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

Abstract

The M r 35,000 ␤-adrenergic receptor mRNA-binding protein, termed ␤ARB protein, is induced by ␤-adrenergic agonists and binds to ␤ 2 -receptor mRNAs that display agonist-induced destabilization. A cognate sequence in the mRNA was identified previously that provides for ␤ARB protein binding in vitro. In the present work, the sequence established in vitro for binding of ␤ARB protein to hamster ␤ 2 -adrenergic receptor mRNA was probed in vivo by site-directed mutagenesis of the 3-untranslated region and expression in Chinese hamster ovary cells. Agonist-induced down-regulation of G protein-linked receptors, such as the ␤ 2 -adrenergic receptor, provides an explanation for long term adaptation to chronic stimuli characteristically observed for members of this receptor family (1-5). For ␤ 2 -adrenergic receptors, steady-state levels of the receptor and its mRNA decline following a challenge with agonist (2, 3). The basis for the decline in receptor mRNA induced by agonist is not transcriptional suppression, but rather post-transcriptional destabilization of receptor mRNAs (6). Recently, we identified a M r 35,000 protein with properties consistent with those expected for an RNA-binding protein selective for mRNAs of receptors that display agonist-induced down-regulation of their messages and protein expression. This M r 35,000 ␤-adrenergic receptor mRNA-binding protein, termed ␤ARB 1 protein (7), demonstrates several RNA binding properties (8) similar to (A ϩ U)-rich element (ARE)-binding proteins reported by others (9 -12).The steady-state levels of highly regulated mRNAs (e.g. mRNAs of granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-␣, and the oncogenes c-myc and c-fos) are markedly influenced by the rate of degradation (13-20). Regulation of mRNA stability and turnover is multifaceted, reflecting not only various cytosolic and nuclear-associated factors and polyadenylation, but also cognate sequences of the 3Ј-untranslated regions (UTR) of mRNA, such as the tandem repeats of AUUUA pentamers (9 -12, 21-23) and nonamers, such as UUAUUUA(U/A)(U/A) (24) and UUAUUUAUU (25).Several classes of RNA-binding proteins have been implicated in regulating mRNA stability and turnover. The heterogeneous, nuclear ribonucleoprotein particles participate in several steps of mRNA maturation including packaging, translocation, and splicing of heterogenous nuclear RNA (26 -28). Splicing and further processing of pre-mRNAs possessing introns, a 5Ј-cap and 3Ј-poly(A) ϩ tract involves the small nuclear RNA-binding proteins (29,30). Cytosolic mRNA-binding proteins include the M r 72,000 poly(A)-binding protein, which binds to long stretches (ϳ25 nucleotides/protein) of poly(A) ϩ and stabilizes the RNA to 3Ј-to 5Ј-nuclease activity (31). A subset of smaller (ranging from M r 30,000 to 40,000), cytosolic mRNA-binding proteins have been identified that display recognition of AU-rich domains in the 3Ј-UTR (7, 9 -12, 32, 33). Although several of these proteins have been purified (32,33), the precise role that these small...

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

“…We previously identified a 20-nucleotide (A ϩ U)-rich region present in the proximal region of the ␤ 2 -AR mRNA 3Ј-UTR that is important in agonist-mediated receptor mRNA destabilization (7,9). In the present work, we demonstrate a major role for 3Ј-UTR sequences in translational control of ␤ 2 -AR mRNA.…”

“…Total RNA extracted was dissolved in RNase-free water and quantified, and the integrity of the RNA was tested directly by agarose gel electrophoresis. The amount of ␤ 2 -AR and luciferase mRNAs was determined by RNase protection assay as described (7,9). An antisense riboprobe corresponding to 285 nucleotides (positions 1201-1485) from the coding region of ␤ 2 -AR mRNA was employed in the RNase protection assay.…”

“…This speculation has been shown to be correct by demonstrating translational suppression of receptor mRNA by a small open reading frame termed the 5Ј-leader cistron (5Ј-LC), which is present in the 5Ј-UTR of all mammalian ␤ 2 -AR mRNAs (5,6). The 3Ј-UTR sequence of this receptor is important in regulating agonist-mediated mRNA destabilization (7)(8)(9).…”

Translational Regulation of Prostaglandin Endoperoxide H Synthase-1 mRNA in Megakaryocytic MEG-01 Cells

“…Similar to previously reported mRNA cis element, there is only one copy of the 20-nt cis element within the PGHS-1 message. A literature search has allowed us to identify other known 20-nt cis elements in the 3Ј-UTRs of c-fos (16), ␤ 2 -adrenergic receptor (17), peptidylglycine ␣-amidating monooxygenase (18), and ribonucleotide reductase R2 (19) that mediate translational regulation. These 20-nt sequences have no homology to the PGHS-1 3Ј-UTR conserved sequence but, like PGHS-1 conserved sequence, all contain stretches of uridylate residues important for the binding activity.…”

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray,Dasyatis sabina

“…Differences in 3ЈUTR length influence subcellular localization and mRNA storage (30,39), which, in turn, affect translational efficiency. Furthermore, the binding of cytosolic regulatory proteins to the 3ЈUTR alters the stability and turnover of mRNA (32,40,41). In elasmobranchs, as well as mammals, differences in 3ЈUTR length are associated with functional diversity.…”