Intact Jurkat cells could be stimulated by
monoclonal antibodies against the Tcell
antigen receptor complex (
OKT3 directed against the
CD3 complex, BMA031 directed against constant framework
epitopes in the alpha/beta heterodimer). The accumulation of
inositol phosphates was inhibited by prior incubation of the cells with
cholera holotoxin. The inhibitory effect of
cholera toxin (CT) was not cAMP mediated because
forskolin (a direct activator of
adenylate cyclase) did not mimic the inhibitory effect. When measuring
phospholipase C (PLC) in a cell-free assay system by using [3H]
inositol-labeled membranes, the
enzyme could be stimulated by the poorly hydrolyzable
GTP analogue
guanosine 5'-O-(thiotriphosphate (
GTP gamma S). Both anti-receptor
antibodies augmented the
GTP gamma S stimulatory effect, while the
antibodies alone had no stimulatory capacity. In membranes from CT-pretreated cells, whereas the
antibodies lost their stimulatory effect on PLC as in untreated cells, whereas the
antibodies lost their stimulatory capacity in the presence of
GTP gamma S. These data imply that CT exerts its inhibitory effect on signaling by acting at the receptor level while the PLC regulating
G protein is not a target for CT-mediated alterations. This assumption is supported by the finding that in intact Jurkat cells CT, which
ADP ribosylated only the alpha-subunit of the stimulatory
G protein of the
adenylate cyclase, led to a loss of the
T cell antigen receptor complex from the cell surface as demonstrated by a decrease of receptor density using flow cytometry analysis. Receptor loss could not be achieved by
forskolin treatment or incubation of the cells with the binding subunit of the toxin alone.