Characterisation of C5a receptor agonists from phage display libraries

C5a des-Arg74 has a 10- to 100-fold lower receptor binding affinity than intact C5a and is only a partial agonist. We have used phage display selection from randomly mutated C5a des-Arg74 libraries to isolate variant proteins that can activate C5a receptors with similar potency to C5a. Here we explore the interactions of three variants (V1–3) with C5aR mutated at residues involved in the differential response. The mutant Asp282Arg-C5aR is preferentially activated by C5a des-Arg74, probably due to repulsion between Arg74 of C5a and the substituent Arg282. In accordance with this hypothesis, V2 (with a polar C-terminus which has no Arg residue) but not V1 (with a C-terminal Arg residue at position 73) could activate Asp282Arg-C5aR. V3, with a very hydrophobic C-terminus, was the most potent agonist at Asp282Arg-C5aR. Arg175 is a potential counterion for the C-terminal carboxylate of C5a. C5aR mutated to either Ala or Asp at this position lost nearly all responsiveness to both C5a and C5a des-Arg74, suggesting that mutation of Arg175 caused a non-specific loss of receptor conformation and a loss of signalling capacity. However, V3 could still activate Arg175Asp/Ala-C5aR with the same potency as wild-type C5aR, demonstrating that the mutant receptors retained high signalling capability and showed a specific loss of responsiveness. Thus C5a des-Arg74 variants produced by phage display are potentially useful tools for the dissection of ligand–receptor interactions. [Copyright &y& Elsevier]