MOLECULAR-STRUCTURE AND FUNCTION OF RAT PLATELET-DERIVED GROWTH-FACTOR BETA-RECEPTOR GENE PROMOTER

Objective To understand the regulatory mechanism of platelet-derived g rowth factor beta-receptor gene expression. Methods A 1.7 kb genomic f ragment was obtained from a rat genomic library. After we had determin ed 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-dir ected 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 f or the CCAAT motif at -67. Effects of the upstream sequence spanning - 310 through -120 on heterologous gene promoters were also investigated . Results Multiple transcription start sites were observed in the 5'-f lanking region, and the 1.7 kb sequence was actually active as a funct ional promoter in vascular smooth muscle cells. Two important sequence s responsible for the basal transcriptonal activity were identified by the functional promoter assay. One was the CCAAT motif at -67 which a cts as a promoter itself, and the other was the upstream region spanni ng -310 through -210 which positively regulates the basal promoter act ivity. Conclusion The basal promoter activity of the rat platelet-deri ved growth factor beta-receptor gene is mainly regulated by the intera ction or coordination of two sequences, the CCAAT motif and the upstre am control element. (C) 1998 Lippincott Raven Publishers.