Ian Zachary

Inhibition of bombesin-induced mitogenesis by pertussis toxin: dissociation from phospholipase C pathway

Prior incubation of quiescent cultures of Swiss 3T3 cells with pertussis toxin selectively inhibited the stimulation of DNA synthesis induced by peptides of the bombesin family. While pertussis toxin blocked mitogenesis at an early stage in the action of the peptide, the toxin did not impair the rapid stimulation of polyphosphoinositide breakdown, Ca2+ mobilization or activation of protein kinase C promoted by bombesin. Thus, inhibition of bombesin-induced mitogenesis by pertussis toxin can be dissociated from inactivation of the phospholipase C signalling pathway.

Increased mitogenic responsiveness of Swiss 3T3 cells expressing constitutively active Gsα

Mutational replacement of glutamine-227 with a leucine residue in the GTP-binding domain of the alpha subunit of GS (Q227L alpha S) reduces its ability to hydrolyse GTP and causes constitutive activation of the mutant protein. Expression in Swiss 3T3 fibroblasts of Q227L alpha S caused markedly increased basal adenylyl cyclase activity, enhanced intracellular cyclic AMP (cAMP) accumulation and increased mitogenic sensitivity in response to forskolin and the potent phosphodiesterase inhibitor Ro 20-1724. These results support a role for cAMP in the regulation of cell proliferation, and suggest that alterations in a G protein can directly modify the ability of cells to respond mitogenically to extracellular factors.

A substance P antagonist also inhibits specific binding and mitogenic effects of vasopressin and bombesin-related peptides in Swiss 3T3 cells

While vasopressin and peptides of the bombesin family bind to different receptors in quiescent Swiss 3T3 cells, the antagonist [D-Arg1,D-pro2,D-Trp7,9,Leu11] substance P blocks the specific binding of both (3H) vasopressin and 125I-gastrin-releasing peptide to these cells. In addition, the antagonist inhibits the mobilization of Ca2+ and induction of DNA synthesis by vasopressin. These results indicate that [D-Arg1,D-Pro2,D-Trp7,9,Leu11] substance P has the ability to interact with the receptors for three structurally unrelated peptide hormones.

Rapid dephosphorylation of a Mr 80000 protein, a specific substrate of protein kinase C upon removal of phorbol esters, bombesin and vasopressin

The mitogens phorbol 12,13-dibutyrate, bombesin and vasopressin stimulate the phosphorylation of an acidic Mr 80,000 cellular protein, a specific substrate of protein kinase C, in intact Swiss 3T3 cells. Phosphorylation of this substrate was rapidly reversed upon the removal of each of these agents. Dephosphorylation occurred with a similar half-life in each of the cases studied (2.2, 1.5 and 2 minutes for phorbol 12,13-dibutyrate, bombesin and vasopressin respectively) and agreed closely with the dissociation of the ligands from their specific high-affinity binding sites in Swiss 3T3 cells.

Characterization of the Bombesin/Gastrin-Releasing Peptide Receptor in Swiss 3T3 Fibroblasts

Publisher Summary The discovery that bombesin and other neuropeptides are potent cellular growth factors not only has important physiological and pathological implications but also has provided a valuable approach for elucidating the molecular mechanisms leading to cell proliferation. In particular, it is now evident that proliferative responses can be initiated through G protein signal transduction pathways. The identification of the G protein(s) coupled to the bombesin/GRP receptor remains a major challenge. Indeed, while a growing number of receptors for neuropeptides have been cloned, the specificities of the G protein–receptor relationship are only beginning to be clarified. This chapter describes some of the techniques that are most relevant to achieve this aim. Direct evidence for a functional association of the bombesin receptor with a G protein has come from biochemical studies using permeabilized cells, membrane preparations, and 125 I-GRP-receptor complexes solubilized from cell membranes. These studies demonstrate that bombesin acts through signal transduction pathways, in which effector stimulation is tightly coupled to the activation of a G protein.