The bombesin-like peptides are a large family of peptides initially characterized in frog skin, but later found to have wide distribution and potent physiologic effects in mammals. The mammalian homologue of bombesin is gastrin-releasing peptide (GRP). GRP is widely distributed in the CNS and GI tract; but of greatest clinical importance, high levels of GRP are expressed by most small cell lung carcinomas (SCLC). Bombesin has been shown to be a growth factor for normal and neoplastic pulmonary cells and blockade of the bombesin receptor expressed by SCLC cell lines with either antibodies or antagonists causes inhibition of tumor growth. Thus characterization of the bombesin receptor expressed by SCLC will provide information on the growth of that tumor and may be of potential diagnostic importance. Receptors for bombesin-like peptides are also expressed throughout the GI tract and CNS, and in those tissues, GRP plays important roles as a neurotransmitter, paracrine regulator and growth factor. In GI tract and CNS there is a second mammalian bombesin-like peptide, neuromedin B (NMB) which will bind to the same receptor as GRP, but appears to also have its own distinct receptor. Thus understanding the role of bombesin- like peptides in those tissues will require characterization of both the GRP and the NMB receptor. Our objective in this proposal is first to characterize, through molecular cloning, the receptors for mammalian bombesin-like peptides and then to analyze receptor expression in normal and neoplastic tissues. Specifically we propose: (1), To first clone the mouse GRP receptor from Swiss 3T3 cells to take advantage of the high number of receptors expressed by those cells. (2), To use the 3T3 bombesin receptor cDNA to identify the human GRP cDNA in order to determine the structure of the human GRP receptor. (3), To use the human GRP receptor cDNA to identify the human NMB receptor cDNA in order to determine the structure of the human NMB receptor. (4), To characterize tissue specific expression of the GRP and NMB receptors in normal and neoplastic tissue and cell lines. Of primary importance will be to determine if either the structure or regulation of the GRP receptor is altered in SCLC. (5), To express GRP and NMB receptor constructs in Xenopus oocytes and SCLC cell lines. Binding of GRP, NMB and antagonists will be determined and the effects of receptor expression on DNA replication and intracellular calcium measured. Structure- function analysis will be performed to identify the ligand bindin and signal transduction domains. The approach to be used to clone the 3T3 bombesin receptor will be by expression in Xenopus oocytes. As described in the body of the grant, substantial progress towards that goal has been made. cDNA's encoding the human GRP and NMB receptors will be identified by hybridization and their identities will be confirmed by expression in Xenopus oocytes.
