In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies
In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies
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使用磷酸位点特异性 GPCR 抗体对阿片类药物和大麻素药物作用进行原位可视化
DOI：
10.1101/2022.06.14.496067
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发表时间：
2023
影响因子：
5.9
通讯作者：
S. Schulz
中科院分区：
文献类型：
--
作者：
S. Fritzwanker;Falko Nagel;A. Kliewer;Viviane Stammer;S. Schulz
在小鼠的常规免疫组织化学程序中，受体的磷酸化显示不稳定，因此磷酸酶抑制剂应与磷酸位点特异性GPCR抗体一起使用。G蛋白偶联受体（GPCR）是重要的信号转导子，其在细胞内丝氨酸和苏氨酸残基激活时被磷酸化。尽管特异性识别GPCR磷酸化状态的抗体已经存在多年，但磷酸化受体在其来源组织中的有效免疫定位一直是不可能的。在这里，我们表明，受体的磷酸化是非常不稳定的，在常规的免疫组织化学程序，需要使用适当的磷酸酶抑制剂，特别是在组织灌注，固定后，冷冻保护，但不是在组织切片的免疫染色。我们使用磷酸化状态特异性μ-阿片受体（MOP）和大麻素受体1（CB 1）抗体提供概念证明。事实上，四种充分表征的磷酸位点特异性MOP抗体中的三种，包括pS375-MOP、pT 376-MOP和pT 379-MOP，仅在包含磷酸酶抑制剂后，在阿片样物质处理的小鼠的脑和脊髓切片中显示出稳健的神经元免疫染色。然后，我们将这种方法扩展到CB 1受体，并证明了三种新产生的磷酸酶特异性CB 1抗体之一，即pS425-CB 1，在大麻素处理的小鼠脑切片中显示出显著的纤维染色和静脉曲张。虽然随后的实验表明，磷酸-CB 1免疫染色是不太敏感的磷酸酶，我们的结论是，使用磷酸酶抑制剂应始终考虑在新的磷酸特异性GPCR抗体的免疫组化程序的发展。总之，我们预计这种改进的方案将促进磷酸化状态特异性抗体的广泛使用，以监测生理和药理条件下内源性GPCR的激活。我们的方法也可能被证明是有用的，以确认天然组织中的GPCR候选药物的目标接合。
Phosphorylation of receptors is shown to be unstable during routine immunohistochemical procedures in mice thus phosphatase inhibitors should be used alongside phosphosite-specific GPCR antibodies. G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues.
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影响因子：
7.3
作者：
Mann, Anika;Mouledous, Lionel;Schulz, Stefan
通讯作者：
Schulz, Stefan
影响因子：
7.3
作者：
Miess, Elke;Gondin, Arisbel B.;Canals, Meritxell
通讯作者：
Canals, Meritxell
影响因子：
3.6
作者：
Just, Sascha;Illing, Susann;Schulz, Stefan
通讯作者：
Schulz, Stefan
奥曲肽治疗患者后神经内分泌肿瘤中 sst2 受体的磷酸化。
DOI：
10.1016/j.ajpath.2012.01.041
发表时间：
2012
期刊：
The American journal of pathology
影响因子：
--
作者：
Waser,Beatrice;Cescato,Renzo;Liu,Qisheng;Kao,YachuJ;Körner,Meike;Christ,Emanuel;Schonbrunn,Agnes;Reubi,JeanClaude
通讯作者：
Reubi,JeanClaude
影响因子：
13.5
作者：
Gurevich, Eugenia V.;Tesmer, John J. G.;Mushegian, Arcady;Gurevich, Vsevolod V.
通讯作者：
Gurevich, Vsevolod V.