Molecular characterization of the VIP receptor transcriptional repressor protein.
The rat type 1 VIP receptor transcriptional repressor protein (VIPR-RP) is a recently isolated novel transcription factor. In the study reported here, the functional domains of VIPR-RP were characterized. To map the DNA binding domain, various regions of VIPR-RP were either transcribed and translated in vitro or expressed in and purified from E. Coli as a glutathione S-transferase (GST) fusion. The ability of the truncated proteins to bind to VIPR-RP specific binding sequence was tested by gel mobility shift assays. The results indicated that the amino acid sequences between 367 and 475 play an essential role for VIPR-RP DNA binding. To determine the amino acid sequences required for transcriptional repression, fusion proteins containing the GAL4 DNA binding domain and various parts of VIPR-RP were constructed, and their ability to repress transcription of the reporter gene containing GAL4 DNA binding sequences were tested in transiently transfected COS7 cells. The results showed that VIPR-RP contains two separate transcriptional repression domains located between amino acids 50 to 101 and 470 to 527.
Pei
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Division of Endocrinology and Metabolism, Cedars-Sinai Research Institute-UCLA School of Medicine, Los Angeles, California 90048, USA. pei@cshs.org
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D066298:In Vitro Techniques; D017434:Protein Structure, Tertiary; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000235:genetics*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C113967:Rfc1 protein, rat; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D051818:Replication Protein C