Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation
Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation
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DOI：
10.1038/15090
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发表时间：
1999-11-01
影响因子：
46.9
通讯作者：
Vogel, H
中科院分区：
文献类型：
--
作者：
Bieri, C;Ernst, OP;Vogel, H
G蛋白偶联受体（GPCR）是一类具有重要药理学意义的膜受体家族。基于细胞的测定是评估GPCR活化的主要手段，但受到其固有复杂性的限制。直接和特异性报告G蛋白受体激活的功能性分子测定可以提供实质性的优势。我们提出了一种方法，以稳定和明确的方向基板的受体。通过表面等离子体共振（SPR），我们能够遵循配体结合，G蛋白活化，和受体失活的一个代表性的GPCR，牛视紫红质。微接触印刷用于产生在受体活性中具有高对比度的微米尺寸的图案。这些模式可用于局部参考，以增强基于芯片的测定的灵敏度。固定化受体在数小时内和几个活化循环期间都是稳定的。光活化激动剂11-顺式-视黄醛的配体剂量-反应曲线在120 nM处显示半最大信号。我们的研究结果可能是有用的，以开发新的检测格式的GPCR的基础上受体固定到固体支持物，特别是传感器表面。
G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.