BIOLOGICALLY-ACTIVE CONFORMER OF THE EFFECTOR REGION OF HUMAN C5A ANDMODULATORY EFFECTS OF N-TERMINAL RECEPTOR-BINDING DETERMINANTS ON ACTIVITY

A conformationally biased decapeptide agonist of human C5a (C5a(65-74) Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational f eatures in the C-terminal effector region of C5a that are important fo r C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious ef fect compared to the natural factor. The maximal efficacy of this anal ogue was 216 +/- 56% that of C5a in stimulating the release of beta-gl ucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, charac teristics not observed with natural C5a or more conformationally flexi ble C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effe ctor template onto which was synthesized various C5aR binding determin ants from the N-terminal core domain of the natural factor. In general , the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector regio n was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptide s used in this study have up to 25% of the potency of C5a in human fet al artery and up to 5% of the activity of C5a in the PMN enzyme releas e assay.