Tran Elizabeth
Biochemical Technologies, Science and Technology Division, Corning Incorporated, Sullivan Park, Corning, NY 14831, USA.
J Biomol Screen. 2008 Dec;13(10):975-85. doi: 10.1177/1087057108326141. Epub 2008 Nov 21.
This article describes duplexed label-free optical biosensor cellular assays for simultaneously assaying 2 endogenous receptors, the G(q)-coupled histamine receptor (H( 1)) and the G(s)-coupled beta(2)-adrenergic receptor (beta(2)AR), in A431 cells. The biosensor cellular assays consist of 2 sequential steps-an initial agonist screening using Sigma LOPAC (Library of Pharmaceutically Active Compounds) and a subsequent antagonist screening using a solution mixture containing the H(1) agonist histamine and the beta(2)AR agonist epinephrine. Results showed that costimulating A431 cells with histamine and epinephrine led to an optical response additive to individual responses. The agonist screening not only identified all full agonists for both the H(1) and beta(2) receptors, but also detected pathway-biased ligands for the beta(2)AR. Furthermore, the succeeding antagonist screening documented all known antagonists in the library for either the H(1) or beta(2) receptors. This is the 1st demonstration of a single cellular assay that is capable of screening ligands against 2 GPCRs coupled to distinct G proteins, and highlights the power of pathway-unbiased and label-free biosensor cellular assays for GPCR screens.
本文描述了用于同时检测A431细胞中2种内源性受体——G(q)偶联的组胺受体（H(1)）和G(s)偶联的β(2)肾上腺素能受体（β(2)AR）的双工无标记光学生物传感器细胞分析方法。该生物传感器细胞分析包括2个连续步骤——首先使用西格玛LOPAC（药物活性化合物库）进行激动剂筛选，随后使用含有H(1)激动剂组胺和β(2)AR激动剂肾上腺素的溶液混合物进行拮抗剂筛选。结果表明，用组胺和肾上腺素共同刺激A431细胞会产生与单个反应相加的光学反应。激动剂筛选不仅鉴定出了H(1)和β(2)受体的所有完全激动剂，还检测到了β(2)AR的偏向信号通路配体。此外，后续的拮抗剂筛选记录了文库中针对H(1)或β(2)受体的所有已知拮抗剂。这是首次证明一种单细胞分析方法能够筛选针对2种与不同G蛋白偶联的GPCR的配体，并突出了无偏向信号通路和无标记生物传感器细胞分析方法在GPCR筛选中的作用。
