Technical study: robust calcium mobilization

As cell surface receptors for a diverse collection of peptides, biogenic amines, proteins, proteases, amino acids, lipids, purines, and even protons, light and calcium ions, G protein-coupled receptors (GPCRs) are a premier class of targets for currently used drugs and for future drug discovery. A key to the successful development and preclinical testing of new GPCR-targeted drugs is the availability of rapid, scaleable assays with high information content for screening and selectivity profiling. GPCRs signal through Gs-mediated increases in cyclic AMP, Gi/o-mediated inhibition of cyclic AMP synthesis, and Gq-mediated elevation of intracellular calcium. The ease of assaying changes in intracellular calcium levels with calcium-sensitive fluorescent dyes and high throughput fluorescence detectors has made Gq-coupled receptors particularly amenable to functional screening. The discovery of ‘promiscuous’ G proteins, G15, G16, and chimeras of Gq and Gi or Gs, which couple Gs and Gi-coupled receptors to the calcium pathway, has broadened the panel of receptors that can be analyzed in such functional, high throughput assays.

Chemicon and Upstate are committed to expanding the availability of tools for effective characterization, screening and profiling of GPCR targets. To this end, Chemicon and Upstate have partnered to expand the ChemiScreen panel of recombinant GPCR-expressing cell lines with high cell surface receptor expression and coupling to the calcium pathway. The ChemiScreen system employs proprietary cell lines that contain endogenous G15 or G16 to enable a broad range of receptors to mobilize calcium. In addition, expression of the GPCR cDNAs is driven by a proprietary promoter optimized for the particular cell lines. As shown in Table 1, the ChemiScreen GPCR expression system has yielded 33 Gs and Gi/o-coupled receptors with robust calcium responses to their respective ligands. In addition, the calicum-optimized cell lines are characterized pharmacologically with selective antagonists. In Figure 1, the Y2 receptor for Neuropeptide Y (NPY), which ordinarily couples to Gi, was stably expressed in the Chem-1 background, and analyzed for calcium flux in response to increasing concentrations of neuropeptide Y; a dose-response with an EC50 of 1.1nM was observed (top panel). Calcium flux induced by a fixed concentration of neuropeptide Y was inhibited by increasing concentrations of the selective antagonist BIIE0246, with an IC50 of 39 nM (bottom panel). The calcium signal in at least one of the ChemiScreen cell lines is amplified by the presence of the Calcium Release Actived Calcium (CRAC) Channel, which causes influx of extracellular calcium when levels of intracellular calcium are increased. With the CRAC channel and high cell surface expression of GPCRs in the ChemiScreen cell lines, Gq-coupled receptors in these cell lines promote particularly robust calcium signals, and 24 Gq-coupled receptors are currently available.

 

Radioligand binding assays with membrane preparations from GPCR-expressing cells and tissues continue to enjoy wide popularity for consistency, strong signal:background, and ease of use. The ChemiScreen GPCR-expressing cell lines are ideal sources for membrane preparations, and Chemicon and Upstate have produced and characterized membrane preparations for a wide selection of GPCRs. The membrane preparations are characterized by saturation binding to determine receptor density/Bmax, and to confirm the Kd. In addition, membranes are titrated to give the optimal signal:background ratio in radioligand binding assays. Many of the membrane preparations have been characterized in GTP?S binding assays, which combine the functional readout of cell-based assays and the ease of use of frozen membranes. In Figure 2, membranes prepared from CCR9-Chem-1 cells or untransfected Chem-1 cells were incubated with [35S]-GTP?S and increasing concentrations of recombinant human TECK, and unbound radioactivity was removed by washing. A dose-dependent increase in [35S]-GTP?S binding was observed with an EC50 of 7nM in CCR9 membranes, whereas no signal was observed in the wild-type membranes.

ChemiScreen calcium-optimized cell lines and membrane preparations offer ideal tools for characterizing GPCR function, and screening and profiling the next generation of GPCR-targeted drugs. Please visit the Chemicon and Upstate web sites for more up-to-date information about our rapidly growing list of GPCR products.

 

TECK induces [35S]-GTP?S binding in CCR9-Chem-1 membrane preparations. 5?g/well CCR9 Membrane Preparation (catalog # HTS036M) and Wild-Type Chem-1 Membrane Preparation (catalog #HTS000MC1) were diluted to a final concentration of 0.1nM [35S]-GTP?S in 20mM HEPES, pH 7.4/100mM NaCl/10mM MgCl2/0.5?M GDP in a 96-well plate. Increasing amounts of unlabeled TECK/CCL25 were added, and the plate was incubated for 30 min at 30ºC. Bound radioactivity was determined by filtration and scintillation counting.

Gs-coupled receptors	i/o-coupled receptors
Adenosine: A2A, A2B	Adenosine: A1, A3
Adrenoceptor: 2	Anaphylotoxin: C3aR, C5aR, ChemR23
Corticotropin-releasing factor: CRF1, CRF2	CXC Chemokine: CXCR1, CXCR2, CXCR3, CXCR4
Parathyroid hormone: PTH1	CC Chemokine: CCR1, CCR2B, CCR6, CCR7, CCR8, CCR9, CCR10
Vasoactive Intestinal Peptide: VPAC1, VPAC2	Cannabinoid: CB1
	Histamine: H3
	Leukotriene: BLT1
	N-formyl peptide: FPR1, FPR2/FPRL1
	Neuropeptide Y: Y1, Y2
	Opioid: NOP/ORL1
	Platelet-activating factor: PAF

Table 1: Non-Gq-coupled receptors linked to calcium signaling pathway in the ChemiScreen cell line system