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3'-Untranslated region of the type 2 bradykinin receptor is a potent regulator of gene expression

Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee

Submitted 12 January 2005 ; accepted in final form 31 August 2005

Regulation of the constitutively expressed type 2 bradykinin (B₂) receptor, which mediates the principal actions of bradykinin, occurs at multiple levels. The goal of the current study was to determine whether the human B₂ 3'-untranslated region (UTR) has effects on gene expression, with particular focus on the variable number of tandem repeats (B₂-VNTR) polymorphic portion of the 3'-UTR and its flanking AU-rich elements (AREs). When inserted downstream of the luciferase coding region of the pGL3-Promoter vector, the B₂-VNTR reduced reporter gene activity by 85% compared with pGL3-Promoter alone (promoter control; P < 0.001), an effect that was not appreciably affected by mutation of the flanking AREs. The negative regulatory effects of the B₂-VNTR region were position and orientation dependent and strongly positively correlated with the number of tandem repeats in the B₂-VNTR region (r = 0.85, P < 0.001). With respect to mechanism, quantitative RT-PCR revealed that the B₂-VNTR mRNA level was 32% of that of promoter control (P = 0.008), whereas the number of polyadenylated transcripts was 4% (P = 0.02). In contrast, the mRNA half-life of the B₂-VNTR was increased (B₂-VNTR: 14.9 vs. promoter control: 12.2 h, P = 0.009). Transient transfection of human kidney-derived tsA201 cells with the B₂-VNTR construct increased transcription of the native B₂ receptor mRNA by 43% (P < 0.05), supporting an endogenous B₂ receptor-regulatory capacity of the B₂-VNTR. In conclusion, these results identify novel pretranslational effects of the B₂-VNTR region to act as a potent negative regulator of heterologous gene expression and support the notion that the bradykinin B₂ 3'-UTR may impact endogenous receptor regulation.

gene regulation; variable number of tandem repeats; polymorphism