THE AMINO-TERMINUS OF THE HUMAN C5A RECEPTOR IS REQUIRED FOR HIGH-AFFINITY C5A BINDING AND FOR RECEPTOR ACTIVATION BY C5A BUT NOT C5A ANALOGS

The binding domain of the human C5a receptor consists of two distinct and physically separable subsites. One of these sites binds the C-term inal 8 amino acids of C5a and is as yet undefined, while the second si te lies in the N terminus of the receptor and interacts with the core of C5a. Two deletion mutants were prepared to probe the importance of this second site. Removal of residues 2-22 decreased the binding affin ity for C5a by 600-fold, while extending the deletion through residue 30 caused a further 75-fold decrease. Thus, the N terminus is responsi ble for at least 45% of the total energy for the binding of C5a. The f ive aspartic acids present in the deleted segments appear to be critic al residues, as their conversion to alanines accounts for most of the affinity lost in the two truncations. Despite its importance for bindi ng, the N terminus is not necessary for signal transduction, as a C-te rminal peptide analog of C5a was able to stimulate G protein activatio n and to generate a Ca2+ flux through a receptor lacking residues 2-22 . However, intact C5a was a very poor activator of this truncated rece ptor. These results imply that interaction between the N terminus of t he receptor and C5a produces a conformational change in C5a that allow s it's C terminus to properly interact with and activate the receptor.