A mammalian equivalent of the amphibian peptide bombesin, designated gastrin-releasing peptide (GRP), has potent biological effects, including hyperglycemia, hypothermia, and release of several gut peptide hormones, consistent with a neuroregulatory role. GRP also stimulates cell proliferation, suggesting a role in growth regulation. Bombesin-like immunoreactivity has been found in mammalian brain, gut, neuroendocrine tumors, and neuroendocrine cells of fetal lung, implicating a role in lung development. Recently, several mRNAs encoding precursors of human GRP(s) (pre-proGRPs) have been isolated from a human pulmonary carcinoid tumor. Sequence studies have shown the mRNAs differ by a 19 base insertion/deletion corresponding to the carboxy-terminal protein-coding sequences, which thus apparently represents a frameshift post-transcriptional splicing event. It is the specific aim of this proposal to characterize rat gene(s) and mRNA(s) encoding GRP using human cDNA and/or synthetic oligonucleotide probes. After decoding the gene and mRNA sequence(s), rat GRP(s) will be synthesized, including different forms of rat proGRP if they exist consistent with human proGRPs. Rabbit antisera will be raised to rat and human GRP(s) and to carboxy terminal regions of proGRP. The development of whole rat embryos will then be studied by 4 methods: (1) Solution and/or solid-phase mRNA hybridization analyses; (2) In situ DNA hybridization of P32 or H-3 labeled probes to embryo sections; (3) Immunohistochemistry for GRP and related peptides on corresponding serial sections; and (4) Radio-receptor localization of I-125 labeled GRP in similar sections. By preliminary RNA blot analyses, at least two major forms of mRNA appear to exist in human fetal lung, in contrast to a single major type in adult lung. Immunoperoxidase analyses indicate that GRP is present in human fetal lung as well as in diseased adult airways. Preliminary in situ hybridization of GRP-encoding oligonucleotide probes to fetal lung suggest the presence of mRNA for GRP(s) in neuroendocrine-like cells. It is postulated that GRP gene(s) may be transiently expressed in developing tissues as part of programmed gene regulation in cellular differentiation. Further, carboxy-terminal peptides resulting from the two reading frames of proGRP may have a biological function. Thus, the long term objective is to determine the role of GRP in embryogenesis by analyses of GRP gene expression in developing tissues. This approach will facilitate subsequent investigation into mechanisms of gene deregulation in carcinogenesis.
