A 20-Nucleotide (A + U)-rich Element of β2-Adrenergic Receptor (β2AR) mRNA Mediates Binding to β2AR-binding Protein and Is Obligate for Agonist-induced Destabilization of Receptor mRNA

Baby G Tholanikunnel,Craig C Malbon

doi:10.1074/jbc.272.17.11471

Abstract

The Mr 35,000 beta-adrenergic receptor mRNA-binding protein, termed betaARB protein, is induced by beta-adrenergic agonists and binds to beta2-receptor mRNAs that display agonist-induced destabilization. A cognate sequence in the mRNA was identified previously that provides for betaARB protein binding in vitro. In the present work, the sequence established in vitro for binding of betaARB protein to hamster beta2-adrenergic receptor mRNA was probed in vivo by site-directed mutagenesis of the 3'-untranslated region and expression in Chinese hamster ovary cells. A 20-nucleotide, (A + U)-rich region in the 3'-untranslated region consisting of an AUUUUA hexamer flanked by defined U-rich regions constitutes the binding domain for betaARB protein. U to G substitution in the hexamer region attenuates the binding of betaARB protein, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of betaARB protein and stabilizes beta2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. These results demonstrate that binding of betaARB protein to the 20-nucleotide, (A + U)-rich domain mediates the agonist and cyclic AMP-induced mRNA decay of G protein-linked receptors, such as the beta2-adrenergic receptor.

Highlights

The steady-state levels of highly regulated mRNAs are markedly influenced by the rate of degradation (13–20)
Both pentamer and hexamer core AREs bind the ␤ARB protein (34), a 20nucleotide, (A ϩ U)-rich sequence consisting of an AUUUUA hexamer flanked by U-rich regions is shown to be obligate for binding of ␤ARB protein and regulation of ␤2-adrenergic receptor mRNA stability in vivo
␤ARB Protein Binding to Full-length Wild-type and Mutant Hamster ␤2AR mRNA—Previous studies using ␤2-adrenergic receptor mRNA and 3Ј-UTRs of highly regulated mRNAs and RNA variants with specific mutations in the AU-rich region demonstrated the importance of AUUUA pentamers flanked by poly(U) regions in the binding of ␤ARB protein (8). 3Ј-UTR of ␤2AR mRNA contains one AUUUA pentamer, which is not flanked by poly(U) regions, and another AUUUUA hexamer, which is flanked immediately on either side by poly(U) regions

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—DDT1MF2 vas deferens smooth muscle cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5%, heat-inactivated fetal bovine serum (HyClone), penicillin (60 ␮g/ml), and streptomycin (100 ␮g/ml) as described by Scarpace et al (35). Plasmid vector pSP70, into which wild-type and various mutants of ␤2AR cDNA were inserted, was used for in vitro transcription after linearization with a restriction enzyme that cleaves the plasmid immediately 3Ј to the receptor cDNA insert. UV Cross-linking and Label Transfer—An aliquot of radiolabeled mRNA (1– 4 ϫ 106 cpm), 5 ␮g of yeast tRNA, and competing unlabeled RNA transcripts (at the molar excess over probe indicated) were each added to a mixture containing the S100 cytosolic fraction (30 –100 ␮g total protein), 4 mM dithiothreitol, 5 ␮g of heparin, and 65 units of RNasin in a total volume of 50 ␮l. Total RNA was extracted from individual culture dishes at the indicated time, and the amount of receptor mRNA was established by use of an RNase protection assay performed as described previously (34). Antisense riboprobes corresponding to 600 (740 –1338) and 285 (1201–1486) nucleotides from the coding region of ␤2AR mRNA were employed for the RNase protection assay

RESULTS

DISCUSSION

Findings

Destabilization of Receptor mRNA