Characterization of the rat vasoactive intestinal polypeptide receptor gene 5' region.
The broad spectrum of vasoactive intestinal polypeptide (VIP) cellular functions are mediated by high-affinity binding sites. To determine regulation of the VIP receptor gene expression, we have isolated and characterized two genomic clones that contain the first three exons and the 5' flanking region of the VIP receptor gene. Using RNase protection assays, receptor gene expression was detected in adult rat lung, liver and intestine, but not in fetal lung, indicating that VIP receptor is expressed in diverse tissues, and its expression is differentially regulated during lung development. The transcription start site of the gene was mapped to a cytosine residue, 76 bp upstream from the ATG initiation codon. Transfection into rat lung cell lines shows that although 126 bp of the VIP receptor 5' DNA sequences are capable of activating VIP receptor gene basal transcription 30-fold over a promoterless control, 488 bp of the 5' sequences further induce this activation to 97-fold over control. However, inclusion of up to 859 bp 5' sequences results in a decrease in basal promoter activity (31-fold over control), indicating that while sequences between -126 and -488 bp contain potential enhancer sequences, sequences between -488 and -859 bp may include a transcriptional repressor sequence. Deletion analysis shows that transcription factor Sp1 plays an important role in activating basal promoter of the VIP receptor gene. DNase I footprinting and gel-mobility-shift assays show that Sp1 binds to its consensus binding sites in the VIP receptor promoter, suggesting that interaction of Sp1 with VIP receptor promoter transactivates this gene.
Pei
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L
Division of Endocrinology, Cedars-Sinai Medical Center-UCLA School of Medicine 90048, USA.
Melmed
S
S
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017930:Genes, Reporter; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics; D012260:Ribonucleases / Q000378:metabolism; D017421:Sequence Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
D004268:DNA-Binding Proteins; D018005:Receptors, Vasoactive Intestinal Peptide; D014157:Transcription Factors; D012260:Ribonucleases; D003850:Deoxyribonuclease I