Molecular structure and function of rat platelet-derived growth factor β-receptor gene promoter

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ObjectiveTo understand the regulatory mechanism of platelet-derived growth factor β-receptor gene expression.MethodsA 1.7 kb genomic fragment was obtained from a rat genomic library. After we had determined an entire sequence of this fragment, transcription start sites were determined both by primer extension analysis and by riboprobe mapping. We performed a functional promoter assay by using a dual-luciferase reporter system. Progressive 5′-deletions of the fragment and site-directed mutagenesis for the CCAAT motif located at −67 or −94 were used for the assay, and their promoter activities in vascular smooth muscle cells were assessed. Gel-mobility shift analysis was also performed for the CCAAT motif at −67. Effects of the upstream sequence spanning −310 through −120 on heterologous gene promoters were also investigated.ResultsMultiple transcription start sites were observed in the 5′-flanking region, and the 1.7 kb sequence was actually active as a functional promoter in vascular smooth muscle cells. Two important sequences responsible for the basal transcriptonal activity were identified by the functional promoter assay. One was the CCAAT motif at −67 which acts as a promoter itself, and the other was the upstream region spanning −310 through −210 which positively regulates the basal promoter activity.ConclusionThe basal promoter activity of the rat platelet-derived growth factor β-receptor gene is mainly regulated by the interaction or coordination of two sequences, the CCAAT motif and the upstream control element.

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