Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native
ligand labeled with a radioisotope of finite lifetime. However, the short half-life and the potential hazard of the radioisotope labled ligand have limited its application in biological systems. Herein,
we have applied the native chemical ligation (NCL) approach to synthesize full-length human C3a specifically modified at the N-terminus by appending diethylenetriaminepentaacetic acid (DTPA),
which is available for chelating europium (Eu−DTPA−hC3a). Time-resolved fluorescence analysis has demonstrated that Eu−DTPA−hC3a binds selectively to its cognate G protein-coupled
receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293
cells that stably expressed C3aR. The potency of Eu−DTPA−hC3a was further validated by competitive binding experiments with a few known C3aR specific ligands (TR16, BR103, SB290157,
BR111) via displacement of Eu−DTPA−hC3a from hC3aR. The macromolecular conjugate Eu−DTPA−hC3a is a novel nonradioactive probe suitable for studying ligand−C3aR interactions with
potential value in accelerating drug development for human C3aR in physiology and disease.