GASTRIN-RELEASING PEPTIDE RECEPTOR-INDUCED INTERNALIZATION, DOWN-REGULATION, DESENSITIZATION, AND GROWTH - POSSIBLE ROLE FOR CYCLIC-AMP

Stimulation of the gastrin-releasing peptide receptor (GRP-R) in Swiss 3T3 cells resembles that of a number of other recently described G pr otein-coupled receptors, insofar as both the phospholipase C and adeny lyl cyclase signal transduction pathways are activated. GRP-R activati on induces numerous alterations in both the cell and the receptor, but because two signal transduction pathways are activated it is difficul t to determine the specific contributions of either pathway. We have f ound that BALB/3T3 fibroblasts transfected with the coding sequence fo r the GRP-R are pharmacologically indistinguishable from native recept or-expressing cells and activate phospholipase C in a manner similar t o that of the native receptor but fail to increase cAMP in response to bombesin; thus, they may be useful cells to explore the role of activ ation of each pathway in altering cell and receptor function. Swiss 3T 3 cells and GRP-R-transfected BALB/3T3 cells expressed identically gly cosylated receptors that bound various agonists and antagonists simila rly. G protein activation, as determined by evaluation of agonist-indu ced activation of phospholipase C and by analysis of the effect of gua nosine-5'(beta,gamma-imido)triphosphate on GRP-R binding affinity, was indistinguishable. Agonist stimulation of GRP-R caused similar recept or changes (internalization and down-regulation) and homologous desens itization in both cell types. Bombesin stimulation of Swiss 3T3 cells that had been preincubated with forskolin increased cAMP levels 9-fold , but no bombesin-specific increase in cAMP levels was detected in tra nsfected cells, even though forskolin and cholera toxin increased cAMP levels in these cells. Quiescent Swiss 3T3 cells treated with bombesi n rapidly increased c-fos mRNA levels and [H-3]thymidine incorporation , whereas both effects were potentiated by forskolin. The specific pro tein kinase A inhibitor H-89 blocked increases in c-fos levels and [H- 3]thymidine incorporation induced by low concentrations of bombesin. G RP-R-transfected BALB/3T3 cells increased c-fos mRNA levels and [H-3]t hymidine incorporation with the addition of serum but not bombesin. Th ese data suggest that bombesin-stimulated increases in cellular levels of cAMP appear not to be an important mediator of GRP-R internalizati on, down-regulation, or desensitization but do play an important role in bombesin-induced mitogenesis.