Charlesworth, Amanda; (1996) Signalling pathways mediated by the bombesin/GRP receptor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neuropeptides, and in particular peptides of the bombesin family, including gastrin releasing peptide, stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as cellular growth factors in a variety of fundamental biological processes including development, tissue regeneration and tumourigenesis. Bombesin elicits multiple signalling pathways in various cell types, including activation of phospholipase C, which leads to activation of protein kinase C and elevation of intracellular calcium; tyrosine phosphorylation of intracellular substrates and reorganisation of the actin cytoskeleton, and cell proliferation. It is not clear however whether these responses are mediated by a single receptor subtype or by different subtypes that couple preferentially to specific pathways. To resolve this the mouse bombesin/GRP receptor was transfected into Rat-1 fibroblasts and the pathways activated by bombesin were investigated. Here it is shown that phospholipase C activation, cell growth and tyrosine phosphorylation emanate from a single class of bombesin receptor. Mitogen Activated Protein Kinase (MAPK) activity is a common point of convergence for mitogenic signals. While the mechanism by which receptor tyrosine kinases activate MAPK is well established, the mechanism by which G protein-coupled receptors activate MAPK is less well understood. The involvement of Ras and Rai in activation of MAPK has not been described in bombesin-induced signalling. This thesis shows that in Rat-1 cells transfected with the bombesin/GRP receptor, bombesin can activate MAPK and Raf-1 in a protein kinase C-independent fashion. Also it is demonstrated for the first time the bombesin stimulates GTP loading of p2lras. Calcium mobilisation from intracellular calcium stores has been observed to be coincident with cell proliferation induced by a variety of growth factors including neuropeptides and especially bombesin. However, a direct role of calcium in this event has not been described. Here it is shown that partial and persistent depletion of intracellular calcium stores, using specific inhibitors of the endoplasmic reticulum calcium pump, thapsigargin and 2,5-di-tert-butylhydroquinone, stimulated reinitiation of DNA synthesis in synergy with either phorbol-12,13-dibutyrate or bombesin in Swiss 3T3 cells. This stimulation of DNA synthesis was dependent on entry of extracellular calcium and hence this suggests a role for calcium in growth control.

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