The M_r 35,000 β-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition

Delineating the molecular basis for agonist-induced destabilization of mRNA of G-protein-linked receptors that contributes to receptor down-regulation is fundamental to our understanding of long-term regulation of receptors by agonist. Previously we identified a prominent, M_r 35,000 cytosolic RNA-binding protein that (i) binds selectively to β₁- and β₂-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind either to α^1b-adrenergic receptor mRNA, which does not undergo agonist-induced down-regulation, or to β-globin mRNA; (iii) displays binding to β₂-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and (iv) its abundance varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA (Port, J. D., Huang, L.-y., and Malbon (1992) J. Biol. Chem. 267, 24103-24108). We demonstrate here that the binding of β-adrenergic receptor mRNA by this protein, termed β-ARB protein, is sensitive to competition by AU-rich domains of the 3′-untranslated regions of c-fos, c-myc, and human granulocyte-macrophage colony-stimulating factor. Using the AU-rich 3′-untranslated regions of wild-type adenovirus IVa2 mRNA and variants with defined mutations in the AUUUA pentamer, AU-rich, and U-rich domains, we were able to define sequences critical to the binding of the β₂-receptor mRNA by the β-ARB protein. Recognition of β-ARB protein requires not only an AUUUA destabilization pentamer, but also a flanking U-rich domain(s). Using radiolabeled 3′-untranslated regions of short-lived mRNA, we were able to identify this same M_r 35,000 cytosolic RNA-binding protein(s), β-ARB protein, as selective for β₂-adrenergic receptor mRNA.