Bombesin receptor regulation of signal transduction pathways and the control of cell growth

BOMBESIN RECEPTOR REGULATION OF SIGNAL TRANSDUCTION PATHWAYS AND THE CONTROL OF CELL GROWTH. Gary L. Johnson, Ph.D. National Jewish Center for Immunology and Respiratory Medicine, Denver. CO 80206. The family of heterotrimeric GTP-binding proteins, referred to as G proteins, function by coupling to cell surface receptors to the control of intracellular signal transduction pathways. G protein-coupled receptors have a characteristic seven transmembrane structure (STM). The extracellular and membrane domain of STM receptors vary in sequence to allow selective binding of different ligands including photons, ions, odorants, small molecules like acetylcholine and catecholamines, peptides and proteases. The STM receptors differentially couple to members of the G protein family. The known G proteins can be. categorized into four families based on sequence and functional homologies. Known effecters for G proteins include adenylyl cyclases, phosphatidylinositol phospholipase Cp, cGMP-phosphodiesterase and specific ion channels. The list of G protein effecters is certain to grow and may include specific tyrosine kinases, phosphatases and the Na+/H+ antiporter. A number of diseases have now been found to involve specific STM receptors and G proteins. In the thyroid and pituitary gain of function mutations in receptors and G proteins result in hyperfunctioning adenomas. STM receptors for bombesin, arginine vasopressin, gastrin, bradykinin, neurotensin and other neuropeptides are involved in the growth stimulation of small cell lung cancers and non-small cell lung cancers which have neuroendocrine characteristics. The bombesin receptor is coupled predominantly to the pertussin toxin-insensitive G proteins, G, and G11. Gq,ll activation catalyzed by bombesin binding to its receptor stimulates phospholipase Cp activity, giving an increase in inositol trisphosphate generation and mobilization of intracellular calcium. In Swiss 3T3 cells, bombesin receptor stimulation also activates p42 and p44 mitogen-activated protein kinase (MAPK) and also generates a strong phospholipase A2 stimulation and arachidonic acid release. MAPK is a threonine/serine protein kinase that regulates a number of cell surface receptors, cytosolic enzymes and nuclear transcription factors. The signal transduction cascade leading to MAPK activation involves several proteins. MAPK is activated, itself, by phosphorylation on both a tyrosine and threonine. This reaction is catalyzed by a specific threonine/tyrosine directed kinase referred to as MEK. MEK in turn is activated by its phosphorylation by the serine/threonine protein kinases Raf- 1, BRaf and MEK kinase (MEKK). Activation of Raf kinases and MEKKs can occur by Rasdependent and -independent mechanisms, and protein kinase C also appears able to regulate Raf1 and MEKK activation. In contrast to growth factor tyrosine kinase receptor stimulation of Ras GTP loading and Raf activation that correlates with MAPK activation, bombesin stimulation of the MAPK pathway did not significantly increase Ras GTP loading. Similarly, bombesin stimulation of Raf-1 is small and significantly different from basal unstimulated levels of activity. Bombesin does significantly activate MEK and MAPK. Incubation of Swiss 3T3 cells with the peptide H2N-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-T~-Leu-Leu-NH2, first described as an inhibitor of small cell lung carcinoma growth, uncouples bombesin stimulation of PLCP activity and calcium mobilization. Strikingly, incubation of cells with this peptide antagonist caused a significant increase in MAPK activation in response to GRP but had no effect on PDGFstimulated activity. The peptide alone had no measurable effect on Ras GTP loading or Raf activity, but in the presence of bombesin caused a modest but significant increase in Raf activation. The influence of the peptide antagonist on Ras GTP loading was small and not statistically different from control basal levels. It should be remembered, however, that the Ras GTP loading assay is not catalytic and is less sensitive than the Raf in vitro kinase assay. The increase in Raf activity in the presence of peptide antagonist was not pertussin toxin-sensitive, indicating that Gl/Gc proteins were not involved in the altered response. The action of the peptide antagonist is to influence the coupling of the bombesin receptor to Gg,ll. The peptide antagonist inhibits the ability of the bombesin receptor to stimulate phosphohpase CD activity, but not the ability of constitutively activated, GTPase-inhibited G, to stimulate phospholipase Cp

activity. Finally, the peptide antagonist inhibits the bombesin-stimulated growth of Swiss 3T3 cells but has no effect on PDGF-stimulated cell growth. Expression of the bombesin receptor in 293 cells also results in bom~sin-stimulated phospholipase Cp and MAPK activation. This result demonstrates that a single bombesin receptor-type is responsible for activating both responses. The peptide antagonist also inhibits phospholipase Cp stimulation but not MAPK activation by the bombesin receptor in 293 cells. Cumulatively, the results demonstrate that the bombesin receptor is coupled to Gq,ll but also coupled to additional signaling proteins that are most likely G proteins. Candidate G proteins are Glz, G13 and GZ, all of which are pertussis toxin-insensitive. The peptide antagonist uncoupled G *$. 11 activation by the bom~sin receptor and enhanced RaflMAPK activation. This finding m Icates that uncoupling of G 11 from the bombesin receptor amplifies the responsiveness of the second pathwAy leading to %AP K activation. The findings also demonstrate that bombesinstimulated cell growth required phospholipase Cp stimulation, and that activation of the MAPK pathway independent of phosphlolipase CD activation was insufficient to stimulate DNA synthesis. Referen= Clapham, D-E. (1994) Cell 75, 1237- 1239. Winitz, S., Russell, M., Qian, N.-X., Gardner, A., Dwyer, L. and Johnson, G.L. (1993) J. Biol. Chem. 268,19196-19199. Battey, J.F., Way, J.M., Cojay, M-H., Shapira, H., Kusano, K., Harkins, R., Wu, J.M., Slattery, T., Mann, E. and Feldman, R.I. (1991) Proc. Natl. Acad. Sci. USA 88,395-399.