Mutation of glutamate 199 of the human C5a receptor defines a binding site for ligand distinct from the receptor N terminus.
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Monk PN, Barker MD, Partridge LJ, Pease JE
Mutation of glutamate 199 of the human C5a receptor defines a binding site for ligand distinct from the receptor N terminus.
J Biol Chem. 1995 Jul 14;270(28):16625-9.
- PubMed ID
- 7622471 [ View in PubMed]
- Abstract
C5a, a potent chemoattractant for monocytes, neutrophils, and other leukocytes, binds to a cell surface receptor of the seven-transmembrane superfamily. Here we report the effects of substituting Gln for Glu199 of the human C5a receptor (hC5aR) expressed in a model cell system for chemoattractant receptor signaling, the rat basophilic leukemia cell line RBL-2H3. Both the binding affinity for hC5a and the EC50 for subsequent cellular signals are reduced 5-10-fold by this substitution. A peptide mimic of the C terminus of C5a also binds to, and activates, hC5aR. The response to this peptide is reduced in cells bearing mutated hC5aR, indicating that the mutation affects interactions with the C terminus of hC5a. The C-terminal peptide contains only two basic residues, a Lys and an Arg (assumed to be analogous to Lys68 and Arg74 of hC5a), which could act as counter-ions for Glu199 of the receptor. If the counter-ion on hC5a was Arg74, then it would be expected that intact hC5a and hC5a des-Arg74 would have identical affinities and potencies when interacting with mutant hC5aR. It was found, however, that the binding affinity and potency (for receptor signaling events) of hC5a des-Arg74 was always lower than for intact hC5a. Furthermore, the equivalent C-terminal peptide to hC5a des-Arg74 (i.e. lacking the C-terminal Arg) could partially activate the wild type but not the mutant receptor, whereas the converse peptide, containing Arg but containing Met instead of Lys, had equal potencies for both wild type and mutant receptors. Taken together these data indicate that Glu199 of hC5aR is not involved in an interaction with Arg74 of hC5a, but may interact with Lys68 of hC5a. Mutation of Glu199 defines a second ligand binding site on hC5aR, distinct from the previously characterized site on the receptor N terminus. Unlike the N-terminal binding site, this second site is associated not just with the interaction with hC5a, but also with receptor activation.