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 CROSS5, 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. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 +/- 0.8 microM 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 CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.
Datta
Amita
A
Department of Chemistry, Indiana University, Bloomington, Indiana 47405-0001, USA.
Stone
Martin J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D054413:Chemokine CCL11; D018925:Chemokines / Q000737:chemistry* / Q000378:metabolism; D019742:Chemokines, CC / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D008024:Ligands; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D011485:Protein Binding; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D054413:Chemokine CCL11; D018925:Chemokines; D019742:Chemokines, CC; D004220:Disulfides; D008024:Ligands; D054397:Receptors, CCR3; D019707:Receptors, Chemokine; D011994:Recombinant Proteins