The G protein coupling T cell antigen receptor/CD3-complex and phospholipase C in the human T cell lymphoma Jurkat is not a target for cholera toxin.
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.
Sommermeyer
H
H
Institut für Molekularpharmakologie, Medizinische Hochschule Hannover.
Schwinzer
R
R
Kaever
V
V
Behl
B
B
Resch
K
K
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis* / Q000502:physiology; D017252:CD3 Complex; D002772:Cholera Toxin / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D016399:Lymphoma, T-Cell / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000032:analysis* / Q000502:physiology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D007295:Inositol Phosphates; D011948:Receptors, Antigen, T-Cell; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins