Different endocytosis pathways of the C5a receptor and the N-formyl peptide receptor.
Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N-formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re-phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand-dependent redistribution of C5aR-EGFP from the plasma membrane to LAMP2-positive compartments, whereas FPR-EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down-regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR-mediated chemotaxis was not observed.
Suvorova
Elena S
ES
Department of Microbiology, Montana State University, Bozeman, MT 59717-3520, USA.
Gripentrog
Jeannie M
JM
Miettinen
Heini M
HM
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002633:Chemotaxis / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004705:Endocytosis; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008024:Ligands; D018613:Microscopy, Confocal; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology; D044087:Receptor, Anaphylatoxin C5a / Q000378:metabolism*; D044042:Receptors, Formyl Peptide / Q000378:metabolism*
D008024:Ligands; D044087:Receptor, Anaphylatoxin C5a; D044042:Receptors, Formyl Peptide; D049452:Green Fluorescent Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine