Evidence that the extracellular N-terminal domain of C5aR contains amino-acid residues crucial for C5a binding.
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.
Mery
L
L
DBMS/Laboratoire de Biochimie (CNRS/URA 1130), Centre d'Etudes Nucléaires, Grenoble, France.
Boulay
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015936:Complement C5a / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D011392:Proline; D019204:GTP-Binding Proteins; D007930:Leucine