MUTATION OF GLUTAMATE-199 OF THE HUMAN C5A RECEPTOR DEFINES A BINDING-SITE FOR LIGAND DISTINCT FROM THE RECEPTOR N-TERMINUS

C5a, a potent chemoattractant for monocytes, neutrophils, and other le ukocytes, binds to a cell surface receptor of the seven-transmembrane superfamily. Here we report the effects of substituting Gin for Glu(19 9) of the human C5a receptor (hC5aR) expressed in a model cell system for chemoattractant receptor signaling, the rat basophilic leukemia ce ll line RBL-2H3. Both the binding affinity for hC5a and the EC(50) for subsequent cellular signals are reduced 5-10-fold by this substitutio n. A peptide mimic of the C terminus of C5a also binds to, and activat es, hC5aR. The response to this peptide is reduced in cells bearing mu tated hC5aR, indicating that the mutation affects interactions with th e C terminus of hC5a. The C-terminal peptide contains only two basic r esidues, a Lys and an Arg (assumed to be analogous to Lys(68) and Arg( 74) of hC5a), which could act as counter-ions for Glu(199) of the rece ptor. If the counter-ion on hC5a was Arg(74), then it would be expecte d that intact hC5a and hC5a des-Arg(74) would have identical affinitie s and potencies when interacting with mutant hC5aR. It was found, howe ver, that the binding affinity and potency (for receptor signaling eve nts) of hC5a des-Arg(74) was always lower than for intact hC5a. Furthe rmore, the equivalent C-terminal peptide to hC5a des-Arg(74) (i.e. lac king the C-terminal Arg) could partially activate the wild type but no t the mutant receptor, whereas the converse peptide, containing Arg bu t containing Met instead of Lys, had equal potencies for both wild typ e and mutant receptors. Taken together these data indicate that Glu(19 9) of hC5aR is not involved in an interaction with Arg(74) Of hC5a, bu t may interact with Lys(68) of hC5a. Mutation of Glu(199) defines a se cond ligand binding site on hC5aR, distinct from the previously charac terized site on the receptor N terminus. Unlike the N-terminal binding site, this second site is associated not just with the interaction wi th hC5a, but also with receptor activation.