Competitive receptor binding assay to probe for agonist binding to CXCR2
Lab/Group: Bernhagen Lab (RWTH Aachen University), Weber Lab (RWTH Aachen)
Related Journal & Article Information
Journal: Nature Medicine
Article Title: MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment
Introduction
Based on an observed apparent structural homology between the monomeric structure of the cytokine MIF and the CXCL8 dimer, we have tested whether MIF can directly interact with the cognate CXCL8 receptor CXCR2. Using a competitive receptor binding assay and other biochemical interaction assays, we found that MIF in fact directly binds to CXCR2 with a nanomolar affinity comparable to that observed for the cognate ligand CXCL8 (ref. 1).
Materials
Reagents
125I -labeled recombinant CXCL8 (IM249, Amersham)
125I-labeled recombinant CXCL12 (IM314, Amersham)
Recombinant CXCL8 (PeproTech EC/CellConcept, Umkirch, Germany)
HEK293 cells stably expressing CXCR2 (from Ben-Baruch Lab, Tel-Aviv, Israel)2
Liquid scintillation cocktail, Aquasafe 500 plus (Zinsser Analytic, Frankfurt)
Equipment
Wallac 1409 DSA liquid scintillation counter (PerkinElmer)
Procedure
Competitive inhibition of CXCL8 binding to CXCR2 receptor by CXCR2 agonist
1. Cultivate cells endogenously expressing or stably overexpressing CXCR2 (i.e. HEK293-CXCR2 cells) (105 cells/mL) in a 24-well plate in DMEM medium plus 10% FCS overnight.
2. Remove the medium and add binding buffer (medium without FCS).
3. Put the plate on ice for 10 min.
4. Add cold agonist, i.e. rMIF or cold CXCL8 (0 – 1 µM) together with the tracer (40 pM of 125I -labeled recombinant CXCL8).
5. Perform equilibrium receptor binding for 90 min at 4 °C.
6. Remove the medium and wash the cells twice with cold medium.
7. Harvest the cells by adding 0.5 M NaOH.
8. Transfer the lysate to a liquid scintillation tube.
9. Add Aquasafe 500 plus LSC cocktail and mix well.
10. Determine the cell-bound radioactivity by liquid scintillation counting.
11. For most agonist-receptor interactions, assume that receptor binding inhibition by agonist (i.e. rMIF or cold CXCL8) follows a one-site model.
12. Calculate the EC50, Ki and Kd values by using the equation of Cheng and Prusoff for heterologous competition experiments, applying the GraphPad software.
Troubleshooting
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Critical Steps
It is advised to use adherent cells. If using such cells, avoid disturbing cell adhesion during washing steps.
Anticipated Results
See for example Bernhagen et al., Nat. Med. 2007.
References
1. Bernhagen, J. et al. MIF is a non-cognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat. Med. Epub ahead of print(2007).
2. Ben-Baruch, A. et al. IL-8 and NAP-2 differ in their capacities to bind and chemoattract 293 cells transfected with either IL-8 receptor type A or type B. Cytokine 9, 37-45 (1997).
Acknowledgements
We sincerely thank Dr. A. Ben-Baruch (Department of Cell Research and Immunology, Tel Aviv University, Israel) for making available the HEK293-CXCR2 cells for us.
Keywords
chemokine receptor, competitive binding, agonist, receptor-ligand interactions, CXCR2