Ligand binding sites on guinea pig C3aR: point and deletion mutations in the large extracellular loop and vicinity.
Human C3a receptor (huC3aR) belongs to the G-protein coupled receptor family chacterized by having seven transmembrane domains. The huC3aR is a unique member of this family having a large extracellular (EC) loop of 175 amino acids between the 4th and 5th transmembrane domains. Based on a comparison of C3aR sequences from several species, a number of charged and conserved amino acids (Asp182, Asp309, Asp310, and Arg331) in and near the large EC loop of guinea pig C3aR were replaced using site-directed mutagenesis. Competitive binding assays showed that changing Arg331 in guinea pig C3aR to Ala (or Gln), but not changing Asp182, Asp309, or Asp310 to Ala, resulted in complete loss of ligand binding activity. These results and major EC loop deletions demonstrated that an essential C3a binding site is present in the transmembrane portion of C3aR, but not in the large EC loop. Replacement of Arg331 by a noncharged residue was sufficient to eliminate ligand-receptor interactions.
Fukuoka
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Department of Immunology, Scripps Research Institute, La Jolla, California, 92037, USA.
Ember
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JA
Hugli
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D000818:Animals; D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D015926:Complement C3a / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D008024:Ligands; D008565:Membrane Proteins; D011951:Receptors, Complement; D011994:Recombinant Proteins; C085567:complement C3a receptor; D015926:Complement C3a; D001120:Arginine