TR-FRET between engineered nanobodies reveals the existence of endogenous CXCR4 oligomers
Joyce Heuninck1, Vladimir Bobkov2,3, Claire M Grison1
1IGF, Univ Montpellier, CNRS, INSERM, Montpellier, France.
Abstract:
Chemokine receptors CXCR4 and ACKR3 are involved in multiple physiological and pathological processes. In overexpression systems, CXCR4 and ACKR3 form oligomers that are important for chemokine recognition and signaling. Whether this holds true in physiological settings is unknown as no evidence of such oligomers are available. To address this gap of knowledge, we develop a new approach for GPCR oligomer detection by time-resolved Förster resonance energy transfer (TR-FRET) between fluorescently labeled nanobodies. Fluorescent probes are conjugated to the C-terminus of extracellularly binding nanobodies using site-directed sortase-mediated labeling, retaining high affinity of the nanobodies for their target receptors. This approach, first validated in transfected cells by detecting the presence of CXCR4 and ACKR3 homo-oligomers and hetero-oligomers, reveals for the first time endogenous CXCR4 homo-oligomers in non-transfected human leukemia/lymphoma-derived cancer cell lines. More importantly, the investigation in peripheral blood mononuclear cells strongly supports the existence of CXCR4 oligomers in native conditions.
Insights
Chemokine receptors CXCR4 and ACKR3 form oligomers in cells. This study developed a new method to detect these CXCR4 oligomers in native conditions, confirming their presence in human blood cells.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Chemokine receptors CXCR4 and ACKR3 play roles in physiological and pathological processes.
- Previous studies showed CXCR4 and ACKR3 form oligomers in overexpression systems, but this was unconfirmed in physiological settings.
Purpose of the Study:
- To develop and validate a novel method for detecting G protein-coupled receptor (GPCR) oligomers in physiological conditions.
- To investigate the presence of endogenous CXCR4 and ACKR3 oligomers in native cellular environments.
Main Methods:
- Development of a time-resolved Förster resonance energy transfer (TR-FRET) assay using fluorescently labeled nanobodies.
- Site-directed sortase-mediated labeling of nanobodies to preserve receptor binding affinity.
- Validation in transfected cells and subsequent application to non-transfected cancer cell lines and primary human blood cells.
Main Results:
- The novel TR-FRET approach successfully detected homo- and hetero-oligomers of CXCR4 and ACKR3 in transfected cells.
- Endogenous CXCR4 homo-oligomers were identified for the first time in human leukemia/lymphoma-derived cancer cell lines.
- Evidence strongly supporting the existence of CXCR4 oligomers in peripheral blood mononuclear cells under native conditions was obtained.
Conclusions:
- The developed TR-FRET nanobody method is effective for detecting GPCR oligomers in physiological settings.
- CXCR4 homo-oligomers exist endogenously in human cancer cells and primary immune cells.
- This finding opens new avenues for understanding chemokine receptor function and developing targeted therapies.
