Coupling of the C5a receptor to Gi in U-937 cells and in cells transfected with C5a receptor cDNA.
The signaling properties of the receptor for the chemoattractant C5a (C5aR) were investigated in differentiated U-937 cells and in NIH/3T3 cells transfected with the C5aR. In both U-937 cells and transfected cells (2A3 cells), C5a induced the mobilization of intracellular calcium, phosphoinositide breakdown, and activation of mitogen-activated protein kinase. In addition, in 2A3 cells C5a induced the inhibition of forskolin-stimulated cAMP generation. Pretreatment with pertussis toxin suppressed all C5a-mediated signal transduction in both cell lines. In the presence of cholera toxin, C5a induced the ribosylation of a 39-40-kDa protein in membranes of both U-937 cells and 2A3 cells. Similar phenomena have been described in other systems, whereby Gi alpha subunits are substrates for cholera toxin-induced ribosylation in the presence of receptor agonists. Moreover, the C5a-induced ribosylation was eliminated in membranes of cells that had been pretreated with pertussis toxin. The G protein alpha subunit G alpha 16, which is insensitive to pertussis toxin, has been reported to couple selectively to C5aR in cells co-transfected with C5aR and G alpha 16 cDNAs. G alpha 16 expression was not detected in U-937 cells or in 2A3 cells, either by reverse transcription-polymerase chain reaction or by immunoblotting. Because pertussis toxin modifies only G alpha subunits of the Gi/o family and all signaling by C5aR was abolished by pertussis toxin pretreatment, the results strongly suggest that, in U-937 and 2A3 cells, C5a-mediated responses can be accounted for entirely through coupling with G proteins of the Gi subtype.
Eisai Research Institute, Andover, Massachusetts 01810.
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Vanek
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Hawkins
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Gusovsky
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D004789:Enzyme Activation / Q000502:physiology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018076:DNA, Complementary; D010716:Phosphatidylinositols; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D012266:Ribose; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium