Molecular cloning of a novel transcriptional repressor protein of the rat type 1 vasoactive intestinal peptide receptor gene.
This study demonstrates that the transcriptional repressor sequence of the rat vasoactive intestinal peptide receptor (VIPR) gene constitutes a 42-base pair core element that is the binding site for a nuclear protein. We showed that this element was able to confer transcriptional repression to a heterologous promoter and that deletion or point mutations within this element resulted in loss of transcriptional repression. Southwestern blot analysis indicated that the VIPR repressor element interacts specifically with a nuclear protein of about 72 kDa. By screening a rat lung expression library coupled with rapid amplification of cDNA ends polymerase chain reactions, we isolated a cDNA clone (designated as VIPR-RP) that contains an open reading frame of 656 amino acids. VIPR-RP is 78% identical to a previously characterized protein, differentiation-specific element-binding protein, which is a member of a family of proteins including components of the DNA replication factor C complex. However, VIPR-RP cDNA encodes for a much smaller protein than differentiation-specific element-binding protein because of a frameshift. VIPR-RP mRNA is expressed in multiple tissues, including lung, liver, brain, heart, kidney, spleen, and testis. VIPR-RP protein specifically interacts with the VIPR repressor element as demonstrated by gel shift assays. Transfection of VIP-RP expression vector into Cos cells resulted in transcriptional repression of a reporter construct containing multiple copies of the VIPR repressor element. These results indicate that VIPR-RP is a novel transcriptional repressor protein that regulates VIPR expression.
Division of Endocrinology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000032:analysis; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000235:genetics*; D051818:Replication Protein C; D012097:Repressor Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018005:Receptors, Vasoactive Intestinal Peptide; D012097:Repressor Proteins; C113967:Rfc1 protein, rat; D051818:Replication Protein C