Mutation of aspartate 82 of the human C5a receptor abolishes the secretory response to human C5a in transfected rat basophilic leukemia cells.
C5a is a potent chemoattractant for monocytes, neutrophils and other leukocytes. The receptor for human C5a is a member of the rhodopsin superfamily of G protein-coupled receptors and contains an aspartate residue (Asp82) within the putative second transmembrane domain conserved in all other G protein-linked receptors. We investigated the role of this residue and also the carboxy-terminal 23 residues of the C5a receptor in ligand binding and signal transduction by expressing wild-type and mutant receptors in the rat basophilic leukemia cell line RBL-2H3. Wild-type and truncated receptors coupled efficiently to effector systems, resulting in the C5a-dependent discharge of granule contents. In contrast RBL cells transfected with receptors in which Asp82 had been mutated to asparagine did not respond to human C5a by secretion despite binding human C5a with high affinity. We conclude therefore that Asp82 is not involved in the interaction with ligand but is essential for the proper transduction of the ligand binding signal.
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield.
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Monk
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Pease
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Marland
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Barker
D000818:Animals; D001224:Aspartic Acid; D002118:Calcium / Q000378:metabolism; D015936:Complement C5a / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism; D009154:Mutation; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D001224:Aspartic Acid; D012701:Serotonin; D015936:Complement C5a; D019204:GTP-Binding Proteins; D002118:Calcium