Identification of the pertussis and cholera toxin substrates in normal and N-ras transformed NIH3T3 fibroblasts and an assessment of their involvement in bombesin-stimulation of inositol phospholipid metabolism.
Cholera and pertussis toxin-sensitive G-proteins were examined using specific immunological probes in wild type NIH3T3 cells and in clones of these cells containing the N-ras gene attached to a promotor where expression either was (T15+) or was not (T15-) induced. The major pertussis toxin sensitive-polypeptide had the immunological characteristics of Gi2. Two distinct forms of Gs alpha (45 and 42 kDa) were identified. Long term over-expression of p21N-ras (T15+ cells) did not alter the levels of Gi2 alpha or of Gs alpha. Pretreatment of NIH3T3 or T15 cells with either pertussis toxin or cholera toxin led to the complete in situ ADP-ribosylation of the respective G-proteins. Modification of Gi2 by pertussis toxin, however, had no inhibitory effect on the ability of bombesin to stimulate the production of inositol phosphates in any of these cells lines. Treatment of these cells with cholera toxin elicited a potent inhibition of the bombesin-stimulated production of inositol phosphates. This could be mimicked, however, by other agents which increase intracellular cyclic AMP concentrations. Cholera toxin treatment did not produce a significant alteration in the number of bombesin receptors on the cell surface. These results suggest that, in the T15 cell line, enhanced coupling of bombesin receptors to a phospholipase C-mediated hydrolysis of inositol phospholipids is either produced directly by p21N-ras or that overexpression of this gene product leads to the enhanced expression or function of a cholera and pertussis toxin-insensitive G-protein which then mediates the effect.
Milligan
G
G
Department of Biochemistry, University of Glasgow, Scotland, UK.
Davies
S A
SA
Houslay
M D
MD
Wakelam
M J
MJ
D001839:Bombesin / Q000494:pharmacology*; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology*; D005347:Fibroblasts; D019204:GTP-Binding Proteins / Q000378:metabolism; D011905:Genes, ras; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000096:biosynthesis*; D037342:Pertussis Toxin; D013403:Sugar Phosphates / Q000096:biosynthesis*; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D007295:Inositol Phosphates; D013403:Sugar Phosphates; D010566:Virulence Factors, Bordetella; D002772:Cholera Toxin; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D001839:Bombesin