SRU Biosystems - Cell-Based Applications >> GPCRs

Cell-Based Applications >> GPCRs

BIND® technology enables cell-based detection of all GPCR subtype activation (Gi, Gq, Gs, G12/13-coupled) using a single assay. BIND is highly sensitive and can be used with both recombinant and endogenous GPCRs, requiring as few as 1000 cells per well in 1536-well format. BIND assays can identify agonist, antagonist, partial agonist and inverse agonists. The use of a single technology for GPCR applications streamlines assay development and screening, while the information-rich nature of BIND kinetic data increases the power and efficiency of compound profiling.

Application Features

Universal Gq, Gi, Gs & G12/13 Assay
Endogenous & Recombinant Receptors
1536-well, Label-free Format
FLIPR Alternative
Identifies Agonist, Antagonist, Partial Agonist
Inverse Agonist and Allosteric Modulators
Capable of Monitoring Dual-Coupled GPCRs and Gα Pathway Switching

GPCR Assay Scheme

The BIND GPCR assay consist of 3 basic steps: Plating of cells on hydrated biosensor, Cell attachment & starvation and Compound addition(s). This method is highly flexible and can be used for adherent, suspension and primary cells and can be performed in a single day or over two days. BIND assays are easily miniaturized and can be run in 96-, 384-, 384lv- and 1536-well format to meet any throughput requirements. The open architecture of the BIND readers allows for biosensor baselines to be read, compounds added and responses measured without having to take the biosensor off the reader, thus simplifying assay protocols and biosensor handling.

BIND GPCR Agonist Assay

BIND GPCR Antagonist Assay

Universal Gα GPCR Assay

GPCR subtype (Gi, Gq, Gs, G12/13 coupled) activation can be measured using a number assay platforms based on various cellular responses, most commonly calcium mobilization, β-arrestin localization or second messenger levels. Many of these technologies require the development of recombinant cell lines that over-express the GPCR target of interest due to sensitivity limitations. In some cases over-expression of promiscuous G proteins is required to drive coupling to calcium mobilization. These artificial assay systems can yield false positives, often identifying leads that do not perform in the in vivo disease models. The BIND GPCR assay is a universal assay which can detect activation of Gi, Gq, Gs, G12/13 alpha subunits using a single, highly flexible yet streamlined protocol.

Gα Subunit Temporal Signatures

GPCR stimulation results in a variety of BIND kinetic responses which fall into one of four signatures, each of which can be attributed to activation of specific Gα pathways. The designation of specific temporal signatures to individual Gα subunits has been supported using known ligands that activate specific Gα subunits (figure 1) as well as using orthogonal assays (figure 2) and pathway inhibitors (figure 3).

Figure 1 - Endogenous GPCRs in HEK293 cells were incubated with a variety of known natural ligands and BIND responses measured over time.

Orthogonal Assay Confirmation - FLIPR®

Figure 2 - Cells were stimulated with agonists and FLIPR or BIND assays performed using standard protocols. BIND PWV changes were measured every 30 seconds for 30 minutes. Inset - BIND responses of cells stimulated with Carbachol. Table - EC50s calculated for 5 agonists that exhibited positive responses in FLIPR and BIND assays

FLIPR is a traditional label-based method for monitoring Ca++ immobilization often used as a measurement of Gq signaling. Studies using both FLIPR and BIND assays were performed in an effort to attribute a specific BIND temporal signature to Gq signaling and further support the use of BIND as a universal GPCR assay.

Cells were stimulated with 11 agonist, several of which were known to signal via Gq, and receptor activation measured using BIND and FLIPR. 5 of 11 compounds tested displayed dose-dependent FLIPR responses (figure 2). These 5 compounds all resulted in similar BIND temporal signatures (figure 2 insert). The calculated EC50 values for these 5 compounds using FLIPR and BIND assays were comparable. The 6 compounds which were negative in FLIPR assays did result in positive BIND responses. The kinetic patterns of these responses, however, were quite distinct from . These data suggest that this temporal signature represents Gq-mediated signaling.

