Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET.
Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET.
复制标题
使用NanoBRET探测白细胞介素-23与活细胞中单个受体组分和IL-23异聚体受体复合物的结合。
DOI：
10.1016/j.chembiol.2021.05.002
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发表时间：
2022-01-20
影响因子：
8.6
通讯作者：
Hill SJ
中科院分区：
文献类型：
--
作者：
Lay CS;Bridges A;Goulding J;Briddon SJ;Soloviev Z;Craggs PD;Hill SJ
白细胞介素-23（IL-23）是一种促炎细胞因子，参与宿主对病原体的防御，但也参与几种自身免疫性疾病的发展。IL-23受体已成为药物发现的关键靶标，但受体配体相互作用的确切机制仍知之甚少。在这项研究中，使用纳米荧光素酶标记的全长蛋白质在活细胞中测量了IL-23对其单个受体组分（IL 23 R和IL 12 R β1）的亲和力以及它们之间形成的异聚体复合物。在这里，我们证明了TAMRA标记的IL-23对IL 12 R β1的亲和力比IL 23 R高7倍以上。然而，在两种受体亚基的存在下，IL-23亲和力增加超过三个数量级至27 pM。此外，我们表明，IL-23诱导的两个受体亚基的N-末端结构域的位置的有力的变化，与异聚体受体结构的构象变化一致。创建NanoBRET测定以监测IL-23与其受体的相互作用。测量IL-23对IL 23 R、IL 12 R β1和异聚体复合物的亲和力。发现在不存在IL-23的情况下，受体单体在复合物中缔合。测量TAMRA标记的IL-23与纳米荧光素酶融合受体亚基的结合，揭示了由IL 23 R和IL 12 R β1的异聚体形成的高亲和力结合位点。NanoBRET还用于测量亚基的相互作用，证明IL-23结合引起预形成的受体异聚体的构象变化。
Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rβ1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rβ1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure. NanoBRET assays were created to monitor the interaction of IL-23 with its receptor The affinities of IL-23 for IL23R, IL12Rβ1 and heteromer complexes were measured Receptor monomers were found to associate in complexes in the absence of IL-23 IL-23 binding induced a rearrangement of the N-terminal domains of the receptor Lay et al. measure the binding of TAMRA-labeled IL-23 to NanoLuciferase fused receptor subunits, revealing a high-affinity binding site formed from a heteromer of IL23R and IL12Rβ1. NanoBRET was also used to measure the interaction of subunits, demonstrating that IL-23 binding causes a conformational change of pre-formed receptor heteromers.
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影响因子：
6
作者：
Floss DM;Moll JM;Scheller J
通讯作者：
Scheller J
DOI：
10.1016/j.bbrc.2012.07.133
发表时间：
2012-08-31
影响因子：
3.1
作者：
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作者：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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