LiveWare

G-Protein Cells & Plasmids

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Hot receptor, no signal

Pharmaceutical companies currently use multiple assay types in their routine screening operations. Recent years have seen an increased interest in functional assays for G-protein-coupled receptors (GPCR).

With a unique imaging design and integrated 96- and 384-well pipettor, the FLIPR fluorometric imaging plate reader performs fluorescence-based functional assays, such as intracellular calcium flux, with excellent accuracy, precision, and fast kinetic updates. With multiple systems in all major pharmaceutical companies, the FLIPR system is now the industry standard for high-throughput screening laboratories using intracellular calcium flux assays. However, over 60% of the known G-protein-coupled receptors signal through pathways other than those which lead to an increase in intracellular calcium (i.e., other than those which couple through G_q), and as genomics reveal more G-protein-coupled receptor targets this trend is continuing to increase.

The LiveWare G-protein solution

LiveWare is a series of cells and expression vectors successfully used in drug discovery¹ and screening environments to aid the study of G-protein-coupled receptors that do not conventionally couple through intracellular calcium. Most are based on a series of G-protein chimeras that have been developed by Bruce Conklin and colleagues at the Gladstone Institute, UCSF. LiveWare also includes the promiscuous G-protein, G_α16. The chimeras consist of the alpha subunit of a G_q-protein complex whose 5 carboxy-terminal amino acids have been replaced with those from one of the other G-proteins (either G_αs, G_αi, G_αo, or G_αz). These amino acids are responsible for the coupling of the receptor to its G-protein. Co-expression of these chimeras with specific non-G_q-coupled receptors may result in the generation of an intracellular calcium signal upon receptor stimulation.

¹Coward, P. et al. Proc. Natl. Acad. Sci. 95(1): 352-357 (1998).

LiveWare applications

LiveWare reagents have been successfully used on the FLIPR system, in conjunction with non-G_q-coupled members of the following receptor families:

α-adrenoceptors Chemokines Glutamate Serotonin

β-adrenoceptors Dopamine Opioid Vasopressin

		Figure 1. Chimeric Gqi5 couples CCR5 chemokine receptor to calcium response.

		Figure 2. Ga16 couples b-adrenergic receptor to calcium response.

		Figure 3. Chimeric and promiscuous G-proteins couple k-opioid receptor to calcium response.

LiveWare products are available in the following configurations (plasmids are available as hygromycin and neomycin (G418)-resistant; cells are hygromycin-resistant*:

• Host Cells: CHO cells stably transfected with one of the G-protein chimera genes or the G_α16 gene.
• Plasmids: Replicating G-protein expression vectors.

Licenses

Purchase of LiveWare products requires the execution of a Biological Materials License Agreement. Execution of the Biological Materials License Agreement conveys to the buyer only the non-transferable right to use the products in research and drug screening activities. These products are not encumbered with any additional licenses. Molecular Devices knows of no patents that would be infringed by the use of these products.

Ordering Information

Host Cells (1 vial, frozen, containing 106 cells)
Order Number	Product Name	Selection Marker
RD-HGA16	Gα16-coupled CHO Cells	hygromycin
RD-HGQO5	Gqo5-coupled CHO Cells	hygromycin
RD-HGQS5	Gqs5-coupled CHO Cells	hygromycin
RD-HGQS5 (G418)	Gqs5-coupled CHO Cells	neomycin
RD-HGQZ5	Gqz5-coupled CHO Cells	hygromycin
RD-HGQZ5 (G418)	Gqz5-coupled CHO Cells	neomycin

Plasmids* (2 vials, 50 µg lyophilized supercoiled DNA ea.)
RD-PGA16	Plasmids containing Gα16 Gene
RD-PGQI5	Plasmids containing Gqi5 Gene
RD-PGQO5	Plasmids containing Gqo5 Gene
RD-PGQS5	Plasmids containing Gqs5 Gene
RD-PGQZ5	Plasmids containing Gqz5 Gene
* Each plasmid order contains one vial of hygromycin-resistance plasmid and one vial of neomycin (G418)-resistance plasmid.

To place an order, call your local Molecular Devices Office or Distributor.