Similar correlation was observed for measurement of cAMP as a read-out of Gs activation and a distinct BIND response (data not shown).

Compound	FLIPR pEC50±sem	SRU BIND pEC50±sem
ATP	5.79 ± 0.11	6.62 ± 0.03
UTP	5.01 ± 0.00	6.02 ± 0.06
ET-1	6.41 ± 0.01	8.11 ± 0.04
Carbachol	6.65 ± 0.03	6.36 ± 0.02
PAR-2	4.85 ± 9.07	5.84 ± 0.01

Inhibitor Confirmation - Pertussis Toxin

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Figure 3 - A). HEK293 cells were treated with a proprietary ligand ± pertusis toxin (PTX) and BIND responses measured. B). Cells were treated with Carbachol, PGE2 or S1P ± PTX and responses measured.

Inhibitors known to block specific cellular signaling pathways are powerful tools for establishing specific modes of agonist signaling. Pertussis Toxin (PTX) is known to inhibit Gi signaling and was used to examine BIND responses upon Gi-coupled GPCR activation. Endogenous GPCRs were stimulated with one of 11 agonists in the presence or absence of PTX and BIND responses measured over time. PGE2 (Gs) and Carbachol (Gq) BIND responses were not affected by PTX. The BIND responses upon stimulation with a proprietary Gi-agonist were abrogated in the presence of PTX, supporting that this agonist signals via Gi. These data also allow us to attribute the temporal signature induced by the proprietary compound to Gi signaling.

The S1P receptor appears to partially signal through Gi. S1P temporal responses shifted from a positive PWV change to a negative PWV change in the presence of PTX. This indicates that while S1P does signal through the Gi pathway, it is able to signal via an alternative Gα subunit as well.

Dual Coupled Receptors

Interpretation of GPCR assays can be complex for receptors that couple via multiple signaling pathways. Traditional assays that measure a single Gα pathway readout such as cAMP or Ca++ flux require multiple assay technologies to examine dual coupling. BIND temporal signatures and the use of pathway inhibitors are a simplified means to identify GPCR coupling to multiple Gα pathways. For the dual-coupled S1P receptor, BIND identified not only the multi-coupling nature of S1PR, but also indicated which Gα pathways contributed to receptor activation (figure 4&5).

Concentration Dependent Pathway Usage

Figure 4 - HEK293 cells were treated with various concentrations of S1P and BIND responses measured over time.

Pathway Identification Using Chemical Inhibitors

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Figure 5 - A). Endogenous GPCRs in HEK293 cells were incubated with 0.3µM S1P in the presence and absence of PTX and a RhoK inhibitor and BIND responses measured over time. B). Dose response curves were calculated for each Gα pathway.

Figure 4 displays kinetic PWV changes upon stimulation of endogenous S1PR with a range of S1P concentrations. Clearly distinct BIND responses were detected for each concentration whose kinetic signatures suggest usage of multiple signaling pathways.

Treatment of cells with PTX and RhoK inhibitors suggest that S1PR signals via G12/13 and Gi pathways. PTX inhibits S1P Gi signaling, while allowing the receptor to signal via another Gα pathway as suggested by the negative PWV shift rather than complete abrogation of a response.

Treatment of cells with S1P and a RhoK inhibitor, known to block G12/13 signaling, shifted receptor signaling to a Gi temporal pattern. Responses to 3 other agonists which signal through Gi, Gs and Gq were not affected by the RhoK inhibitor suggesting that the alteration of the S1P response in the presence of RhoK inhibitor was due to inhibition of G12/13 signaling (figure 5A).

Neither inhibitor alone abrogates BIND responses suggesting that if one signaling pathway is blocked, the second Gα signaling pathway can be used. Taken together, these data suggest that stimulation of cells with 0.3mM S1P activates a combination of Gi and G12/13 pathways each of which can be independently inhibited. S1P dose response curves can be Independently calculated for the 2 signaling pathways using PTX and RhoK pathway inhibitors (figure 5B).