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Cell lines expressing recombinant aequorin and a G-protein coupled receptor for functional screening

s: only 25, 000 cells per well are required, this is an approximate cost of 0.01 US$/well.

Total cost per measurement is:

ADVANTAGES OF THE AEQUORIN-BASED ASSAY:

High signal-to-noise ratio (≥ 50).

Robust assay: everything at room temperature, a single preparation of cells can be used for several hours or even several days.

Homogeneous assay (no separation step) thus limiting the handling time and device requirements to perform the assay.

Low cost (10 times less expensive than luciferase assay).

Universal coupling to calcium for most of the receptors through co-expression of an adequate signalling protein. This allows screening of a target without previous knowledge of the specific signalling pathway.

Short exposure time of cells to compounds (≤ 30 seconds compared to several hours for transcription-based assays): allows testing of the activity of cytotoxic compounds. avoids metabolization of the tested compounds.

Ca++ mobilization is an early event following the binding of an agonist. No artefact due to the transcription of a reporter gene (no bell-shape curve for dose-effect measurements).

EC50 values are similar to those obtained by classical functional assays.

APPLICATIONS

This functional screening assay has been tested with success on receptors belonging to different families including

Cannabinoid receptor CB1

Chemokine receptors: CCR1, CCR2, CCR3, CCR5, CCR6, CCR8, CXCR4

Corticotropin-releasing factor CRF2 Histamine H1 receptor

Melanin-concentrating hormone (MCH) receptor (SLC-1, HNOP801)

Melanocortin receptor MC4

Motilin receptor

Nociceptin re
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