Oligomerization of G-protein-coupled receptors is independent of ligand binding in living cells: an analysis using single molecule imaging techniques

GPCRs (G-Protein-Coupled Receptors) play critical roles on homeostasis in mammals as receptors for neurotransmitters, hormones, chemokines etc. GPCRs are considered as targets for similar to45% of the medicines for clinical treatments. GPCRs were considered to exist as a monomer on plasma membrane. In the last few years, however, increasing evidence obtained by biochemical analyses implied that GPCRs form homo and hetero-dimers or oligomers. To examine this hypothesis, several chemokine receptors of the GPCR family fused with EGFP at the C terminus were expressed in CHO cells and observed using an objective type total internal reflection fluorescence microscope to facilitate single fluorescent molecule imaging. We demonstrate that constitutive and ligand-independent oligomerization is the relevant feature of GPCRs classified into HIV, chemokine- and chemoattractant-receptors, such as CCR5, CXCR4, CXCR1 and FPR1. These GPCRs exist as constitutive oligomers in a living cell. Each singe oligomer of the GPCR consists of various number of the GPCR molecules. The distribution of the number of GPCR molecules in the oligomers occupied by their ligand is statistically the same as those under absence of the ligand. These results unambiguously support the hypothesis that GPCRs form constitutive oligomers independent of ligand binding.