Regulation of Opioid Signaling by Tyr-Phosphorylation

这项申请寻求支持，以继续成功的赠款，旨在研究 磷酸化对G蛋白偶联钾通道(Kit3)阿片受体激活的影响 以前资助的时期，丝氨酸/苏氨酸磷酸化和酪氨酸磷酸化的影响 关于阿片受体的脱敏和Kir3通道的激活。我们建议将这些措施延长 通过集中精力研究酪氨酸磷酸化(Y-PO4)对MU阿片类药物的特异性影响 Kir3的受体(MOR)激活。我们先前的定点突变研究确定了潜在的Y-PO4 受体和通道内的ITs可能控制对阿片类激动剂的反应。最初， 这一调节的机制将利用MoR和Kir3.1在AtT20细胞和 原代海马区培养。体外电生理记录OI将扩大研究范围 DAMGO激活Kir3反应以及受体和通道转运的共聚焦成像。我们会使用 用于鉴定Y-PO4介导效应的激酶和磷酸酶的药物抑制剂 在这些可延展的体外系统中。这一目标的结果将检验特定位置的Y-PO4的假设 在MOR和Kir3.1内，调节阿片信号的效率。特异蛋白的磷酸化状态 MOR和Kir3.1序列中的位点将通过~(32)P掺入和通过与 新的磷酸专一性抗体。酪氨酸磷酸化对阿片受体信号的调节 对理解生理应激期间阿片类药物反应的重要意义；因此，从 对更复杂的体内系统进行简单的体外分析将是优先考虑的。在描述了 磷酸化特异性抗体(MOR-YP和Kir3.1-YP)在体外系统中的特异性和实用性 将检验神经损伤导致生长因子诱导的酪氨酸变化的假设 从MOR-YP和Kir3.1-YP免疫染色的变化检测MOR和Kir3.1的磷酸化 在脊髓和大脑的伤害性环路内。拟议的研究结果可能会提供 对阿片类药物信号可塑性的调节机制的进一步理解。

This application seeks support for the continuation of a successful grant designed to study the effects of phosphorylation on opioid receptor activation of G-protein coupled potassium channels (Kit3) In the previously funded period, the effects of both serine/threonine phosphorylation and tyrosine phosphorylation on opioid receptor desensitization and Kir3 channel activation were described. We propose to extend these studies by focusing our effort on the specific effects of tyrosine phosphorylation (Y-PO4) on mu opioid receptor (MOR) activation of Kir3. Our previous site directed mutagenesis studies identified potential Y-PO4 Isites within the receptor and channel likely to control the response to opioid agonists. Initially, the mechanisms of this regulation will be studied using cDNA expression of MOR and Kir3.1 in AtT20 cells and primary hippocampal cultures. Studies would be extended by in vitro electrophysiological recording oi DAMGO activated Kir3 responses and confocal imaging of receptor and channel trafficking. We would use pharmacological inhibitors to identify the kinases and phosphatases responsible the Y-PO4 mediated effects in these malleable in vitro systems. Results of this aim would test the hypothesis that Y-PO4 of specific sites within MOR and Kir3.1 regulates the efficiency of opioid signaling. The phosphorylation state of specific sites within the MOR and Kir3.1 sequences would be assessed by 32p-incorporation and by probing with novel phosphospecific antibodies. Regulation of opioid receptor signaling by tyrosine phosphorylation has important implications for understanding opioid responses during physiological stress; thus, moving from simple in vitro analyses to more complex, in vivo systems would be a priority. After characterizing the specificity and utility of the phosphospecific antibodies (MOR-YP and Kir3.1-YP) in the in vitro systems, we will test the hypothesis that nerve trauma results in growth factor-induced changes in tyrosine phosphorylation of MOR and Kir3.1 detectable by changes in MOR-YP and Kir3.1-YP immunostaining within nociceptive circuits in spinal cord and brain. Results of the proposed studies are likely to provide additional understanding of the mechanisms mediating the plasticity of opioid signaling.