GQ IN BOMBESIN RECEPTOR SIGNALLING AND HUMAN TUMORS

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Reconstitution studies using purified proteins in lipid vesicles indicate that the Gq sub-family of alpha subunits, in contrast to the Gs, Gi, and Go subfamilies, activate phospholipase C-beta 1 (Berstein et al., J. Biol. Chem. 267: 8081-8088, 1992) after ligand receptor interaction. Based on these observations, we postulate that Gq, or structurally similar G11, is important for bombesin receptor signal transduction. We plan to test this hypothesis by observing the effects of Gq-specific antisense molecules on bombesin-mediated responses in two different biological contexts, Xenopus oocytes and Swiss 3T3 fibroblasts. We have isolated and sequenced cDNA clones for mouse and Xenopus Gq and G11. The Xenopus proteins are over 90% identical in amino acid sequence to their mammalian counterparts. These clones will be used to design the synthesis of antisense oligonucleotides which will be injected into Xenopus oocytes expressing either GRP-R or NMB-R to observe their effects on bombesin signalling. The mouse cDNAs will be used to generate stably transfected Swiss 3T3 fibroblasts, where expression of antisense Gq or G11 RNA is directed by a dexamethasone-inducible MMTV promoter. Antisera specific for Gq/G11 will be used to determine if protein levels are diminished by antisense induction, and the effects on GRP-R signal transduction correlated with protein levels. Mutations in G-alpha subunits have been implicated in the transformation of human pituitary and thyroid tumors. Given the importance of phospholipase C activation in cell growth, we postulate that analagous mutations altering the function of Gq may also be important in other solid tumors. We have identified a structurally modified Gq protein by Western blot analysis in small cell lung carcinoma H82. We plan to characterize this abnormal Gq by molecular cloning, and investigate the functional consequences of Gq mutations found in H82, as well as Gq mutations found in other lung cancer cells.