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Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7
Kathryn Luker1, Mudit Gupta, Gary Luker
1Center for Molecular Imaging, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA.
Abstract:
Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine[#x02013]chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gaussia luciferase (GL), an ATP-independent enzyme that is the smallest known luciferase. Fusing CXCL12 to Gaussia luciferase (CXCL12-GL) did not alter the bioluminescence emission spectrum and only minimally affected enzyme function under varying conditions of pH, temperature, and NaCl concentration. CXCL12-GL also activated CXCR4-dependent signaling to a comparable extent as unfused CXCL12. Using multiwell plate assays, we established that CXCR7 increases cell-associated CXCL12 to a significantly greater extent than CXCR4. We also showed that CXCL12-GL can be used to quantify inhibition of chemokine receptor binding by compounds that specifically target CXCR7. These data validate CXCL12-GL as a bioluminescent probe to investigate molecular functions of CXCR4 and CXCR7 and screen for compounds that modulate ligand-receptor binding.
Insights
Researchers developed a novel bioluminescent probe, CXCL12-GL, to study chemokine CXCL12 and its receptors CXCR4/CXCR7. This tool aids in understanding disease roles and drug development for conditions like cancer and HIV.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Chemokine CXCL12 and its receptors CXCR4/CXCR7 are implicated in diseases such as cancer metastasis, atherosclerosis, and HIV.
- These molecules represent potential therapeutic targets for drug development.
- Current drug discovery screening relies on luminescent assays, but a bioluminescent assay for chemokine-receptor binding is lacking.
Purpose of the Study:
- To develop a novel bioluminescent probe for studying chemokine CXCL12 binding and cellular uptake.
- To create a tool for investigating the molecular functions of CXCR4 and CXCR7.
- To enable high-throughput screening for compounds modulating CXCL12-receptor interactions.
Main Methods:
- Chemokine CXCL12 was genetically fused to Gaussia luciferase (GL) to create the CXCL12-GL probe.
- The functional properties of CXCL12-GL, including bioluminescence and receptor activation, were assessed.
- Cell-associated CXCL12 levels mediated by CXCR4 and CXCR7 were quantified using multiwell plate assays.
Main Results:
- The CXCL12-GL probe retained bioluminescence properties and minimally affected enzyme function.
- CXCL12-GL effectively activated CXCR4-dependent signaling.
- CXCR7 significantly increased cell-associated CXCL12 compared to CXCR4.
- The probe successfully quantified the inhibition of CXCR7 binding by specific compounds.
Conclusions:
- CXCL12-GL is a validated bioluminescent probe for studying chemokine-receptor interactions.
- This probe facilitates research into the roles of CXCR4 and CXCR7 in disease.
- CXCL12-GL serves as a valuable tool for screening potential therapeutic compounds targeting these receptors.
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