Cellular Pharmacology of Kappa Opiod Receptor

描述（由申请人提供）：阿片受体（KOPR）是三种主要类型之一。,吗?和？)的阿片受体介导阿片样物质在体内的作用。KOPR的激活会产生镇痛、烦躁、水利尿、体温过低和免疫反应调节。KOPR拮抗剂可能对抑制可卡因渴望和作为抗抑郁剂和抗焦虑剂有潜在的作用。KOPR是7TMR家族的一员，通过百日咳毒素敏感的G蛋白与多种效应物偶联。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7TMRs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，人们对生物合成途径的调控知之甚少。本资助申请的重点是描述KOPR沿生物合成途径运输的调控，特别是通过蛋白质GEC1， sortilin和14-3-3，我们发现它们与KOPR相互作用并参与KOPR出口运输。核心假设是出口贩运是由与KOPR相互作用的分子调节的。具体目的如下。(1)阐明gec1促进KOPR表达和转运的机制。GEC1通过增强NSF的atp酶活性而不是通过脂质偶联膜结合来增强KOPR表达的假设将被验证。(2)探讨14-3-3在KOPR转运及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，掩盖COPI结合，使KOPR分类到质膜。(3)研究KOPR与sortilin的相互作用及其功能后果。待验证的假设是sortilin结合KOPR将受体分类到核内体和溶酶体，这代表了一种新的er后质量控制机制。我们还将研究KOPR与这些蛋白的相互作用是否影响信号传导和调控。提出的研究将更好地了解KOPR的细胞生物学，并为KOPR的输出调节提供机制见解。此外，这种理解将对其他膜结合受体具有重要意义，因为这些输出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的数量是如何调节的，它在对外部刺激（包括药物和神经递质）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.