GASTRIN-RELEASING PEPTIDE GENE EXPRESSION

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A mammalian equivalent of the amphibian peptide bombesin, designated gastrin-releasing peptide (GRP), has potent biological effects, including release of several gut peptide hormones, consistent with a neuroregulatory role. GRP also induces cell proliferation, suggesting a role in growth regulation. GRP has been found in mammalian brain, gut, fetal lung, and neuroendocrine (NE) tumors. Three mRNAs for human GRP(s) have been isolated from human pulmonary NE tumors, all of which encode GRP but differ in the C-terminal extension peptide of proGRP, due to a 19 or 21 base insertion/deletion splicing event. The major objective of this project is to determine the sequence and developmental expression of the rat gene encoding GRP. This will allow analyses of tissue-specific GRP gene expression in rat embryogenesis, which should clarify GRP's role in normal mammalian growth and development, as well as in neoplasia. Initial studies of human fetal lung development have demonstrated peak immunoreactive GRP in NE cells at 18 to 24 weeks of gestation, which follows peak mRNA levels at 16 to 22 weeks and correlates with the canalicular period of lung growth. My specific aims are: (1) to complete sequencing of the rat GRP gene, to clarify the exact structure of possible RNA splicing variants. (2) To analyze patterns of tissue-specific gene levels in normal rat embryogenesis: in whole tissue homogenates by RNA blotting and S1 mapping, and in embryo tissue sections by in situ hybridization to precisely localize cell-specific GRP mRNA. Antisera to rat GRP and proGRP(s) will be raised and used for immunoperoxidase studies to be correlated with in situ hybridization on serial embryo sections. It will be important to determine whether C-terminal peptides are also expressed, as these may have biological functions. GRP-immunoelectron microscopy and GRP receptor autoradiography will be similarly carried out to further clarify GRP's physiological role. (3) to determine GRP's involvement in pathological processes by in situ hybridization and immunohistochemical analyses for both GRP and C-terminal peptides. The major focus will be on lung tumors, where GRP has been implicated as a potential atuocrine growth factor. Thus, GRP gene(s) may be transiently expressed in developing tissues as part of programmed gene regulation in cellular differentiation, and analyses of this expression may elucidate mechanisms of gene deregulation in oncogenesis.