Human growth hormone enhances pertussis toxin-stimulated ADP-ribosylation of Gi in Nb2 cell membrane.
The Nb2 node lymphoma cell line has been widely used as a model for investigating lactogen cellular actions. Both pertussis (PTX) and cholera (CTX) toxins modulate lactogen-stimulated Nb2 cell mitogenesis, suggesting G protein involvement in lactogen signal transduction. The following studies were performed to further investigate this possibility. Both PTX-sensitive (41 kDa) and CTX-sensitive substrates (42 and 45 kDa) were identified in Nb2 cell membrane and recognized by specific anti-Gi and anti-Gs antibodies, respectively. Equal numbers of Nb2 cells were then incubated with the lactogen human growth hormone (hGH, 10 ng/ml) for 0-72 h. Membrane protein prepared from each time point (50 micrograms) was compared in toxin-stimulated ADP-ribosylation studies. CTX-stimulated ADP-ribosylation was unaffected by prior hGH incubation. PTX-stimulated ADP-ribosylation increased 237 +/- 69% (X +/- S.E.) compared with 0-h controls (n = 11; p less than 0.01) after 4-7 h of hGH incubation then decreased toward 0-h samples by 24 and 72 h. No change in Gi alpha concentration was observed, but beta subunit concentration increased (145 +/- 14% at 7 h; p less than 0.01; n = 3) in a time course that paralleled the changes in PTX-stimulated ADP-ribosylation. In summary, 1) both Gi and Gs were present in Nb2 cell membrane, 2) incubation of cells with a lactogen, hGH, for 4-7 h markedly enhanced PTX-stimulated ADP-ribosylation of Gi alpha in vitro, whereas CTX-stimulated ADP-ribosylation of Gs alpha was unchanged, and 3) although no change in Gi alpha concentration was observed, beta subunit concentration increased in parallel with the increase in PTX-stimulated ADP-ribosylation of Gi alpha. These results suggest that hGH may modify PTX-stimulated ADP-ribosylation of Gi not by changing Gi alpha concentration, perhaps by increasing beta subunit concentration, enhancing association of Gi alpha by beta gamma subunits, which, in turn, is preferentially ADP-ribosylated. This may represent a late signal transduction event and may also have implications for other effectors dependent on Gi-mediated events.
Larsen
J L
JL
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198-3010.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D015153:Blotting, Western; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D004357:Drug Synergism; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D008223:Lymphoma; D037342:Pertussis Toxin; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D013006:Growth Hormone; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins