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.