Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein

R Plevin, S Palmer, S D Gardner, M J Wakelam

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 (GTP[S]) 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 GTP[S] 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 GTP[S] 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 GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] 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.

Original languageEnglish
Pages (from-to)605-610
Number of pages6
JournalBiochemical journal
Volume268
Issue number3
Publication statusPublished - 15 Jun 1990

Keywords

  • animals
  • bombesin
  • cell membrane permeability
  • cells, cultured
  • drug synergism
  • fibroblasts
  • GTP-binding proteins
  • guanosine 5'-O-(3-Thiotriphosphate)
  • guanosine triphosphate
  • inositol 1,4,5-trisphosphate
  • mice
  • stimulation, chemical
  • tetradecanoylphorbol acetate
  • thionucleotides

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