MECHANISMS IN THE TRANSCRIPTIONAL REGULATION OF BRADYKININ B-1 RECEPTOR GENE-EXPRESSION - IDENTIFICATION OF A MINIMUM CELL-TYPE-SPECIFIC ENHANCER

To investigate the mechanisms of bradykinin B-1 (BRB1) receptor gene e xpression, transient DNA transfection analyses of human BKB1 receptor gene promoter were performed in SV-40 transformed IMR90 cells. A posit ive regulatory element (PRE) located at position -604 to -448 base pai r (bp) upstream of the transcription start site consistently exhibited , by far, the highest level of relative luciferase activity. A negativ e regulatory element, at position -682 to -604 bp, was able to complet ely ablate the function of the PRE. Transfection combined with deletio n and mutation analyses illustrated that the PRE contains a classic, p owerful enhancer. This enhancer was minimized to a 100-bp element at p osition -548 to -448 bp, A 78-bp fragment of negative regulatory eleme nt functioned as a silencer. Transient transfection of the enhancer co nstruct, driven by heterologous herpes simplex thymidine kinase promot er, into a variety of cell types, showed that this enhancer presents a cell-type specific feature. In the characterization of the enhancer, motifs A (-548 to -532) and B (-483 to -477) were found to be essentia l for full enhancer activity. Motif D (-472 to -467) played a smaller role in enhancer activation. Gel shift and antibody supershift assays determined that an AP-1 factor binds with motif B. The nuclear protein which binds to motif A has yet to be identified. Both factors are the critical regulators for this enhancer activation.