Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation

G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacol. interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional mol. 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. [on SciFinder (R)]


Published in:
Nature Biotechnology, 17, 11, 1105-1108
Year:
1999
Laboratories:




 Record created 2006-02-27, last modified 2018-03-17


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