Mutating the four extracellular cysteines in the chemokine receptor CCR6 reveals their differing roles in receptor trafficking, ligand binding, and signaling.
CCR6 is the receptor for the chemokine MIP-3 alpha/CCL20. Almost all chemokine receptors contain cysteine residues in the N-terminal domain and in the first, second, and third extracellular loops. In this report, we have studied the importance of all cysteine residues in the CCR6 sequence using site-directed mutagenesis and biochemical techniques. Like all G protein-coupled receptors, mutating disulfide bond-forming cysteines in the first (Cys118) and second (Cys197) extracellular loops in CCR6 led to complete elimination of receptor activity, which for CCR6 was also associated with the accumulation of the receptor intracellularly. Although two additional cysteines in the N-terminal region and the third extracellular loop, which are present in almost all chemokine receptors, are presumed to form a disulfide bond, this has not been demonstrated experimentally for any of these receptors. We found that mutating the cysteines in the N-terminal domain (Cys36) and the third extracellular loop (Cys288) neither significantly affected receptor surface expression nor completely abolished receptor function. Importantly, contrary to several previous reports, we demonstrated directly that instead of forming a disulfide bond, the N-terminal cysteine (Cys36) and the third extracellular loop cysteine (Cys288) contain free SH groups. The cysteine residues (Cys36 and Cys288), rather than forming a disulfide bond, may be important per se. We propose that CCR6 forms only a disulfide bond between the first (Cys118) and second (Cys197) extracellular loops, which confines a helical bundle together with the N-terminus adjacent to the third extracellular loop, creating the structural organization critical for ligand binding and therefore for receptor signaling.
Ai
Li-Shaung
LS
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Liao
Fang
F
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D002522:Chlorocebus aethiops; D003545:Cysteine; D017931:DNA Primers; D005822:Genetic Vectors; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054399:Receptors, CCR6; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection
C106311:CCR6 protein, human; D017931:DNA Primers; D008024:Ligands; D054399:Receptors, CCR6; D019707:Receptors, Chemokine; D003545:Cysteine; D002118:Calcium