Multiplexing G protein-coupled receptors in microarrays: A radioligand-binding assay

Bruce Posner, Yulong Hong, Eric Benvenuti, Michael Potchoiba, Dave Nettleton, Li Lui, Ann Ferrie, Fang Lai, Ye Fang, Juan Miret, Chris Wielis, Brian Webb

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Multiplexing of G protein-coupled receptors (GPCRs) in microarrays promises to increase the efficiency, reduce the costs, and improve the quality of high-throughput assays. However, this technology is still nascent and has not yet achieved the status of "high throughput" or laid claim to handling a large set of receptors. In addition, the technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to a subset of GPCRs. To expand the impact of multiplexing on this receptor class, we have developed a radiometric approach to the microarray assay. In these studies, we considered two receptors in the α-adrenergic receptor family, α2A and α2C, and the 125I-labeled agonist clonidine. We demonstrate that microarrays of these receptors can be readily detected (signal/noise ratio ∼ 160) using a Typhoon 9210 PhosphorImager. In addition, biochemical characterization shows that ligand-binding profiles and selectivity are preserved with the selective antagonists BRL44408 and ARC239. Importantly, these microarrays use approximately 200- to 400-fold less membrane preparation required by conventional assay methods and allow two or more receptors to be assayed in an area equivalent to a standard well of a microtiter plate. The impact of this approach on screening in drug discovery is discussed.

Original languageEnglish (US)
Pages (from-to)266-273
Number of pages8
JournalAnalytical biochemistry
Volume365
Issue number2
DOIs
StatePublished - Jun 15 2007

Keywords

  • Adrenergic
  • GPCR
  • High-throughput screening
  • Microarrays
  • Radioligands
  • Typhoon

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Posner, B., Hong, Y., Benvenuti, E., Potchoiba, M., Nettleton, D., Lui, L., Ferrie, A., Lai, F., Fang, Y., Miret, J., Wielis, C., & Webb, B. (2007). Multiplexing G protein-coupled receptors in microarrays: A radioligand-binding assay. Analytical biochemistry, 365(2), 266-273. https://doi.org/10.1016/j.ab.2007.03.014