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 Citations (Scopus)

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

Fingerprint

Radioligand Assay
Microarrays
G-Protein-Coupled Receptors
Multiplexing
Assays
Ligands
Technology
Cyclonic Storms
Clonidine
Drug Discovery
Adrenergic Receptors
Throughput
Costs and Cost Analysis
Membranes
Screening
Costs

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Posner, B., Hong, Y., Benvenuti, E., Potchoiba, M., Nettleton, D., Lui, L., ... 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

Multiplexing G protein-coupled receptors in microarrays : A radioligand-binding assay. / Posner, Bruce; Hong, Yulong; Benvenuti, Eric; Potchoiba, Michael; Nettleton, Dave; Lui, Li; Ferrie, Ann; Lai, Fang; Fang, Ye; Miret, Juan; Wielis, Chris; Webb, Brian.

In: Analytical Biochemistry, Vol. 365, No. 2, 15.06.2007, p. 266-273.

Research output: Contribution to journalArticle

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, vol. 365, no. 2, pp. 266-273. https://doi.org/10.1016/j.ab.2007.03.014
Posner, Bruce ; Hong, Yulong ; Benvenuti, Eric ; Potchoiba, Michael ; Nettleton, Dave ; Lui, Li ; Ferrie, Ann ; Lai, Fang ; Fang, Ye ; Miret, Juan ; Wielis, Chris ; Webb, Brian. / Multiplexing G protein-coupled receptors in microarrays : A radioligand-binding assay. In: Analytical Biochemistry. 2007 ; Vol. 365, No. 2. pp. 266-273.
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