Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

Kusumam Joseph,Baby G. Tholanikunnel,Daniel J. Fernandes,Aleksander Baldys,Karthikeyan Kandasamy,Louis M. Luttrell,Paul J. McDermott,John R. Raymond

doi:10.1074/jbc.m110.168229

Kusumam Joseph, Baby G. Tholanikunnel + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m110.168229

Copy DOI

Abstract

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Highlights

On the contrary, translation-independent mRNA localization usually requires the recognition of the transcript by RNA-binding proteins within the nucleus, which renders the mRNA-protein complex translationally inactive when the complex reaches the cytoplasm [9]
ShRNA-mediated Knockdown of HuR Reduces Steady State Levels of ␤2-AR mRNA While Increasing Receptor Expression— Earlier studies have identified the 3Ј-untranslated region (UTR) of ␤2-AR mRNA as a target for HuR protein [17, 23]
Our earlier studies demonstrated that the 3Ј-UTR sequence and their binding proteins regulate the translational control of ␤2-AR mRNA [16, 17]

Summary

Introduction

Translation-independent mRNA localization usually requires the recognition of the transcript by RNA-binding proteins within the nucleus, which renders the mRNA-protein complex translationally inactive when the complex reaches the cytoplasm [9]. Additional evidence for HuR association with ␤2-AR mRNA was obtained by UV-cross-linking of cytosolic lysates from DDT1-MF2 cells with in vitro transcribed 32P-labeled receptor 3Ј-UTR RNA, followed by IP of the resulting complexes in the presence or absence of specific anti-HuR or control IgG1. On the contrary, when cytoplasmic extracts of HuR knockdown DDT1-MF2 cells were used for polysome analysis, a clear shift in receptor mRNA to heavier polyribosomal fractions was observed (Fig. 4B, upper and lower panels).

Results

Conclusion