Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase
Introduction
G protein-coupled receptors (GPCRs) are a superfamily of cell-surface receptors which couple to heterotrimeric G proteins and regulate multiple downstream effectors such as adenylyl cyclases, phospholipases, protein kinases and ion channels [1], [2], [3], [4]. All GPCRs share common structural features with a hydrophobic core of seven transmembrane-spanning α-helices, three intracellular loops, three extracellular loops, an N-terminus outside the cell and a C-terminus inside the cell. It has been well documented that the magnitude of receptor-elicited cellular response to a given signal is modulated by elaborately regulated intracellular trafficking which dictates the level of the receptor expression at the plasma membrane and the targeting of the receptor to specific subcellular compartments [5], [6], [7], [8]. The life of GPCRs begins at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. Properly folded receptors are transported from the ER to the ER–Golgi intermediate complex (ERGIC), the Golgi apparatus and the trans-Golgi network (TGN). During their migration, receptors undergo post-translational modifications to attain mature status [5]. Mature receptors then move from the TGN to the plasma membrane, their functional destination. At the plasma membrane GPCRs may undergo internalization upon stimulation by their ligands. The internalization involves phosphorylation of GPCRs by G protein receptor kinases and subsequent binding of the phosphorylated receptors to arrestins, which serve as adaptor proteins recruiting components of the transport machinery to the clathrin-coated pits, initiating formation of the early endosome [9]. The internalized receptors in the early endosome may be sorted to the lysosome for degradation or to the recycling endosome for return to the plasma membrane [10].
Protein export from the ER is mediated through COPII-coated vesicles containing of a small GTPase Sar1 and the heterodimeric Sec23/24 and Sec13/31 complexes [11], [12], [13], [14]. It has been well demonstrated that GDP/GTP exchange and GTP hydrolysis by Sar1 GTPase play a crucial role in the regulation of formation and budding of COPII-coated vesicles on the ER membrane. Assembly of the COPII coat takes place on the ER membrane at discrete locations called ER exit sites and is initiated by the exchange of GDP for GTP on Sar1 GTPase, which is catalyzed by the ER-localized transmembrane guanine nucleotide exchange factor Sec12. GTP activation of Sar1 GTPase then recruits the Sec23/24 complex to the ER membrane forming a functional prebudding complex. The prebudding complex containing GTP-bound Sar1 and Sec23/24 are then clustered by Sec13/31 forming the COPII vesicles. Hydrolysis of GTP to GDP by Sar1 GTPase results in the dissociation of Sar1 GTPase from the prebudding complex and facilitates the release of the COPII vesicles from the ER membrane [14], [15], [16], [17], [18].
ER export of GPCRs represents the first step in intracellular trafficking of the receptors and influences the kinetics of receptor maturation and cell-surface targeting [19], [20]. Interestingly, recent studies have demonstrated that β2-adrenergic receptor (β2-AR) may form signaling complexes with G protein βγ subunits and adenylyl cyclase II in the ER [21], [22]. However, compared with extensive studies on the events involved in the endocytic and recycling pathways, molecular mechanism underlying the ER export and targeting to the cell surface of GPCRs and regulation of receptor signaling by export trafficking are relatively less understood. The progress achieved over the past few years indicates that GPCR export from the ER is a highly regulated process. First, GPCR export from the ER is directed by highly conserved motifs identified exclusively in the membrane-proximal C-termini [23], [24], [25], [26], [27]. Second, GPCR dimerization (homo- and heterodimerization) plays an important role in proper receptor folding and assembly to pass through the ER quality control mechanism, in addition to regulating ligand binding, signal transduction and internalization [28], [29]. Third, GPCR export from the ER is modulated by direct interactions with multiple regulatory proteins such as ER chaperones, accessory proteins and receptor activity modifying proteins, which may stabilize receptor conformation, facilitate receptor maturation and promote receptor delivery to the plasma membrane [8], [19].
