Analysis of the AU-Rich Elements in the 3‘-Untranslated Region of β2-Adrenergic Receptor mRNA by Mutagenesis and Identification of the Homologous AU-Rich Region from Different Species

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Abstract
The 35000-Mr β-adrenergic receptor mRNA binding protein (βARB) is induced by β-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3‘-untranslated region of the hamster β2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for βARB. U to G substitution in the hexamer region attenuates the binding of βARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of βARB and stabilizes β2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for βARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of βARB correlates with the extent of destabilization of β2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to βARB was revealed by SDS−polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, βARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for β2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3‘-untranslated regions of receptor mRNAs.

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