Analysis of post-translational CCR8 modifications and their influence on receptor activity.
Post-translational modifications of the extracellular portions of receptors located in the cell membrane can contribute to modulating their biological activity. Using a mutagenesis approach in which single or multiple Tyr-to-Phe, Thr-to-Ala, Ser-to-Ala, and Asn-to-Gln substitutions were made at the appropriate positions, we analyzed the sulfation and glycosylation state of the murine CCR8 chemokine receptor, and the way in which these post-translational modifications affect CCR8 activity. A Y14Y15-to-F14F15 CCR8 mutant was less sulfated than the wild-type receptor. An N8-to-Q8 mutant was less glycosylated than wild-type, and a double T10T12-to-A10A12 mutant showed even less glycosylation. We established a flow cytometric analysis with an Fc-fused form of mouse CCL1 to determine precisely the ligand-binding activity of these mutants. Single mutants at amino acid positions 8, 10 or 12 bound CCL1-Fc similarly to wild-type CCR8, whereas the F14F15 double mutant was essentially inactive and the A10A12 double mutant showed about 65% of wild-type ligand-binding activity. Calcium flux activity assays were performed with these mutants, yielding results consistent with those from the ligand binding assays. These data indicate that sulfation at specific positions of the N-terminal domain of mouse CCR8 is critical for its biological activity, whereas glycosylation has a minor influence.
Gutiérrez
Julio
J
Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
Kremer
Leonor
L
Zaballos
Angel
A
Goya
Iñigo
I
Martínez-A
Carlos
C
Márquez
Gabriel
G
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009439:Neuraminidase / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D054401:Receptors, CCR8; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D014162:Transfection
C103330:CCR8 protein, human; D008024:Ligands; D054401:Receptors, CCR8; D019707:Receptors, Chemokine; D013431:Sulfates; D009439:Neuraminidase; D002118:Calcium