To define the mechanism underlying the export from the ER and transport from the ER to the cell surface of GPCRs, we focused our effort on angiotensin II type 1 receptor (AT1R), β2-AR, α1-AR and α2-AR, representative members of the GPCR superfamily, which couple to different G proteins and initiate distinct signaling pathways. We previously determined the role of Rab1, a Ras-like small GTPase that specifically coordinates protein transport from the ER to the Golgi [30], [31], in the transport of these receptors. We demonstrated that the transport from the ER to the cell surface of AT1R, β2-AR and α1-AR is dependent on Rab1, whereas the transport of α2B-AR is independent of Rab1 [32], [33], [34]. We have recently reported that the transport of α2B-AR from the ER to the cell surface is also independent of Rab6 [35], a small GTPase involved in regulation of retrograde protein transport from the Golgi to the ER. These data indicate that the cell-surface targeting of different GPCRs may be mediated through distinct pathways and suggest a novel pathway mediating α2B-AR targeting to the cell surface. We have identified the highly conserved F(X)6LL motif which is required for the export of AT1R and α2B-AR from the ER [26], [27] and the YS motif in the N-termini close to the first transmembrane domain which is essential for α2-AR export from the Golgi [36]. In this manuscript we sought to further define the mechanism underlying the export of GPCRs from the ER by investigating the role of the ER-derived COPII transport vesicles in mediating GPCR export from the ER through manipulating the function of Sar1 GTPase. We demonstrated that expression of the constitutively active GTP-bound mutant Sar1H79G similarly inhibits the cell-surface expression and signaling of α2B-AR, β2-AR and AT1R, but differentially modifies their subcellular localization. These data indicate that efficient GTP hydrolysis by Sar1 GTPase selectively regulates GPCR export from the ER and provide the first evidence indicating different regulatory roles for Sar1-coordinated COPII vesicle assembly on the ER membrane in the export of distinct GPCRs.
Section snippets
Materials
Rat α2B-AR in vector pcDNA3, human β2-AR in vector pBC and rat AT1R in vector pCDM8 were kindly provided by Dr. Stephen M. Lanier, Dr. John D. Hildebrandt (Department of Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC) and Kenneth E. Bernstein (Department of Pathology, Emory University, Atlanta, GA), respectively. AT1R, α2B-AR and β2-AR tagged with green fluorescent protein (GFP) at their C-termini and α2B-AR tagged with the HA epitope at its
Effect of transient expression of Sar1H79G on the cell-surface expression and signaling of AT1R, α2B-AR and β2-AR
Sar1 GTPase, through its abilities to undergo GDP/GTP exchange and hydrolyze GTP to GDP, plays a crucial role in regulating the assembly of the COPII-coated vesicles, which mediate the transport of newly synthesized proteins exclusively from the ER to the ERGIC. The function of Sar1 GTPase was manipulated by transiently expressing the constitutively active GTP-bound mutant Sar1H79G, which has been well characterized to inhibit protein transport from the ER [14], [15], [16], [17], [18]. We first
Discussion
The molecular mechanism underlying the recruitment and package of GPCRs into the ER-derived COPII-coated vesicles, which transport cargo proteins exclusively from the ER to the ERGIC, remains undefined. To address this issue we investigated the function of Sar1 GTPase, which plays a crucial role in the assembly and budding of the COPII-coated vesicles on the ER membrane, in regulating the export from the ER and transport to the cell surface of α2B-AR, β2-AR and AT1R. Loss-of-function of Sar1
Acknowledgments
This work was supported by the National Institutes of Health grant GM076167 (to G. W.) and an American Heart Association, Southeast Affiliate postdoctoral fellowship (to C. D.). We are grateful to Drs. Stephen M. Lanier, William E. Balch, Kenneth E. Bernstein and John D. Hildebrandt for sharing reagents.
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These authors contributed equally to this work.