Cloning and characterization of rabbit Rgs4 promoter in gut smooth muscle

Regulator of G-protein signaling 4 (Rgs4) regulates the strength and duration of G-protein signaling, and plays an important role in cardiac development, smooth muscle contraction and psychiatric disorders. Rgs4 expression is regulated at both mRNA and protein levels. In order to examine the transcriptional mechanism of Rgs4 expression, we have cloned and characterized rabbit Rgs4 promoter. The coding sequence of rabbit Rgs4 was obtained by degenerative RT-PCR and used for Northern blot and 5'-RACE analysis. A single transcript was identified in rabbit colonic smooth muscle cells. The 5'-untranslated region (UTR) extended 120 bp nucleotides upstream of the Rgs4 start codon. A putative promoter sequence (1389 bp) showed a consensus TATA box and cis-acting binding sites for several potential transcriptional factors. Reporter gene assay identified strong promoter activity in various cell types. Further analysis by deletion mutagenesis suggested that the proximal region had a highest core promoter activity while the distal region is suppressive. IL-1beta significantly increased the promoter activity. The in vitro and in vivo binding activities for NF-kappaB transcription factor were validated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay respectively. Mutation of NF-kappaB site reduced the promoter activity. These data suggest that the cloned rabbit Rgs4 promoter is functionally active and NF-kappaB binding site possesses enhancer activity in regulating Rgs4 transcription. Our studies provide an important basis for further understanding of Rgs4 regulation and function in different diseases.