REGULATION OF THE PREPRO GRP GENE

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The mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), are important regulatory neuropeptides which mediate a range of biologic responses including smooth muscle contraction, stimulation of secretion, modulation of neuronal activity, and control of cell proliferation. Previous studies from this laboratory have shown that: 1) in tumor cell lines, the prepro-GRP gene is regulated at the level of primary transcription; and 2) detectable expression is restricted to a subset of neurons in the central and peripheral nervous system, neuroendocrine cells, and derivative tumor cell lines. We plan to define the molecular mechanisms important in prepro-GRP transcriptional activation in expressing cells, and suppression in nonexpressing cells. The first step in this analysis is the identification of cis-acting prepro-GRP promoter elements important for transcription. A series of fusion genes were assembled to assay the transcriptional effects of sequence elements in the prepro-GRP promoter. In these constructs, the firefly luciferase reporter gene is placed under transcriptional control of varying lengths of the human prepro-GRP promoter region. After transfection and subsequent transient expression in various host cell lines, luciferase activity is determined for each construct containing different prepro-GRP promoter elements. Using this assay system, we have made the following observations: 1) Promoter sequences between -5000 and -1600 (the origin is defined as the initiation site for transcription) exert an approximately two-fold negative effect on transcription, while sequences between -1600 and -400 exert a weak positive effect. In this positive domain, a several hundred base long 5' upstream sequence is very highly conserved between the rat and human prepro-GRP gene (-720 to -450); and 2) The constructs behave the same in host cells that do or do not normally express the prepro-GRP gene. We conclude that promoter elements between -5000 and + 100 alone do not determine cell-type specificity of transcription regulation.