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.