Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization. Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 12-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO4/mol of receptor at Ser314, Ser317, Ser327, Ser332, Ser334, and Ser338. Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser332 to Ser338 is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.

Myeloid cells are attracted and activated by a variety of chemoattractants that bind to G protein-coupled receptors. In the past few years, the receptors for the classical chemoattractants (fMLF, C5a, PAF) and the chemotactic cytokines, known as C-X-C and C-C chemokines, have been cloned from myeloid cells. This review briefly describes recent advances in structure-function relationships of chemotactic receptors in human leukocytes as well as activation of signaling pathways and regulation of receptor function. In neutrophils, the binding of chemoattractants mainly activates the Gi2 protein inducing PIP2 hydrolysis and activation of the MAP kinase pathway. The C-C chemokine receptor, CC CKR5, and a chemokine receptor homologue, named fusin, have been shown to be the major cofactors for HIV-1 entry in macrophages and T cells. Recent studies suggest that the phosphorylation of chemoattractant receptors is a key event that regulates their biological function.