Translational Control of β2-Adrenergic Receptor mRNA by T-cell-restricted Intracellular Antigen-related Protein

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

doi:10.1074/jbc.m405937200

Karthikeyan Kandasamy, Kusumam Joseph + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m405937200

Copy DOI

Abstract

Cellular expression of the beta(2)-adrenergic receptor (beta(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 beta(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 beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(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 beta(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 beta(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 beta(2)-AR mRNA by TIAR.

Highlights

␤2-Adrenergic receptors (␤2-AR)1 are low abundance membrane-integrated G-protein-coupled receptors that are activated by catecholamines at the cell surface. ␤2-AR mRNA is transcribed from a single intronless gene [1,2,3], and both transcriptional [4] and post-transcriptional mechanisms [5,6,7,8,9,10] are known to regulate receptor expression
Expression of 3Ј-untranslated region (UTR) Sequences Leads to Reversal of ␤2Adrenergic Receptor Translational Repression in DDT1-MF2 and A431 Cells—Our previous work demonstrated that 3Ј-UTR sequences of the ␤2-AR mRNA suppress the translation of receptor mRNA [7]
We reported that 3Ј-UTR sequences of ␤2-AR mRNA participate in the post-transcriptional regulation of receptor mRNA, including translational suppression and agonist-mediated mRNA destabilization [7, 11, 13]

Summary

Introduction

␤2-Adrenergic receptors (␤2-AR)1 are low abundance membrane-integrated G-protein-coupled receptors that are activated by catecholamines at the cell surface. ␤2-AR mRNA is transcribed from a single intronless gene [1,2,3], and both transcriptional [4] and post-transcriptional mechanisms [5,6,7,8,9,10] are known to regulate receptor expression. In vitro transcribed radiolabeled receptor 3ЈUTR transcripts were incubated and UV-cross-linked with cytosolic extracts from DDT1-MF2 cells and subjected to IP with TIAR- and HuR-specific antibodies.

Results

Conclusion