Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species.
The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-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 beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-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 beta ARB 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 beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB 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 beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.
Tholanikunnel
B G
BG
Department of Medicine (Division of Nephrology), Medical University of South Carolina, Charleston 29425-2221, USA. tholanik@musc.edu
Raymond
J R
JR
Malbon
C C
CC
D020413:3' Untranslated Regions / Q000378:metabolism*; D000225:Adenine / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D008647:Mesocricetus; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011063:Poly A-U / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D014498:Uracil / Q000378:metabolism
D020413:3' Untranslated Regions; D002352:Carrier Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018343:Receptors, Adrenergic, beta-2; C076141:adenosine-uridine binding factor; D011063:Poly A-U; D014498:Uracil; D000225:Adenine