Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases

Three Rab GTPases, Rab1, Rab2 and Rab6, are involved in protein transport between the endoplasmic reticulum (ER) and the Golgi. Whereas Rab1 regulates the anterograde ER-to-Golgi transport, Rab2 and Rab6 coordinate the retrograde Golgi-to-ER transport. We have previously demonstrated that Rab1 differentially modulates the export trafficking of distinct G protein-coupled receptors (GPCRs). In this report, we determined the role of Rab2 and Rab6 in the cell-surface expression and signaling of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). Expression of the GTP-bound mutant Rab2Q65L significantly attenuated the cell-surface expression of both alpha(2B)-AR and beta(2)-AR, whereas the GTP-bound mutant Rab6Q72L selectively inhibited the transport of beta(2)-AR, but not alpha(2B)-AR. Similar results were obtained by siRNA-mediated selective knockdown of endogenous Rab2 and Rab6. Consistently, Rab2Q65L and Rab2 siRNA inhibited alpha(2B)-AR and beta(2)-AR signaling measured as ERK1/2 activation and cAMP production, respectively, whereas Rab6Q72L and Rab6 siRNA reduced signaling of beta(2)-AR, but not alpha(2B)-AR. Similar to the beta(2)-AR, AT1R expression at the cell surface and AT1R-promoted inositol phosphate accumulation were inhibited by Rab6Q72L. Furthermore, the nucleotide-free mutant Rab6N126I selectively attenuated the cell-surface expression of beta(2)-AR and AT1R, but not alpha(2B)-AR. These data demonstrate that Rab2 and Rab6 differentially influence anterograde transport and signaling of GPCRs. These data also provide the first evidence indicating that Rab6-coordinated retrograde transport selectively modulates intracellular trafficking and signaling of GPCRs.     

Distinct pathways for the trafficking of angiotensin II and adrenergic receptors from the endoplasmic reticulum to the cell surface: Rab1-independent transport of a G protein-coupled receptor

The molecular mechanism underlying the transport of G protein-coupled receptors from the endoplasmic reticulum (ER) to the cell surface is poorly understood. This issue was addressed by determining the role of Rab1, a Ras-related small GTPase that coordinates vesicular protein transport in the early secretory pathway, in the subcellular distribution and function of the angiotensin II type 1A receptor (AT1R), beta2-adrenergic receptor (AR), and alpha2B-AR in HEK293T cells. Inhibition of endogenous Rab1 function by transient expression of dominant-negative Rab1 mutants or Rab1 small interfering RNA (siRNA) induced a marked perinuclear accumulation and a significant reduction in cell-surface expression of AT1R and beta2-AR. The accumulated receptors were colocalized with calregulin (an ER marker) and GM130 (a Golgi marker), consistent with Rab1 function in regulating protein transport from the ER to the Golgi. In contrast, dominant-negative Rab1 mutants and siRNA had no effect on the subcellular distribution of alpha2B-AR. Similarly, expression of dominant-negative Rab1 mutants and siRNA depletion of Rab1 significantly attenuated AT1R-mediated inositol phosphate accumulation and ERK1/2 activation and beta2-AR-mediated ERK1/2 activation, but not alpha2B-AR-stimulated ERK1/2 activation. These data indicate that Rab1 GTPase selectively regulates intracellular trafficking and signaling of G protein-coupled receptors and suggest a novel, as yet undefined pathway for movement of G protein-coupled receptors from the ER to the cell surface.     

A conserved motif for the transport of G protein-coupled receptors from the endoplasmic reticulum to the cell surface

