Phosphorylation, dephosphorylation, and recycling of the C5a receptor in differentiated HL60 cells.
Binding of activation peptide from the fifth component of C (C5a) to its receptor triggers events leading to both stimulation of cellular responses and receptor desensitization in myeloid cells. However, although transmission of a signal to pertussis toxin-sensitive G proteins is a prerequisite to neutrophil activation, we show that the process of receptor phosphorylation is mainly independent from activation of this pathway. Treatment of cells with pertussis toxin did not modify the incorporation of phosphate mediated by a saturating concentration of C5a, indicating that agonist-occupied C5aR can be fully phosphorylated, presumably by a specific G protein-coupled receptor kinase, in the absence of activation of the Gi protein. Receptor phosphorylation was transient, with a half-life of 30 to 40 min, which suggested a role for protein phosphatases in the regulation of the state of phosphorylation of C5aR in dHL60 cells. Pretreatment of cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, increased the basal phosphorylation of unoccupied receptor and extended the phosphorylation mediated by C5a binding. Okadaic acid delayed, but did not suppress, the dephosphorylation process, which suggests either the involvement of additional phosphatase(s) or the degradation of nondephosphorylated receptors in the endocytic pathway. The data strongly suggest that internalized C5aR are recycled to the plasma membrane with a time course consistent with the kinetics of dephosphorylation. Dephosphorylation of C5aR might be essential to receptor recycling and resensitization during chemotaxis.
Giannini
E
E
Biochemistry Laboratory (CNRS/URA 1130), DBMS Center for Nuclear Research, Grenoble, France.
Boulay
F
F
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015936:Complement C5a / Q000378:metabolism; D004705:Endocytosis; D004988:Ethers, Cyclic / Q000494:pharmacology; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000502:physiology; D019833:Genistein; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*; D015398:Signal Transduction
D004988:Ethers, Cyclic; D007529:Isoflavones; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D019319:Okadaic Acid; D015936:Complement C5a; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D010749:Phosphoprotein Phosphatases; D019204:GTP-Binding Proteins