Elsevier

Analytical Biochemistry

Volume 270, Issue 2, 1 June 1999, Pages 242-248
Analytical Biochemistry

Regular Article
Chimeric G Proteins Allow a High-Throughput Signaling Assay of Gi-Coupled Receptors

https://doi.org/10.1006/abio.1999.4061Get rights and content

Abstract

G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the Gipathway, generating signals that are difficult to detect. We have combined chimeric G proteins, automated sample handling, and simultaneous 96-well fluorometric imaging to develop a high-throughput assay system for Gisignaling. The chimeric G proteins alter receptor coupling so that signaling can occur through Gqand result in mobilization of intracellular calcium stores. An automated signaling assay device, the fluorometric imaging plate reader (FLIPR), can simultaneously measure this response in real time in 96-well microplates, allowing two people to process more than 10,000 points per day. We used the chimeric G protein/FLIPR system to characterize signaling by the Gi-coupled human opioid receptors. We show that the μ, δ, and κ opioid receptors and the related nociceptin receptor, ORL1, each couple to Gαqi5, Gαqo5, and Gα16(Gαqi5and Gαqo5refer to Gαqproteins containing the five carboxyl-terminal amino acids from Gαiand Gαo, respectively) and that different receptor/G protein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand–receptor combinations); all 31 activated at least one receptor type, and several activated multiple receptors with differing potencies. This high-throughput assay could be useful for dissecting the complex ligand–receptor relationships that are common in nature.

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  • Cited by (0)

    Hardman, J. G.Limbird, L. E.Molinoff, P. B.Ruddon, R. W.Gilman, A. G.

    1

    The first two authors contributed equally to this work.

    2

    Current address: Caliper Technologies, 605 Fairchild Drive, Mountain View, CA 94043-2234.

    3

    To whom correspondence should be addressed. Fax: (415) 285-5632. E-mail:[email protected]. Web:http://gladstone.ucsf.edu/conklin.html.

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