The structural determinants for the export trafficking of G protein-coupled receptors are poorly defined. In this report, we determined the role of carboxyl termini (CTs) of alpha2B-adrenergic receptor (AR) and angiotensin II type 1A receptor (AT1R) in their transport from the endoplasmic reticulum (ER) to the cell surface. The alpha2B-AR and AT1R mutants lacking the CTs were completely unable to transport to the cell surface and were trapped in the ER. Alanine-scanning mutagenesis revealed that residues Phe436 and Ile433-Leu444 in the CT were required for alpha2B-AR export. Insertion or deletion between Phe436 and Ile443-Leu444 as well as Ile443-Leu444 mutation to FF severely disrupted alpha2B-AR transport, indicating there is a defined spatial requirement, which is essential for their function as a single motif regulating receptor transport from the ER. Furthermore, the carboxyl-terminally truncated as well as Phe436 and Ile443-Leu444 mutants were unable to bind ligand and the alpha2B-AR CT conferred its transport properties to the AT1R mutant without the CT in a Phe436-Ile443-Leu444-dependent manner. These data suggest that the Phe436 and Ile443-Leu444 may be involved in both proper folding and export from the ER of the receptor. Similarly, residues Phe309 and Leu316-Leu317 in the CT were identified as essential for AT1R export. The sequence F(X)6LL (where X can be any residue, and L is leucine or isoleucine) is highly conserved in the membrane-proximal CTs of many G protein-coupled receptors and may function as a common motif mediating receptor transport from the ER to the cell surface.     

Regulation of anterograde transport of alpha2-adrenergic receptors by the N termini at multiple intracellular compartments

The studies on the intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly focused on the C termini of the receptors. In this report we determined the role of the extracellular N termini of alpha(2)-adrenergic receptors (alpha(2)-ARs) in the anterograde transport from the endoplasmic reticulum (ER) through the Golgi to the cell surface. The N-terminal-truncated alpha(2B)-AR mutant is completely unable to target to the cell surface. A single Met-6 residue is essential for the export of alpha(2B)-AR from the ER, likely through modulating correct alpha(2B)-AR folding in the ER. The Tyr-Ser motif, highly conserved in the membrane-proximal N termini of all alpha(2)-AR subtypes, is required for the exit of alpha(2A)-AR and alpha(2B)-AR from the Golgi apparatus, thus representing a novel Tyr-based motif modulating GPCR transport at the Golgi level. These data provide the first evidence indicating an essential role of the N termini of GPCRs in the export from distinct intracellular compartments along the secretory pathway.     

Rab1 GTPase promotes expression of β-adrenergic receptors in rat pulmonary microvascular endothelial cells

It is known that Rab1 regulates the expression and function of beta-adrenoceptors (β-ARs) in many cells. However, the effect of these changes in rat pulmonary microvascular endothelial cells (RPMVECs) is not known. In the present study, we investigated the role of Rab1, a Ras-like GTPase that coordinates protein transport from the endoplasmic reticulum (ER) to the Golgi body and regulates the cell-surface targeting and function of endogenous β-ARs in RPMVECs in the presence of lipopolysaccharide (LPS).We found that lentivirus-driven expression of wild-type Rab1 (Rab1WT) in RPMVECs strongly enhanced the amount of β-ARs on the cell surface, whereas the dominant-negative mutant Rab1N124I significantly attenuated β-ARs expression on the cell surface. In addition, LPS stimulation significantly reduced β-ARs expression on the cell surface in RPMVECs; however, this effect was reversed by over-expression of wild-type Rab1WT. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA (siRNA) significantly induced the accumulation of green fluorescent protein (GFP)-tagged β₂-AR in the ER. Consistent with their effects on β-ARs export, Rab1WT and Rab1N124I differentially modified the β-AR-mediated activation of extracellular signal-regulated kinase1/2 (ERK1/2). Importantly, over-expression of Rab1WT markedly reduced LPS-induced hyper-permeability of RPMVECs by increasing the expression of β₂-AR on the cell surface. These data reveal that β-ARs function in RPMVECs could be modulated by manipulating β-ARs traffic from the ER to the Golgi body. We propose the ER-to-Golgi transport as a regulatory site for control of permeability of RPMVECs.     

A Single Lys Residue on the First Intracellular Loop Modulates the Endoplasmic Reticulum Export and Cell-Surface Expression of α2A-Adrenergic Receptor

Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein-coupled receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue Lys65also modulates the cell-surface transport of α_2A-adrenergic receptor (α_2A-AR). Mutation of Lys65 to Ala, Glu and Gln significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented α_2A-AR expression at the cell surface. Consistent with the effects on the cell-surface expression of α_2A-AR, mutation of Lys65 to Ala and Arg produced opposing effects on α_2A-AR-mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the α_2A-AR mutant K65A displayed a strong intracellular expression pattern and was extensively co-localized with the ER marker DsRed2-ER, suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys residue on the ICL1 in the ER export and cell-surface expression of α_2A-AR. These data also suggest that the ICL1 may possess multiple signals that control the cell-surface targeting of GPCRs via distinct mechanisms.     

Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors

The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.     

Agonist-dependent trafficking of alpha2-adrenoceptor subtypes: dependence on receptor subtype and employed agonist

Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-L?hdesm?ki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.     

A triple arg motif mediates α(2B)-adrenergic receptor interaction with Sec24C/D and export

Recent studies have demonstrated that cargo exit from the endoplasmic reticulum (ER) may be directed by ER export motifs recognized by components of the coat protein II (COPII) vesicles. However, little is known about ER export motifs and vesicle targeting of the G protein-coupled receptor (GPCR) superfamily. Here, we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α(2B)-adrenergic receptor (α(2B)-AR). The 3R motif mediates α(2B)-AR interaction with Sec24C/D and modulates ER exit, cell surface transport and function of α(2B)-AR. Furthermore, export function of the 3R motif is independent of its position within α(2B)-AR and can be conferred to CD8 glycoprotein. These data provide the first evidence implicating that export of GPCRs is controlled by code-directed interactions with selective components of the COPII transport machinery.     

Dominance of the alpha1B-adrenergic receptor and its subcellular localization in human and TRAMP prostate cancer cell lines

The function and distribution of alpha1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the alpha1-AR subtype and suggested that the alpha1A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of alpha1-ARs was determined by saturation binding and the distribution of the different alpha1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major alpha1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the alpha1B-AR. DU145 cells contained 100% of the alpha1B-AR subtype, whereas PC3 cells were composed of 21% alpha1 A-AR and 79% alpha1B-AR. TRAMP cell lines contained between 66% and 79% of the alpha1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing alpha 1B-AR and increasing alpha1 A- and alpha1D-AR densities. Transfection with EGFP-tagged alpha1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of alpha 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that alpha1B-ARs are the major alpha1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α(2B)-adrenergic receptor (α(2B)-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α(2B)-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α(2B)-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α(2B)-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α(2B)-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.     

Regulation of subcellular localization of alpha1-adrenoceptor subtypes

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.     

A Single Conserved Leucine Residue on the First Intracellular Loop Regulates ER Export of G Protein‐Coupled Receptors

The intrinsic structural determinants for export trafficking of G protein‐coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The α_2B‐adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates α_2B‐AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of α_2B‐AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of β₂‐AR, α_1B‐AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.     

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α₂-adrenergic receptors (α₂-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α_2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α_2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α_2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α_2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.     

Regulation of G protein-coupled receptor export trafficking

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.     

Regulation of G protein-coupled receptor export trafficking

G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling.     

alpha 1-Adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1

Three distinct subtypes of alpha(1)-adrenergic receptors (alpha(1)A-, alpha(1)B-, and alpha(1)D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of alpha(1)A- or alpha(1)B-AR inhibits serum-promoted cell proliferation, whereas alpha(1)D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G(1)/S transition after activation of alpha(1)A/alpha(1)B-AR but not of alpha(1)D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR, whereas all alpha(1)-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27(Kip1), was blocked after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR. These results show that alpha(1)-AR subtypes differentially activate the cAMP/p27(Kip1) pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where alpha(1)-AR subtypes are generally co-expressed.     

The regulatory mechanisms of export trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are a superfamily of cell-surface receptors that regulate a variety of cell functions by responding to a myriad of ligands. The magnitude of the response elicited by a ligand is dictated by the level of receptor available at the plasma membrane. GPCR expression levels at the cell surface are a balance of three highly regulated, dynamic intracellular trafficking processes, namely export, internalization and degradation. This review will cover recent advances in understanding the mechanism underlying GPCR export trafficking by focusing on specific motifs required for ER export and the role of the Ras-like Rab1 GTPase and glycosylation in regulating ER–Golgi-cell-surface transport. The manifestation of diseases due to the disruption of GPCR export is also discussed.