Characterization of CHO Cells Stably Expressing a Gα_16/zChimera for High Throughput Screening of GPCRs

Abstract

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.

Activation of the Human FPRL-1 Receptor Promotes Ca2+ Mobilization in U87 Astrocytoma Cells

“…A number of G i -coupled receptors have recently been shown to be able to elevate intracellular Ca 2+ level by themselves. These include l-opioid [35], GABA B [27], muscarinic M 2 and M 4 [24], somatostatin SST 2 [36], and a 2 -adrenrgic [37] receptors. However, the underlying signaling mechanism utilized by these receptors is controversial.…”

“…It has been demonstrated that functional FPRL-1 receptors are expressed in U87 human astrocytoma cells [15], and we have previously observed that several G i/o -coupled receptors including FPRL-1 can promote Ca 2+ mobilization upon overexpression in CHO cells [24]. We thus examined the ability of FPRL-1 to trigger increase in intracellular Ca 2+ as well as its downstream signaling pathway in U87 cells.…”

“…U87 cells were grown in Minimum Essential Medium (MEM) adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, and supplemented with 10% (v/v) FBS, 50 U/ml penicillin and 50 lg/ml streptomycin. The establishment of CHO cells stably expressing the FPRL-1 receptor (FPRL-1/CHO) has been described previously [24].…”

“…The following day, the medium was removed and the cells were labeled with labeling medium consisting of 1:1 (v/v) Opti-MEM/Hanks' balanced salt solution (HBSS), 2.5% (v/v) FBS, 20 mM HEPES, pH 7.4, 2.5 nM probenecid, and 2 lM Fluo-4 AM. After incubation with labeling medium for 60 min, the cells were assayed using an optimized FLIPR protocol [24]. Where indicated, pharmacological inhibitors were added at appropriate time points before the assay.…”

A Hematopoietic Perspective on the Promiscuity and Specificity of Gα₁₆ Signaling

“…The same strategy worked equally well for G s -coupled receptors [27] . Chimeric G ␣ 16 subunits with broadened receptor-coupling capability have been successfully employed in high throughput screening platforms [23,[28][29][30] . Further efforts have been made to decipher the minimal requirements for switching the receptor-coupling specificity.…”