Cell-surface targeting of α2-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

doi:10.1016/j.cellsig.2005.05.014

Cellular Signalling

Volume 18, Issue 3, March 2006, Pages 318-327

https://doi.org/10.1016/j.cellsig.2005.05.014 Get rights and content

Abstract

We previously demonstrated that the α_2B-adrenergic receptor mutant, in which the F(x)₆IL motif in the membrane-proximal carboxyl terminus were mutated to alanines (α_2B-ARm), is deficient in export from the endoplasmic reticulum (ER). In this report, we determined if α_2B-ARm could modulate transport from the ER to the cell surface and signaling of its wild-type counterpart. Transient expression of α_2B-ARm in HEK293T cells markedly inhibited cell-surface expression of wild-type α_2B-AR, as measured by radioligand binding. Subcellular localization demonstrated that α_2B-ARm trapped α_2B-AR in the ER. The α_2B-AR was shown to form homodimers and heterodimers with α_2B-ARm as measured by co-immunoprecipitation of the receptors tagged with green fluorescent protein and hemagglutinin epitopes. In addition to α_2B-AR, the transport of α_2A-AR and α_2C-AR to the cell surface was also inhibited by α_2B-ARm. Furthermore, transient expression of α_2B-ARm significantly reduced cell-surface expression of endogenous α₂-AR in NG108-15 and HT29 cells. Consistent with its effect on α₂-AR cell-surface expression, α_2B-ARm attenuated α_2A-AR- and α_2B-AR-mediated ERK1/2 activation. These data demonstrated that the ER-retained mutant α_2B-ARm conferred a dominant negative effect on the cell-surface expression of wild-type α₂-AR, which is likely mediated through heterodimerization. These data indicate a crucial role of ER export in the regulation of cell-surface targeting and signaling of G protein-coupled receptors.

Introduction

Cell-surface receptors coupled to heterotrimeric G proteins represent a superfamily of membrane proteins that respond to a vast array of sensory and chemical stimuli [1], [2]. G protein-coupled receptors are synthesized in the endoplasmic reticulum (ER)², transported to the Golgi apparatus for posttranslational modification (e.g. glycosylation), and then moved on to their functional destinations at the plasma membrane. Therefore, ER export represents the first step in intracellular trafficking of G protein-coupled receptors. There are several events associated with the ER that critically regulate G protein-coupled receptor export. First, similar to many other membrane proteins, receptors must be correctly folded in order to pass the ER quality control mechanism [3], [4]. Second, export from the ER and further transport to the cell surface of some G protein-coupled receptors requires specific accessory proteins or chaperones [5], [6], [7], [8]. Interaction of the receptors with chaperones may regulate correct folding/assembly of the receptors within the ER and facilitates receptor transport from the ER and on to the cell surface. Third, dimerization of the receptors (homo-and/or hetero-dimerization) in the ER may be required for ER export and further cell-surface targeting. Recently, Bouvier's group has demonstrated that mutation of the putative dimerization motif GxxxGxxxL in the β₂-adrenergic receptor (AR) prevents normal trafficking of the receptor to the plasma membrane [9]. Although dimerization has been demonstrated for a variety of G protein-coupled receptors [10], [11], [12], [13], [14], [15], whether α₂-AR is able to homodimerize and the role of dimerization in the trafficking of α₂-AR remain unknown. Fourth, receptor exit from the ER may be directed by specific signals or motifs embedded in the receptors. Recent studies have identified two classes of ER-export signals in the cytoplasmic carboxyl termini of a variety of membrane proteins. The diacidic motif (DxE) was found in the cytoplasmic carboxyl termini of vesicular stomatitis viral glycoprotein, cystic fibrosis transmembrane conductance regulator and potassium channels [16], [17], [18], [19]. The double phenylalanine motif (FF) is required for efficient ER-to-Golgi transport of the p24 family proteins and ER-Golgi intermediate compartment-53 [20]. These ER-export motifs mediate the interaction of transported proteins with the COP II vesicles, directing the export of proteins from the ER. In regards to G protein-coupled receptors, a triple phenylalanine sequence (FxxxFxxxF) seems to act as an ER-exit motif for the dopamine D1 receptor [21].

We demonstrated that a phenylalanine and double leucine spaced by 6 residues [F(x)₆LL] in the membrane-proximal carboxyl termini of α_2B-AR and angiotensin II type 1 receptor (AT1R) are required for exit from the ER [22]. Mutation of the F(x)₆LL motif to alanines abolishes export of the receptors out of the ER and further transport to the cell surface. We report here that, similar to many other G protein-coupled receptors, α_2B-AR apparently homodimerizes in the ER, and that the α_2B-AR mutant deficient in ER export confers an inhibitory effect on the cell-surface targeting and function of its wild-type (WT) counterpart through heterodimerization in the ER. These data indicate a crucial role of ER export in the regulation of cell-surface targeting and signaling of G protein-coupled receptors.

Section snippets

Materials

Rat α_2A-, α_2B- and α_2C-AR in vector pcDNA3 were kindly provided by Dr. Stephen M. Lanier (Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA) and rat AT1R in vector pCDM8 by Dr. Kenneth E. Bernstein (Department of Pathology, Emory University, Atlanta, GA). Antibodies against phospho-ERK1/2, green fluorescent protein (GFP), HA (conjugated with rhodamine) and calregulin were purchased from Santa Cruz Biotechnology, Inc.

Effect of α_2B-ARm on the cell-surface expression of α_2B-AR

We have recently demonstrated that α_2B-ARm, in which Phe⁴³⁶Ile⁴⁴³Leu⁴⁴⁴ in α_2B-AR were mutated alanines is unable to export from the ER [22]. To determine whether α_2B-ARm could function as a dominant negative mutant for the transport of its WT counterpart from the ER to the cell surface, we first determined the effect of α_2B-ARm on the transport of α_2B-AR to the cell surface by radioligand binding of membrane preparations and intact cells. The membrane fractions prepared from cells expressing α

Discussion

The molecular mechanisms underlying the transport of G protein-coupled receptors from the ER to the cell surface and its role in regulation of receptor signaling remain poorly understood. To address these issues, we previously determined the role of Rab1, a Ras-like GTPase that regulates vesicle-mediated protein transport in the early secretory pathway, specifically from the ER to the Golgi, in the export trafficking of α_2B-AR, β₂-AR and AT1R [23], [24]. We have demonstrated that, whereas the

Acknowledgements

This work was supported in part by the National Institutes of Health Award “Mentoring in Cardiovascular Biology” 1P20RR018766 (Program Director: Stephen M. Lanier, Ph.D.-Louisiana State University Health Sciences Center) and by the Louisiana Board of Regents Grant LEQSF (2002-05)-RD-A-18 (to G. W.). Mathew T. Duvernay is a recipient of Louisiana Board Regents Graduate Fellowship. We acknowledge Stephen M. Lanier (Department of Pharmacology and Experimental Therapeutics) for review of this work

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¹: These authors contributed equally to this work.

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Cell-surface targeting of α2-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

Abstract

Introduction

Section snippets

Materials

Effect of α2B-ARm on the cell-surface expression of α2B-AR

Discussion

Acknowledgements

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J. Biol. Chem.

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Cell-surface targeting of α₂-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

Effect of α_2B-ARm on the cell-surface expression of α_2B-AR