Mutational scans of the chemokine receptors CXCR4 and CCR5 based on bimolecular fluorescence complementation reveal multiple mechanisms for receptor association

Many G protein-coupled receptors (GPCRs) are found to homo- or hetero-oligomerize, but how and why GPCRs associate remains controversial. The class A chemokine receptors CCR5 and CXCR4 both homo- and heterodimerize with each other, and crystal structures of CXCR4 in inactive conformations have captured dimeric organization. By selecting libraries of CCR5 and CXCR4 variants based on bimolecular fluorescence complementation, we determine how nearly every single amino acid substitution effects homo-associations. We find three mechanisms for chemokine receptor association: (i) non-specific aggregation in the membrane phase enhanced by destabilizing structural mutations, (ii) specific protein-protein dimerization interfaces, and (iii) motifs in the cytoplasmic tails that are hypothesized to promote lipid raft localization, thereby bringing receptors close together at high density. We identify mutations that tease apart the effects of these respective mechanisms, and show that specific dimer organization, despite reducing agonist binding, is necessary for active signaling within the cell. Overall design: Single-site saturation mutagenesis libraries were constructed of myc-tagged CXCR4 and CCR5 fused at their cytosolic C-termini with VC (Venus I152L a.a. D155-K238). The libraries were transfected into human Expi293F lines over-expressing wildtype FLAG-tagged CXCR4 or CCR5 fused to VN (Venus I152L a.a. V1-A154) at the receptors' cytosolic C-termini. Cells were evolved by FACS for surface expression based on the extracellular myc tag, and for receptor oligomerization based on complementation of split Venus. Frequencies of variants in the sorted population (measured from RNA transcripts) were compared to frequencies in the DNA libraries to calculate log(base2) enrichment ratios for all amino acid substitutions. All evolution experiments were in duplicate. The library construction protocol and data processing details are also provided in the readme.txt file.