COUPLING OF THE C5A RECEPTOR TO G(I) 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 c ells transfected with the C5aR. In both U-937 cells and transfected ce lls (2A3 cells), C5a induced the mobilization of intracellular calcium , phosphoinositide breakdown, and activation of mitogen-activated prot ein kinase. In addition, in 2A3 cells C5a induced the inhibition of fo rskolin-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 39-4 0-kDa protein in membranes of both U-937 cells and 2A3 cells. Similar phenomena have been described in other systems, whereby G(ia) subunits are substrates for cholera toxin-induced ribosylation in the presence of receptor agonists. Moreover, the C5a-induced ribosylation was elim inated in membranes of cells that had been pretreated with pertussis t oxin. The G protein alpha subunit G(alpha 16), which is insensitive to pertussis toxin, has been reported to couple selectively to C5aR in c ells co-transfected with C5aR and G(alpha 16) cDNAs. G(alpha 16) expre ssion was not detected in U-937 cells or in 2A3 cells, either by rever se transcription-polymerase chain reaction or by immunoblotting. Becau se pertussis toxin modifies only G(alpha) subunits of the G(i/o), fami ly and all signaling by C5aR was abolished by pertussis toxin pretreat ment, the results strongly suggest that, in U-937 and 2A3 cells, C5a-m ediated responses can be accounted for entirely through coupling with G proteins of the G(i) subtype.