Evidence that the extracellular N-terminal domain of C5aR contains amino-acid residues crucial for C5a binding

Abstract: The human C5a anaphylatoxin is a cationic 74 amino-acid long glycopeptide which derives from proteolysis of the fifth component of complement. It interacts with high affinity with a receptor that belongs to the G protein-coupled receptor superfamily. Several studies have previously suggested that multiple contact points between C5a and the receptor are required to achieve high-affinity interaction. However, at the receptor level little is known about the sites of interaction with C5a. We have investigated by in vitro mutagenesis whether the N-terminal extracellular sequence of the C5a receptor, which is rich in aspartic acid residues, could play some role in C5a binding. Conversion of Asp¹⁰ into asparagine did not impair the level of expression at the plasma membrane, nor did it alter the affinity for C5a. However, we consistently observed a discrepancy between an apparent high level of surface expression and a weak capacity to bind C5a with high affinity, suggesting that many receptor molecules, although present on the cell surface, might be misfolded and unable to bind C5a. Replacement of Pro⁹ by an isoleucine had little effect, if any, on either the affinity or the C5a-binding capacity, whereas the conversion of Pro³⁶ into leucine dramatically reduced the expression of high-affinity receptor at the cell surface. N-glycosylation of human C5a receptor was found to be dispensable for the function of the receptor.