Molecular Analysis of a Novel Opioid Peptide Transporter

描述(申请人提供)：参与阿片能神经传递的内源性阿片类药物是由5个或更多氨基酸组成的多肽。一般认为，这些多肽从突触中清除并随后终止神经传递是由多肽酶介导的。最近，我们发现了一种新的钠(Na)-氯(Cl)偶联转运蛋白，它是阿片肽的特异性转运蛋白。这样的转运体在文献中没有描述过这类神经递质。这种转运蛋白在大鼠大脑和人类神经细胞系中都有明显的表达。该转运蛋白的特性与以前已知的其他神经递质转运蛋白相似。我们推测，这种转运蛋白代表了阿片能神经传递的一种新的、迄今尚未被认识的成分，因此是阿片类药物功能和阿片成瘾的关键决定因素。人类免疫缺陷病毒HIV-1编码的反式激活蛋白Tat显著上调了细胞系中的这种转运蛋白。由于阿片类药物具有免疫调节功能，并影响艾滋病的发生和发展，我们预测TAT诱导的阿片肽转运体表达的变化可能与HIV-1感染的患者有临床意义。尽管转运蛋白存在的功能证据是明确的，但目前对负责转运蛋白功能的蛋白质的分子性质一无所知。在这里，我们建议使用功能表达策略克隆转运蛋白，并建立转运蛋白的分子同源性。然后，我们将利用哺乳动物细胞和非洲爪哇卵母细胞的异源表达系统来阐明转运蛋白的功能特征。转运蛋白的成功克隆将导致负责新的转运蛋白功能的基因和蛋白质的分子同一性。这将有助于开发必要的工具，如核苷酸探针和抗体，以对转运蛋白在完整动物和培养细胞中的表达和定位进行分子研究。我们还将比较对照小鼠和TAT转基因小鼠中转运蛋白的表达和活性，以评估HIV-1 TAT对完整动物的阿片类药物功能的影响。这些研究将导致发现一种以前没有特征的新蛋白质，它不仅可能在阿片类药物生物学和阿片成瘾领域发挥关键作用，而且还可能在阿片成瘾者艾滋病毒-1感染和艾滋病的进展中发挥关键作用。

DESCRIPTION (provided by applicant): Endogenous opioids involved in opioidergic neurotransmission are peptides consisting of 5 or more amino acids. It is generally assumed that clearance of these peptides from the synapse and consequent termination of neurotransmission are mediated by peptidases. Recently, we have identified a novel sodium (Na)-and chlorine (Cl)-coupled transporter that is specific for opioid peptides. Such a transporter has not been described in the literature for this class of neurotransmitters. The expression of this transporter is demonstrable in the rat brain and in human neuronal cell lines. The characteristics of this transporter are similar to those of previously known other neurotransmitter transporters. We hypothesize that this transporter represents a new, hitherto unrecognized, component of opioidergic neurotransmission and hence a key determinant of opioid function and opiate addiction. Tat, the transactivator protein coded by the human immunodeficiency virus HIV-1, markedly up-regulates this transporter in cell lines. Since opioids exhibit immunomodulatory function and influence the pathogenesis and progression of AIDS, we predict that Tat-induced changes in the expression of the opioid peptide transporter may have clinical relevance to patients with HIV-1 infection. Even though the functional evidence for the existence of the transporter is unequivocal, nothing is known at present on the molecular nature of the protein responsible for the transport function. Here we propose to clone the transporter using functional expression strategy and establish the molecular identity of the transporter. We will then elucidate the functional features of the transporter using heterologous expression systems with mammalian cells and Xenopus oocytes. Successful cloning of the transporter will lead to the molecular identity of the gene and the protein responsible for the novel transport function. This will facilitate development of tools such as nucleotide probes and antibodies necessary to perform molecular studies on the expression and localization of the transporter in intact animals and in cultured cells. We will also compare the expression and activity of the transporter in control mice and in Tat-transgenic mice to assess the influence of HIV-1 Tat on opioid function in intact animals. These studies will lead to the discovery of a previously uncharacterized novel protein which might function as a key player not only in the area of opioid biology and opiate addiction but also in the progression of HIV-1 infection and AIDS in opiate addicts.