Di-acidic Motifs in the Membrane-distal C Termini Modulate the Transport of Angiotensin II Receptors from the Endoplasmic Reticulum to the Cell Surface

Xiaoping Zhang,Chunmin Dong,Qiong J Wu,William E Balch,Guangyu Wu

doi:10.1074/jbc.m111.222034

Abstract

The molecular mechanisms underlying the endoplasmic reticulum (ER) export and cell surface transport of nascent G protein-coupled receptors (GPCRs) have just begun to be revealed and previous studies have shown that hydrophobic motifs in the putative amphipathic 8(th) α-helical region within the membrane-proximal C termini play an important role. In this study, we demonstrate that di-acidic motifs in the membrane-distal, nonstructural C-terminal portions are required for the exit from the ER and transport to the plasma membrane of angiotensin II receptors, but not adrenergic receptors. More interestingly, distinct di-acidic motifs dictate optimal export trafficking of different angiotensin II receptors and export ability of each acidic residue in the di-acidic motifs cannot be fully substituted by other acidic residue. Moreover, the function of the di-acidic motifs is likely mediated through facilitating the recruitment of the receptors onto the ER-derived COPII transport vesicles. Therefore, the di-acidic motifs located in the membrane-distal C termini may represent the first linear motifs which recruit selective GPCRs onto the COPII vesicles to control their export from the ER.

Highlights

We found that two di-acidic motifs, ExD and ExE, in the membrane-distal C-terminal regions of rat and human Ang II type 2 receptors (AT2R), respectively, play an obligatory role in their forward trafficking toward the cell surface and the function of the di-acidic motif in AT2R export is likely mediated through facilitating the recruitment of the cargo receptors onto the COPII vesicles
AT2R Transport to the Cell Surface Depends on Its Membrane-distal C Terminus—A number of studies have demonstrated that the C termini play an important role in the cell surface transport of G protein-coupled receptors (GPCRs)
Consistent with the cell surface expression of AT2R measured by flow cytometry, fluorescence microcopy analysis of the subcellular distribution of AT2R showed that, similar to the C-terminal truncation mutants, the ExD mutant tagged with green fluorescent protein (GFP) or HA was expressed in the perinuclear region unable to transport to the cell surface (Fig. 2B)

Summary

Introduction

We found that two di-acidic motifs, ExD and ExE, in the membrane-distal C-terminal regions of rat and human AT2R, respectively, play an obligatory role in their forward trafficking toward the cell surface and the function of the di-acidic motif in AT2R export is likely mediated through facilitating the recruitment of the cargo receptors onto the COPII vesicles. To determine the effect of treatment with PD123319, a specific AT2R antagonist, on the cell surface expression of wild-type AT2R and its mutant ExD-AxA, HEK293 cells were transfected with AT2R for 6 h and incubated with PD123319 at a concentration of 10 ␮M for 30 –36 h.

Results

Conclusion