The 3′-Untranslated Region of the β2-Adrenergic Receptor mRNA Regulates Receptor Synthesis

Abstract

␤ 2 -Adrenergic receptors (␤ 2 -ARs) are low abundance integral membrane proteins that mediate the effects of catecholamines at the cell surface. Post-transcriptional regulation of ␤ 2 -AR is dependent, in part, on sequences within the 5-and 3-untranslated regions (UTRs) of the receptor mRNA. In this work, we demonstrate that 3-UTR sequences regulate the translation of the receptor mRNA. Deletion of the 3-UTR sequences resulted in 2-2.5-fold increases in receptor expression. The steadystate levels of ␤ 2 -AR mRNA did not change significantly in the presence or absence of the 3-UTR, suggesting that the translation of the receptor mRNA is suppressed by 3-UTR sequences. Introduction of the receptor 3-UTR sequences into the 3-UTR of a heterologous reporter gene (luciferase) resulted in a 70% decrease in reporter gene expression without significant changes in luciferase mRNA levels. Sucrose density gradient fractionation of cytoplasmic extracts from Chinese hamster ovary cells transfected with full-length receptor cDNA demonstrated that the receptor transcripts were distributed between polysomal and non-polysomal fractions. Deletion of 3-UTR sequences from the receptor cDNA resulted in a clear shift in the distribution of receptor mRNA toward the polysomal fractions, favoring increased translation. The 3-UTR sequences of the receptor mRNA were sufficient to shift the distribution of luciferase mRNA from predominantly polysomal fractions toward non-polysomal fractions in cells transfected with the chimeric luciferase construct. Taken together, our results provide the first evidence for translational control of ␤ 2 -AR expression by 3-UTR sequences. Presumably, this occurs by affecting the receptor mRNA localization.

Complex haplotypes derived from noncoding polymorphisms of the intronless α 2A -adrenergic gene diversify receptor expression

“…For the M 1 -muscarinic receptor, an Ϸ260-bp region in the 3Ј UTR, thought to form a stable stem-loop, is critical for mRNA stability (32). For the ␤ 2 AR subtype, 3Ј UTR sequences have been shown to negatively regulate translation of the receptor (33)(34)(35). T cellrestricted intracellular antigen-1 is one RNA binding protein that binds to ␤ 2 AR 3Ј UTR and acts to repress expression at the translational level (35).…”

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

“…In contrast, when these cells were transfected with GFP NLS / myr without ␤ 2 -AR 3Ј-UTR, the GFP distribution was localized to the perinuclear region of the cells suggesting an important role for the receptor 3Ј-UTR sequences in limiting the distribution of GFP to the cell periphery. In addition, as expected from the inhibitory effect of ␤ 2 -AR 3Ј-UTR on translation (16,17), the fusion of ␤ 2 -AR 3Ј-UTR to GFP caused a reduction in GFP expression. We isolated heavy (plasma membrane) and light membrane fractions from HuR knockdown and control cells and measured the distribution of ␤ 2 -AR by radioligand binding in these fractions.…”

“…We recently reported that cellular expression of ␤ 2 -AR is suppressed at the translational level by 3Ј-untranslated region (UTR)-binding proteins (16,17). The current study was designed to address the importance of translational silencing and mRNA localization in ␤ 2 -AR trafficking to the plasma membrane.…”

“…Because the deletion of 3Ј-UTR from ␤ 2 -AR cDNA results in increased receptor expression (16), CHO cells also were co-transfected separately with 5 g each of ␤ 2 -AR cDNA, with and without 3Ј-UTR sequences to serve as proper controls. The control cells were co-transfected with empty vector in place of HuR cDNA.…”

“…Cytoplasmic extracts of cells expressing control shRNA and HuR-specific shRNA were used. Sucrose density gradient fractionation and RNA extractions were performed as described previously (16,17). Monosomes and polysomes were well separated as identified by absorbance at 254 nm (Fig.…”