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Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

Key words: CRF1 GPCR receptor binding assay LEADseeker SPA Imaging Beads


In the mammalian brain, the corticotrophin-releasing factor (CRF) system comprises four CRF-related peptides (CRF and urocortin 1, 2, and 3) and two G protein-coupled CRF receptor subtypes (CRF1 and CRF2, which includes CRF2α and CRF2β) (1). These receptor subtypes are positively coupled to adenylyl cyclase and show distinct expression patterns and binding characteristics. CRF1 receptors, for instance, are widely expressed in the brain, the anterior pituitary, and the GI tract (2). Strong evidence suggests that CRF1 receptors play an important role in stress responses and stress-related behaviors mediated through the hypothalamic-pituitary-adrenal (HPA) axis and through extra-hypothalamic systems (3).

This application note describes a 384-well CRF1 neuropeptide receptor binding assay developed using LEADseeker™ Multimodality Imaging System. The miniaturized assay is robust, achieving a Z’ value of 0.64, facilitating its adaptation to automated screening formats.


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) PS SPA RPNQ0260
Imaging Beads

[125I]Tyr0-CRF (ovine) IMQ20237


Other materials required
Human recombinant CRF1 receptor membrane preparation (Euroscreen)

Human urocortin 1 (Bachem)

Astressin (Tocris)

Costar™ solid white 384-well NBS™ coated microplate (Corning)

Protease-free BSA (Fluka)

EDTA-free protease inhibitor cocktail tablets (Roche)

Buffer: 25 mM Hepes pH 7.4
5 mM MgCl2
1 mM CaCl2
0.2% BSA (w/v) plus protease inhibitor cocktail

GraphPad Prism™ software v4.0 (GraphPad Software)


Protocol
Human recombinant CRF1 receptor membrane preparation was used in conjunction with [125I]Tyr0-CRF ligand and WGA PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 200 nM urocortin. The standard assay format was as follows:

1) Reagents were added in the following order: buffer, unlabeled ligand (NSB wells) labeled ligand, membrane, and bead. Total assay volume was 50 µl.

2) Wells contained 10 µl of 1.5 nM [125I]Tyr0-CRF (final concentration 0.3 nM) unless otherwise stated. Wells also contained 0.3 µg of membrane and 30 µg of beads, which were added in 10 µl volumes.

3) NSB wells contained 10 µl of 1000 nM urocortin (final concentration 200 nM) in addition to the above.

4) Plates were sealed and incubated in darkness for 18 h at room temperature (20–25 °C).

5) Following incubation, plates were imaged on LEADseeker Multimodality Imaging System for 5 min using quasi-coincident averaging and 3 ×3 binning.


Results
Kd determination
Saturation binding was performed with dilutions of [125I]Tyr0-CRF to give a range of concentrations from 0.06 to 4.96 nM in the wells. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis s oftware package GraphPad Prism v4.0. A Kd value of 0.4 nM (95% confidence intervals 0.3–0.5 nM) was estimated directly from the curve.

IC50 determination
Competitive binding of 0.3 nM [125I]Tyr0-CRF with urocortin (Fig 2A) and astressin (Fig 2B) was assessed and the IC50 value of each ligand calculated. Final concentrations in the wells were 0.039–160 nM and 0.005–500 nM respectively. Two separate batches of [125I]Tyr0-CRF were used for these evaluations, each with significantly different specific activities. This is demonstrated by the increase in specific IODs shown in Figure 2B. The IC50 value for urocortin was determined to be 3.5 nM (95% confidence interval range 2.7–4.5 nM) and the Ki value was 1.9 nM (95% confidence intervals 1.5–2.5 nM). The 50 value for astressin was 1.2 nM (95% confidence interval range 1.1–1.3 nM) and the Ki value was 0.6 nM (95% confidence intervals 0.6–0.7 nM).

Z’ analysis
Finally, a Z’ analysis was performed using 66 replicate values for “total” and NSB wells (4). The Z’ value indicated by Figure 3 is 0.64, which was well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.


References
1. Steckler, T. and Holsboer, F. Biological Psychiatry 46, 1480–1508 (1999).

2. Hauger, R. L. et al. Pharmacological Review 55, 21–26 (2003).

3. Timpl, P. et al. Nature Genetics 19, 162–166 (1998).

4. Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).



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