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J. Biol. Chem., Vol. 280, Issue 3, 1931-1943, January 21, 2005

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Translational Control of ₂-Adrenergic Receptor mRNA by T-cell-restricted Intracellular Antigen-related Protein^*

Karthikeyan Kandasamy, Kusumam Joseph, Kothandharaman Subramaniam, John R. Raymond, and Baby G. Tholanikunnel¶

From the Department of Medicine and Division of Nephrology and Division of Pulmonary, Critical Care Medicine, Allergy and Clinical Immunology, Medical University of South Carolina, Charleston, South Carolina 29425-2221

Cellular expression of the ₂-adrenergic receptor (₂-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of ₂-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of ₂-AR in cell lines that endogenously express this receptor. Reversal of ₂-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of ₂-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on ₂-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed ₂-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of ₂-AR mRNA by TIAR.

Received for publication, May 27, 2004 , and in revised form, October 29, 2004.

^* This work was supported by United States Public Health Service Grant RO1 GM 58740 (to B. G. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Medicine/Division of Nephrology, Medical University of South Carolina, Charleston, SC 29425. Tel.: 843-789-6773; Fax: 843-876-5129; E-mail: tholanik{at}musc.edu.

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