Subtle conformational changes between CX3CR1 genetic variants as revealed by resonance energy transfer assays

The chemokine CX3CL1 is expressed as a membrane protein that forms a potent adhesive pair with its unique receptor CX3CR1. This receptor has 3 natural variants, V249-T280 (VT), I249-T280 (IT), and I249-M280 (IM), whose relative frequencies are significantly associated with the incidence of various inflammatory diseases. To assess the adhesive potency of CX3CR1 and the molecular diversity of its variants, we assayed their clustering status and their possible structural differences by fluorescence/bioluminescence resonance energy transfer (FRET or BRET) techniques. FRET assays by flow cytometry showed that the CX3CR1 variants cluster, in comparison with appropriate controls. BRET assays showed low nonspecific signals for VT and IT variants and high specific signals for IM, and thus pointed out a structural difference in this variant. We used molecular modeling to show how natural point mutations of CX3CR1 affect the packing of the 6th and 7th helices of this G-protein coupled receptor. Moreover, we found that the BRET technique is sensitive enough to detect these tiny changes. Consistently with our previous finding that CX3CL1 aggregates, our data here indicate that CX3CR1 clustering may contribute to the adhesiveness of the CX3CL1-CX3CR1 pair and may thus represent a new target for anti-inflammatory therapies.