Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein. |
| |
Authors: | R Plevin S Palmer S D Gardner and M J Wakelam |
| |
Institution: | Department of Biochemistry, University of Glasgow, Scotland, U.K. |
| |
Abstract: | The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5'-gamma-thio]triphosphate (GTPS]) and inhibited by guanosine 5'-beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated 3H]InsP3 generation in 3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTPS] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTPS] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTPS]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTPS] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C. |
| |
Keywords: | |
|
|