INTERNALIZATION AND RECYCLING OF THE C5A ANAPHYLATOXIN RECEPTOR - EVIDENCE THAT THE AGONIST-MEDIATED INTERNALIZATION IS MODULATED BY PHOSPHORYLATION OF THE C-TERMINAL DOMAIN

The C5a anaphylatoxin receptor is a member of the G protein-coupled re ceptor family involved in chemoattraction and activation of myeloid ce lls, as well as in host defence against infection by Pseudomonas aerug inosa. Upon challenge by C5a, the C5a receptor undergoes a rapid phosp horylation on serine residues in the carboxyl-terminal region, In this study, we used cells stably transfected with either the wild-type C5a receptor, or mutants affected in their capacity to be phosphorylated, to examine the role played by phosphorylation in the intracellular tr afficking of the C5a receptor, Upon agonist binding, the wildtype rece ptor was rapidly internalized into endosomes that cluster near the nuc leus after 10 minutes. Internalization of a non-phosphorylable mutant was severely impaired relative to wild-type receptor, whereas a mutant phosphorylated on serine 327 and/or serine 338, showed a rate of inte rnalization intermediate between that of wild-type receptor and that o f the non-phosphorylable mutant, Under continuous exposure to C5a and in the absence of protein synthesis, the C5a receptor was maintained i n a highly phosphorylated state but was not degraded, Confocal microsc opy and ligand-binding studies indicated that internalized receptors w ere recycled to the plasma membrane. During this process, receptors we re dephosphorylated with kinetics that correlated with the kinetics of receptor recovery on the cell surface, Altogether, our data suggest t hat phosphorylation plays a key role in the intracellular trafficking of the C5a receptor, Phosphorylated receptors might be recognized by a n adaptor protein that interacts with the endocytic machinery.