|Home | About | Journals | Submit | Contact Us | Français|
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 α2B-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 α2B-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 α2B-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, α1B-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.
G protein-coupled receptors (GPCRs) constitute a superfamily of integral membrane proteins that regulate the cellular responses to a broad spectrum of extracellular signals, such as hormones, neurotransmitters, chemokines, proteinases, odorants, light and calcium ion (1,2). GPCRs translate extracelluar stimulation by their respective ligand through coupling to a specific catalogue of heterotrimeric G proteins, which in turn, regulate a specific set of downstream effectors such as adenylyl cyclases, phospholipases, protein kinases and ion channels (1–3). The proper function of GPCRs relies on the dynamic and highly regulated intracellular trafficking of the receptors. The life of GPCRs begins at the endoplasmic reticulum (ER), where they are synthesized. Properly folded receptors are recruited from the ER and packaged into transport vesicles that are targeted to the ER–Golgi intermediate complex (ERGIC). Receptors then move from the ERGIC through the Golgi apparatus and trans-Golgi network (TGN), undergoing various post-translational modifications (e.g. glycosylation), before being delivered to the plasma membrane (4). At the plasma membrane, GPCRs are susceptible to stimulation by their ligands, which induces an endocytosis event. The internalized receptors are either recycled back to the plasma membrane or targeted to the lysosomal compartment for degradation (5–10). The delicate balance of these trafficking forces dictates the level of receptor available at the plasma membrane and influences the magnitude of the cellular response to a given signal. Compared with the extensive efforts dedicated to understanding the events involved in the endocytic and recycling pathway (5–10), the molecular mechanism underlying the export trafficking of GPCRs from the ER through the Golgi to the plasma membrane and the regulation of receptor function by these processes are relatively less well defined (11).
As the initial step in post-translational receptor biogenesis, the efficiency of ER export of nascent GPCRs plays a crucial role in the regulation of receptor maturation and signaling at the plasma membrane. Indeed, ER export has been shown to be a rate-limiting step in the transport of receptors to the cell surface (12). A number of studies have demonstrated that, similar to their endocytic trafficking, ER export of GPCRs is coordinated by many regulatory factors. First, as with all other proteins, GPCRs must attain native conformation in order to exit from the ER. Incompletely folded or misfolded receptors are excluded from ER-derived transport vesicles by the ER quality control mechanism (11,13,14). Importantly, accumulation of misfolded GPCRs in the ER caused by naturally occurring mutations or truncations in the receptors is clearly associated with the pathogenesis of human disease (11,15–17). Second, GPCR export from the ER is modulated by direct interactions with a multitude of regulatory proteins such as ER chaperones and receptor activity modifying proteins, which may stabilize receptor conformation, facilitate receptor maturation and promote receptor delivery to the plasma membrane (18–20). Third, GPCR dimerization (homo- and heterodimerization) may also play an important role in proper receptor assembly in the ER (21–24). Fourth, recent studies have indicated that GPCR export from the ER is directed by highly specific sequences or motifs embedded within the receptors (25–30).
GPCRs share a common molecular topology characterized by a core of seven membrane-spanning α-helices, three intracellular loops, three extracellular loops, an extracellular N-terminus and an intracellular C-terminus. Efforts to define structural determinants of GPCR export from the ER have mainly focused on the C- and N-terminal tails. The requirement of the membrane proximal C-terminal region for ER export has been demonstrated for a number of GPCRs and alanine-scanning mutagenesis studies have identified several highly conserved motifs essential for GPCR export from the ER (25–30). We have reported a Phe and di-Leu spaced by six residues in the membrane proximal C-termini, which are required for ER export of angiotensin II type 1 (AT1R) and α2B-adrenergic receptors (α2B-AR) (25). We have also recently identified the YS motif located in the N-termini of α2-ARs, which modulates receptor export at the level of the Golgi (31). Here we have expanded these studies to define the role of the first intracellular loop in the traffic of GPCRs from the ER to the cell surface. In this manuscript we report that a single Leu residue in the center of the first intracellular loops (ICL1s) plays an essential role in ER export of α2B-AR, β2-AR, AT1R and α1B-AR. This isolated Leu residue is remarkably conserved among the family A GPCRs and thus, may coordinate a general mechanism for ER export of GPCRs.
