MicroRNA let-7 establishes expression of β 2 -adrenergic receptors and dynamically down-regulates agonist-promoted down-regulation

• Published in: Proceedings of the National Academy of Sciences - PNAS 2011-04, Vol.108 (15), p.6246-6251
• Main Authors: Wang, Wayne C. H., Juan, Aster H., Panebra, Alfredo, Liggett, Stephen B.
• Format: Article
• Language: English
• Subjects: Adrenergic receptors; Argonaute 2 protein; Feedback; G protein-coupled receptors; Gene expression; Lung; miRNA; nucleic acids; Seeds; Tachyphylaxis; Transfection; Translation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1101439108

Although β 2 -adrenergic receptors (β 2 AR) are expressed on most cell types, mechanisms that establish expression levels and regulate expression by chronic agonist remain unclear. The 3′ UTR of ADRB2 has a conserved 8-nucleotide seed region that we hypothesized is targeted by the let-7 family of miRNAs leading to translational repression. In luciferase assays with transfected cells, luc-β 2 WT3′UTR had decreased expression when cotransfected with let-7f , but a mutated luc-β 2 3′UTR lacking the seed was unaffected by let-7f ; a mutated let-7f also had no effect on luc-β 2 WT3′UTR expression. ADRB2 mRNA was in greater abundance in immunoprecipitates of Ago2, a core component of the miRNA-induced silencing complex, when cells were transfected with let-7f , but not with a mutated let-7f , indicating a direct interaction with the silencing mechanism. H292 cells transfected with let-7f caused ∼60% decrease in native β 2 AR expression, but transfection with let-7f –specific locked nucleic acid anti-miRNA increased β 2 AR expression by ∼twofold. We considered that an increase in let-7f leading to greater repression of translation contributes to agonist-promoted down-regulation. Paradoxically, in cells and in lungs from mice treated in vivo, an ∼50% decrease in let-7f occurs during long-term agonist exposure, indicating a counterregulatory event. Consistent with this notion, let-7f locked nucleic acid transfection caused depressed agonist-promoted down-regulation. Thus, let-7f miRNA regulates baseline β 2 AR expression and decreases in let-7f evoked by agonist attenuate down-regulation. This positive feedback loop has not previously been described for a G protein-coupled receptor and its miRNA. Methods to decrease let-7f expression in targeted cells may increase therapeutic responses to β-agonist by increasing β 2 AR expression or minimizing tachyphylaxis.