Exploring functions of mu opioid receptor carboxyl termini by gene targeting

描述(申请人提供)：广泛用于止痛也被滥用的MU阿片类药物，通过MU阿片受体起作用。许多这些药物作用的临床差异增加了Mu受体亚型的可能性，从Mu阿片受体(OPRM1)基因的大量3‘和5’剪接变体的鉴定中证实了这一概念。5‘剪接的重要性最近在一个外显子11基因敲除的小鼠中得到了证明。外显子11相关突变体的缺失对吗啡的镇痛作用几乎没有影响，但降低了海洛因和吗啡-62-葡萄糖醛酸(M6G)的活性。相反，外显子1的中断导致吗啡作用完全丧失，而海洛因和M6G仍具有活性。3‘剪接是从外显子1/2/3剪接到外显子3下游的替代外显子(3’外显子)，在小鼠体内产生15种不同的羧基末端变体。在大鼠和人类中也发现了类似的C末端变体。这些C端变异体在mRNA和蛋白水平的区域特异性表达、突触前后的定位以及Mu激动剂诱导的G蛋白偶联、磷酸化和受体内化的差异表明了这些C末端变体的功能意义。然而，这些C末端的体内功能在很大程度上仍不清楚。这一建议的主要目的是通过基因打靶在小鼠中更好地了解OPRM1基因3‘端选择性剪接产生的羧基末端的药理功能。我们的目标不是消除受体，而只是破坏由3‘剪接产生的C末端尾巴。我们的靶向策略是在外显子中插入终止密码子，在不干扰3‘剪接的情况下产生截短的受体。我们对微型基因构建的初步研究表明，这些设计的突变终止密码子可以在P19和NIE-115细胞中产生预测的缺乏C末端尾巴的突变受体，有望在靶向小鼠中获得成功。第一个目标是通过在外显子3的末端创建一个终止密码子，将所有MOR-1剪接变异体转换为相同的截断受体。第二个目标是通过在各自外显子的开头创建终止密码子，将截断限制在含有由外显子4或外显子7编码的C末端尾部的变体。虽然这种方法可以用于所有的变异体，但我们选择关注外显子4和外显子7，因为这些外显子是最保守的3‘外显子，是小鼠大脑中绝大多数MOR-1变异体的原因。第三个目标将通过建立突变受体的分布和表达，并确定它们的阿片结合和G蛋白偶联图谱来表征这些突变小鼠。通过建立这些模型，我们希望为研究C末端尾巴的体内功能提供有用的工具。了解C末端在体内的功能将为深入了解u阿片受体的结构和功能关系以及阿片类药物在动物和人类中的复杂作用提供新的见解，可能会为开发用于控制疼痛和药物滥用的药物提供新的靶点。 与公共卫生相关：这项建议的主要目标是通过基因靶向在小鼠中更好地了解OPRM1基因3‘端选择性剪接产生的羧基末端的药理功能。所提出的靶向小鼠模型将为研究C末端尾巴的体内功能提供有用的工具。了解C末端在体内的功能将为深入了解u阿片受体的结构和功能关系以及阿片类药物在动物和人类中的复杂作用提供新的见解，可能会为开发用于控制疼痛和药物滥用的药物提供新的靶点。

DESCRIPTION (provided by applicant): Mu opioids, widely used for pain management and also abused, act through mu opioid receptors. Clinical differences in the actions of many these drugs raised the possibility of mu receptor subtypes, a concept confirmed by the identification of a vast array of both 3' and 5' splice variants from the mu opioid receptor (OPRM1) gene. The importance of 5' splicing has recently been demonstrated in an exon 11 knockout mouse. Loss of exon 11-associated variants had little effect upon morphine analgesia but diminished the activity of both heroin and morphine-62-glucuronide (M6G). Conversely, disruption of exon 1 led to complete loss of morphine action while heroin and M6G retained activity. 3' splicing, defined by splicing from exons 1/2/3 to alternative exons downstream of exon 3 (3' exons), generates 15 different carboxyl terminal variants in mouse. Similar C-terminal variants were identified in rat and human. The functional significance of these C-terminal variants has been suggested by differences in region-specific expressions at both mRNA and protein levels, pre- and post-synaptic localization and mu agonist-induced G protein coupling, phosphorylation and receptor internalization. However, the in vivo function of these C- terminal tails remains largely unknown. The primary goal of this proposal is to obtain a better understanding of the pharmacological function of carboxyl terminal tails generated by 3' alternative splicing in the OPRM1 gene through gene targeting in mice. Our objective is not to eliminate receptors, but only to disrupt the C-terminal tails produced by 3' splicing. Our targeting strategy is to insert stop codons into exons to generate truncated receptors without interfering with the 3' splicing. Our preliminary studies with minigene constructs indicated that these designed mutant stop codons are feasible to generate predicted mutant receptors lacking C-terminal tails in P19 and NIE-115 cells, promising success in targeted mice. The first aim will convert all the MOR-1 splice variants into identical truncated receptors by creating a stop codon at the end of exon 3. The second aim will limit truncation to variants containing C-terminal tails encoded by either exon 4 or exon 7 by creating the stop codon at the beginning of the respective exon. Although the approach could be used on all the variants, we choose to focus on exon 4 and exon 7 because these exons are the most highly conserved 3' exons and responsible for the vast majority of MOR-1 variants in the mouse brain. The third aim will characterize these mutant mice through establishing distribution and expression of the mutant receptors, and determining their opioid binding and G protein coupling profiles. By generating these models, we hope to provide useful tools to investigate the in vivo functions of the C-terminal tails. Understanding the in vivo function of the C-terminal tails will provide new insights into the structural and functional relationships of the mu opioid receptors, and into the complex actions of opioids in animals and humans, possibly leading to new targets for developing drugs used in control of pain and drug of abuse. PUBLIC HEALTH RELEVANCE: The primary goal of this proposal is to obtain a better understanding of the pharmacological function of carboxyl terminal tails generated by 3' alternative splicing in the OPRM1 gene through gene targeting in mice. The proposed targeted mouse models will provide useful tools to investigate the in vivo functions of the C-terminal tails. Understanding the in vivo function of the C-terminal tails will provide new insights into the structural and functional relationships of the mu opioid receptors, and into the complex actions of opioids in animals and humans, possibly leading to new targets for developing drugs used in control of pain and drug of abuse.