Soluble mimics of a chemokine receptor: Chemokine binding by receptor elements juxtaposed on a soluble scaffold

Despite the broad biological importance of G protein–coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS 5 , consists of the N‐terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS 5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 ± 0.8 μM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS 5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS 5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS 5 could be applied to many other GPCRs, with a variety of potential applications.