M. Vanek et al., COUPLING OF THE C5A RECEPTOR TO G(I) IN U-937 CELLS AND IN CELLS TRANSFECTED WITH C5A RECEPTOR CDNA, Molecular pharmacology, 46(5), 1994, pp. 832-839
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.