We have previously demonstrated an obligatory role for the C- and N-termini in α2B-AR export to the cell surface (25,31). Here we have expanded these studies to define the role of the intracellular loops in regulating GPCR trafficking. We first determined the effect of deleting six residues within the ICL1 (45SRSLRA50) (Figure 1A) on the transport of α2B-AR to the cell surface. GFP-tagged α2B-AR and its mutant lacking the ICL1 (α2B-ARΔICL1) were transiently expressed in HEK293T cells and their expression was first determined by western blot analysis of total cell lysate using anti-GFP antibodies. The expression levels of α2B-AR and α2B-ARΔICL1 were comparable (Figure 1B). Total receptor expression as measured by flow cytometric analysis of the GFP signal also showed no significant difference between α2B-AR and α2B-ARΔICL1 (Figure 1C). The amount of receptor transported to the cell surface was quantified by binding of the ligand [3H]-RX821002 to intact live cells. The ligand binding of the α2B-ARΔICL1 mutant was abolished, indicating that the mutated receptor was unable to transport to the cell surface (Figure 1C). Consistent with the ligand binding data, subcellular distribution analysis of the receptors at steady state revealed that α2B-ARΔICL1 displayed an entirely intracellular distribution pattern, whereas wild-type α2B-AR showed a robust cell-surface expression (Figure 1D), which was confirmed by colocalization with tetramethylrhodamine-conjugated concanavalin A, a plasma membrane marker (data not shown).
To further confirm the inhibitory effect of deleting the ICL1 on α2B-AR transport to the cell surface as observed by intact-cell ligand binding and subcellular localization, we compared the abilities of α2B-AR and α2B-ARΔICL1 to initiate signaling by measuring ERK1/2 activation in response to stimulation with the α2B-AR agonist UK14304. ERK1/2 activation by UK14304 was almost completely lost in cells transfected with α2B-ARΔICL1 (Figure 1E). In contrast, ERK1/2 activation was markedly stimulated by UK14304 in cells transiently transfected with α2B-AR (Figure 1E). These data strongly indicate that the ICL1 is required for the cell-surface transport of α2B-AR.
To identify specific residues within the ICL1 that are essential for the cell-surface expression of α2B-AR, we performed site-directed alanine mutagenesis on the ICL1 and determined the effect of the mutation on α2B-AR expression at the cell surface by intact-cell ligand binding. The α2B-AR mutants S45A, R46A, S47A and R49A transported to the cell surface at levels similar to wild-type α2B-AR (Figure 2A). In contrast, transport to the cell surface of L48A was drastically reduced by 97% compared to wild-type α2B-AR (Figure 2A). To exclude the possibility that mutation of L48 influences α2B-AR binding to ligand, α2B-AR and its mutant L48A were tagged with three HA at their N-termini and their expression at the cell surface was then measured by flow cytometry following staining with anti-HA antibodies in unpermeabilized cells. Consistent with the data obtained in intact-cell ligand binding, cell-surface expression of the mutant L48A was blocked by 97% as compared to wild-type receptor (Figure 2A). Subcellular distribution analysis of each α2B-AR mutant showed that the L48A mutant was intracellularly accumulated and unable to transport to the cell surface, whereas S45A, R46A, S47A and R49A mutants were normally expressed at the cell surface (Figure 2B). Consistently, ERK1/2 activation in response to UK14304 stimulation was significantly inhibited in cells transfected with the mutant L48A when compared with cells transfected with α2B-AR (Figure 2C). These data indicate that a single Leu48 within the ICL1 regulates cell-surface expression of α2B-AR.
