THE NH2-TERMINAL REGION OF C5AR BUT NOT THAT OF FPR IS CRITICAL FOR BOTH PROTEIN-TRANSPORT AND LIGAND-BINDING

The N-formylated tripeptide, formylmethionylleucyl-phenylalanine (fMLP ), and the 74-amino-acid long human C5a anaphylatoxin activate phagocy tic cells via two structurally related G protein-coupled receptors (FP R and C5aR), which are 34% identical in amino acid sequence. C5aR chim eras were constructed in which the entire NH2-terminal extracellular s equence or part of it was replaced by the counterparts from FPR or FPR H. Although the NH2-terminal region of C5aR presents an extremely high interspecies variability, substitution of the entire NH2-terminal seq uence of C5aR by that of FPR or FPRH surprisingly resulted in chimeras that were apparently retained in the endoplasmic reticulum. In contra st, when the NH2-terminal domain of FPR was replaced by the correspond ing region from C5aR or FPRH normal expression to the plasma membrane and high affinity binding of N-formylated peptides were observed. Thus , the NH2-terminal region of C5aR, in contradistinction to that of FPR , seems to be required either for the translocation of C5aR through th e ER membrane or for correct folding and sorting of C5aR to the plasma membrane. Replacement of the first 8 residues of C5aR by the correspo nding region of FPR did not alter the cellular transport and the C5a b inding capacity, whereas the exchange of the first 13 residues resulte d in a chimera that was readily transported to the plasma membrane but showed no capability to bind C5a. Mutations of Asp into Asn in the NH 2-terminal segment of C5aR further indicated that negative charges are required to endow the receptor with a C5a binding capacity. The resid ues critical for binding are either involved directly by interacting w ith cationic residues of C5a, or indirectly by influencing the overall structure of the ligand-binding site.