Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function.
The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.
Chao
T H
TH
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
Ember
J A
JA
Wang
M
M
Bayon
Y
Y
Hugli
T E
TE
Ye
R D
RD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006168:Guinea Pigs; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011951:Receptors, Complement / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D011951:Receptors, Complement; C085567:complement C3a receptor