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-G(i) and anti-G(s) 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 μg) 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 < 0.01) after 4-7 h of hGH incubation then decreased toward 0-h samples by 24 and 72 h. No change in G(iα) concentration was observed, but β subunit concentration increased (145 ± 14% at 7 h; p < 0.01; n = 3) in a time course that paralleled the changes in PTX-stimulated ADP-ribosylation. In summary, 1) both G(i) and G(s) 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 G(iα) in vitro, whereas CTX-stimulated ADP-ribosylation of G(sα) was unchanged, and 3) although no change in G(iα) concentration was observed, β subunit concentration increased in parallel with the increase in PTX-stimulated ADP- ribosylation of G(iα). These results suggest that hGH may modify PTX- stimulated ADP-ribosylation of G(i) not by changing G(iα) concentration, perhaps by increasing β subunit concentration, enhancing association of G(iα) by βγ 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 G(i)-mediated events.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology