Chemokine receptor-ligand interactions measured using time-resolved fluorescence

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• G protein-coupled receptor subtypes CXCR1 and CXCR2 mediate Interleukin-8 (IL8) signaling.
• Accurate measurement of chemokine receptor-ligand interactions is crucial for understanding cellular responses.

Purpose of the Study:

• To develop a nonradioactive, lanthanide-chelate derivatized IL8 ligand for measuring binding activity to CXCR1 and CXCR2.
• To assess the utility of lanthanide-labeled proteins in studying receptor-ligand interactions.

Main Methods:

• Site-specific mutagenesis of IL8 to create IL8-S72C with a single free sulfhydryl group.
• Conjugation of an iodoacetamide derivative of the Eu3+-chelate of DTTA to IL8-S72C, yielding [Eu3+]IL8-S72C.
• Measurement of dissociation constants using time-resolved fluorometry on CHO cells expressing CXCR1 or CXCR2.

Main Results:

• Successfully synthesized a europium-labeled IL8 conjugate ([Eu3+]IL8-S72C) with one Eu3+ per IL8 molecule.
• The dissociation constant of [Eu3+]IL8-S72C was comparable to radiolabeled IL8 (approximately 2 nM).
• Demonstrated high sensitivity, stability, and specific activity of the europium-labeled IL8.

Conclusions:

• Lanthanide-labeled proteins, exemplified by europium-labeled IL8, are effective tools for measuring receptor-ligand interactions.
• This nonradioactive approach offers a viable alternative to radioligands for studying chemokine receptor binding.
• The methodology can potentially be extended to other peptide ligands and receptor systems.