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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 physiologically relevant myeloid HL-60 cells. Ser334 was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser332 and Ser334 or Ser334 and Ser338, was critical 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 allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser334 and Ser338 is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For 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 defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.

N-formyl-methionyl peptides are powerful chemoattractants which bind to specific receptors on the neutrophil plasma membrane. A cDNA library from HL-60 cells, differentiated into granulocytes highly responsive to N-formyl-methionyl peptides, was constructed in the COS cell expression vector CDM8. A cDNA clone was isolated that conferred to COS cells the ability to bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys. The transfected COS cells displayed two classes of binding sites with Kd values of 1–5nM and 5–10 nM, respectively. The cDNA was 1.9 kb long with a 1050 bp open reading frame encoding a 350 residue protein. The hydropathy plot analysis revealed seven hydrophobic segments, a pattern quite similar to that of G protein-coupled receptors.