Cellular Pharmacology of Kappa Opiod Receptor

描述（由申请人提供）：该？阿片受体（KOPR）是三种主要类型之一（？，?和？）介导阿片类药物在体内作用的阿片受体。KOPR的激活产生镇痛、烦躁、利尿、体温过低和免疫应答的调节。KOPR拮抗剂可能用于抑制可卡因渴望以及作为抗抑郁剂和抗焦虑剂。KOPR是7 TMR家族的成员，通过百日咳毒素敏感的G蛋白与多种效应物偶联。对于7 TMR，激动剂调节下游信号传导分子的能力取决于细胞表面上受体的可用性。细胞表面7 TMR的数量反映了生物合成和内吞途径之间的平衡。活化后的内吞事件已被充分记录，然而，调控沿着生物合成途径是少得多的理解。本授权申请的重点是表征KOPR运输沿着生物合成途径的调节，特别是通过蛋白质GEC 1、分拣蛋白和14-3-3，我们发现这些蛋白质与KOPR相互作用并参与KOPR输出运输。中心假设是，出口贩运是由与KOPR相互作用的分子调节的。具体目标如下。(1)阐明GEC 1促进KOPR表达和运输的机制。GEC 1通过增强NSF的ATP酶活性而不是通过脂质缀合的膜缔合来增强KOPR表达的假设将被检验。(2)研究14-3-3在KOPR的转运和细胞药理学中的作用。假设14-3-3蛋白结合KOPR C-末端结构域和/或i3环，其掩盖COPI结合，允许KOPR分选到质膜。(3)研究KOPR与分拣蛋白的相互作用及其功能后果。待检验的假设是分拣蛋白结合KOPR将受体分类至内体和溶酶体，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传导和调节。拟议的研究将提供更好地了解KOPR的细胞生物学，并提供机制的见解调节输出的KOPR。此外，这种理解将有重要的影响，其他膜结合受体，因为这些出口的调节机制可能适用于其他一些蛋白质。公共卫生相关性：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，这些受体在对包括药物和神经递质在内的外部刺激的反应中起着重要作用。

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.