasma membranes. (3) To examine the interaction of the KOPR with sortilin and its functional consequences. The hypothesis to be tested is that sortilin binding to the KOPR sorts the receptor to endosomes and lysosomes and this represents a novel post-ER quality control mechanism. We will also examine if the interactions of the KOPR with these proteins affect signaling and regulation. The proposed studies will provide better understanding of cell biology of the KOPR and provide mechanistic insights into regulation of export of the KOPR. In addition, such understanding will have important implications for other membrane bound receptors since these regulatory mechanisms of export are likely to be applicable to some other proteins. PUBLIC HEALTH RELEVANCE: The proposed research will provide better understanding on how cell surface receptor numbers are regulated, which play important roles in response to external stimuli, including drugs and neurotransmitters.