Abstract
beta(2)-Adrenergic receptors (beta(2)-ARs) are low abundance integral membrane proteins that mediate the effects of catecholamines at the cell surface. Post-transcriptional regulation of beta(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 beta(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 beta(2)-AR expression by 3'-UTR sequences. Presumably, this occurs by affecting the receptor mRNA localization.
Highlights
2-Adrenergic receptor (2-AR)1 mRNA, which is transcribed from a single intronless gene, makes low abundance membrane
Because the cDNA used is of hamster origin, the results reported here are those obtained from stable transfections into Chinese hamster ovary (CHO) cells. 2-AR expression was quantitated by a radioligand binding assay using (Ϫ)-125I-cyanopindolol performed in triplicate
This study provides evidence in support of a role for 3Ј-untranslated regions (UTRs) sequences of 2-AR mRNA in translational suppression
Summary
2-Adrenergic receptor (2-AR)1 mRNA, which is transcribed from a single intronless gene, makes low abundance membrane-. To distinguish between these two possibilities, we measured the steady-state levels of receptor mRNA in CHO cells expressing the wild-type receptor and different deletion constructs by RNase protection assay.
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