Functional studies with membrane-bound and detergent-solubilized 脦脦₂-adrenergic receptors expressed in Sf9 cells

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• 作者：Sen ; Saurabh ; Jaakola ; Veli-Pekka ; Piril脙脙 ; P脙脙ivi ; Finel ; Moshe ; Goldman ; Adrian
• 关键词：G protein-coupled receptor ; Baculovirus ; Over-expression ; Localization ; Spodoptera frugiperda Sf9 cell ; Surface plasmon resonance
• 刊名：BBA - Biomembranes
• 出版年：2005
• 期刊代码：14_00052736
• 类别：bio
• 出版时间：June 15, 2005
• 卷：1712
• 期：1
• 页码：62-70
• 文件大小：301 K

摘要

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.

We have expressed the three subtypes of α₂-adrenergic receptor (α₂-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α_2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K_d values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α₂-AR is active even in a lipid-free environment. The K_d values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α₂-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.