Two variants of the human N-formylpeptide chemoattractant receptor have been isolated from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with Bt2cAMP. Both recombinant receptors, fMLP-R26 and fMLP-R98, are 350 amino acids long (Mr 38,420); they differ from each other by two residue changes at positions 101 and 346 and by significant differences in the 5' and 3' untranslated regions. Both clones were able to transfer to COS-7 cells the capacity to specifically bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys, referred to as fMLPK-Pep12. Photolabeling experiments revealed that the glycosylated form of the fMLP receptor in COS cells has a molecular weight (Mr 50,000-70,000) similar to that observed for the native receptor in differentiated HL-60 cells. Northern blot analysis revealed a major transcript of 1.6-1.7 kb and two minor hybridization signals of 2.3 and 3.1 kb, suggesting a related family of receptors. The complex hybridization pattern obtained with restricted genomic DNA was consistent with either two genes encoding fMLP receptor isoforms or a single gene with at least one intron in the coding sequence. Sequence comparison established that the fMLP receptor belongs to the G-protein-coupled receptor superfamily. The structural similarities observed with RDC1, a receptor isolated from a dog thyroid cDNA library, which shares weak homologies with other members of the family, suggests that the fMLP receptor is representative of a new subfamily.

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A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.

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.