Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.
Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.
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DOI：
10.1111/bph.14504
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发表时间：
2018-12
影响因子：
7.3
通讯作者：
Lambert DG
中科院分区：
文献类型：
--
作者：
Bird MF;Guerrini R;Willets JM;Thompson JP;Caló G;Lambert DG
伤害素/孤啡肽FQ(N/OFQ)受体(NOP)是阿片受体家族的一员，参与许多生理反应，如疼痛和免疫调节。在这项研究中，我们将红色荧光团ATTO594连接到NOP受体的多肽配体N/OFQ(N/OFQATTO594)上，并探讨了NOP受体在高表达(在重组系统中)和低表达(在免疫细胞上)的功能。我们评估了N/OFQATTO594受体在重组CHO细胞系中的结合、选择性和功能活性。在(I)表达NOP和NOPGFP受体的HEK细胞，(Ii)表达hNOPGαQI5嵌合体的CHO细胞(强制偶联可测量的钙反应)和(Iii)新鲜分离的人多形核细胞(PMN)中，检测到活细胞N/OFQATTO594结合。N/OFQATTO594与NOP受体结合具有NM亲和力和高选择性。N/OFQATTO594通过减少cAMP的形成和增加αQI5细胞内的钙离子水平来激活一氧化氮合酶受体。N/OFQATTO594也能在PMN细胞上观察到低表达水平的NOP受体。在NOP-GFP标记的受体中，N/OFQATTO594用于FRET协议，其中GFP发射激活ATTO，可视化配体-受体相互作用。当NOPGFP受体被N/OFQATTO594激活时，可以测量到配体和受体从细胞表面到胞浆的移动。在缺乏有效的NOP受体抗体和围绕使用放射性标记的问题(特别是在低表达系统中)的情况下，这些数据表明N/OFQATTO594用于研究N/OFQ驱动的广泛的细胞反应。
The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore‐ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. We assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). N/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP‐GFP‐tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand–receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594, movement of ligand and receptor from the cell surface to the cytosol can be measured. In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ‐driven cellular responses.
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