Probing the "message:address" sites for chemoattractant binding to the C5a receptor. Mutagenesis of hydrophilic and proline residues within the transmembrane segments.
The C5a anaphylatoxin ligand-receptor interaction on polymorphonuclear granulocytes stimulates chemotaxis, degranulation, and the oxidative burst. The receptor is a member of the large G-protein-coupled family. The ligand is a cationic peptide of 72 amino acids derived from the C5 component of complement and has been shown to have a number of structural requirements for interaction with the receptor. In order to probe the potential interaction sites between ligand and receptor, we constructed a series of mutated receptor molecules, targeting cysteines, prolines, and additional amino acids of interest because of combinations of charge or hydrophobicity and putative location with respect to the membrane. Transfected mutant receptors were analyzed for cell surface expression, ligand binding, and ligand-activated phospholipase C activity. The receptors created can be placed generally in four distinct classes: those which bind and signal like the natural receptor; those which bind but fail to transduce signals; those which are expressed but neither bind nor transduce signal; and those which are not expressed at the cell surface.
Kolakowski
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Ina Sue Perlmutter Laboratory, Children's Hospital, Boston, Massachusetts 02115, USA.
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Gerard
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Gerard
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D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002630:Chemotactic Factors / Q000378:metabolism*; D015936:Complement C5a / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D002630:Chemotactic Factors; D008565:Membrane Proteins; D015335:Molecular Probes; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D011392:Proline; D003545:Cysteine