Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses - Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. ( 1995) J. Biol. Chem. 270, 19166-19172), To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiol ogically relevant myeloid HL-60 cells. Ser(334) was found to be a key resid ue that controls receptor phosphorylation, Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was c ritical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allow ed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findin gs and the ability of the S314A/S317A/S327A/S532A mutant receptor to underg o desensitization indicate that the phosphorylation of Ser(334) and Ser(338 ) is critical and sufficient for C5aR desensitization. The lack of phosphor ylation was found to result not only in a sustained calcium mobilization an d extracellular signal-regulated kinase 2 activity but also in the enhancem ent of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. F or instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was d efective, it was sequestered with the same time course and the same efficie ncy as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitizatio n and sequestration of C5aR appear to occur through divergent molecular mec hanisms.