We next attempted to define the precise subcellular location of the mutant L48A by colocalization with various organelle markers. The mutant L48A extensively colocalized with the ER marker DsRed2-ER (Figure 3A), but only very weakly colocalized with the cis-Golgi marker GM130 (Figure 3B) and the TGN marker p230 (Figure 3C). To further confirm whether the mutant L48A was arrested in the ER, we performed sucrose gradient centrifugation to biochemically isolate the ER fraction. The mutant L48A was predominantly localized in fractions 7–10 and exclusively overlapped with calnexin, an ER resident protein (Figure 3D). These data indicate that the mutant L48A was mainly retained in the ER compartment and suggest that the Leu48 residue in the ICL1 modulates α2B-AR export at the level of the ER.
To define the physiochemical properties of the Leu48 residue that are essential to its role in coordinating α2B-AR export from the ER, the Leu48 residue was mutated to several hydrophobic residues including Ile, Val, Phe, Tyr and Trp. Each of these mutants was expressed in HEK293T cells and their abilities to functionally substitute for Leu were then assessed by intact-cell ligand binding, subcellular localization and ERK1/2 activation. Similar to mutation to Ala, Leu48 mutation to Ile, Val, Tyr and Trp markedly attenuated α2B-AR transport to the cell surface (Figure 4A). The mutants L48I, L48V, L48Y and L48W were expressed extensively in the perinuclear region, presumably the ER (Figure 4B). Surprisingly, mutation of Leu48 to Phe did not significantly alter α2B-AR transport to the cell surface. The cell-surface expression of the L48F mutant was comparable to that of wild-type α2B-AR as measured by intact-cell ligand binding (Figure 4A). Consistently, there was no clear difference in the subcellular distribution between α2B-AR and its mutant L48F, both displaying a robust cell-surface expression pattern (Figure 4B).
We then measured the ability of wild-type α2B-AR and its mutants L48F and L48W to stimulate ERK1/2 activation. ERK1/2 were activated dose-dependently by UK14304 in cells expressing wild-type α2B-AR or the L48F mutant. In contrast ERK1/2 activation by UK14304 was markedly inhibited in cells transfected with the mutant L48W when compared to cells transfected with α2B-AR or the mutant L48F. These data are consistent with the differences in the cell-surface expression of wild-type α2B-AR and its mutants as determined by ligand binding and subcellular localization (Figure 4C).
To define the molecular mechanism underlying the function of the Leu48 residue in regulating GPCR export from the ER, we first determined whether Leu48 was involved in proper folding of α2B-AR in the ER. In the first series of experiments, we analyzed the ability of low-temperature culture and treatment with the chemical chaperone DMSO to rescue cell-surface expression of the mutant L48A. Both treatment conditions are known to promote the cell-surface expression of certain misfolded proteins, including GPCRs, by virtue of either relaxing the quality control system or promoting tighter protein conformations within the ER (31–33). HEK293T cells were transfected with α2B-AR and its mutant L48A and then either treated with 2% DMSO or cultured at 30°C for 24 h. The DMSO treatment significantly increased the cell-surface expression of both wild-type α2B-AR and the mutant L48A (Figure 5A). However, the increases in the cell-surface expression of both wild-type and mutated receptors by the DMSO treatment were equivalent and the absolute amount of L48A expressed at the cell surface was less than 6% compared with wild-type α2B-AR (Figure 5A), indicating that the DMSO treatment did not specifically facilitate the cell-surface expression of the mutated receptor L48A. Furthermore, the low-temperature culture did not significantly increase the cell-surface expression of the mutant L48A (Figure 5B).
Consistent with the ligand binding data, the DMSO treatment and reduced temperature culture did not clearly alter the subcellular localization of α2B-AR and its mutant F48A (Figure 5C). In contrast, under the same conditions the DMSO treatment and reduced temperature culture partially rescued the cell-surface expression of another α2B-AR F436A (Figure 5A, B). F436 has been demonstrated to regulate α2B-AR export from the ER likely through intramolecular interaction with other hydrophobic residues to modulate receptor folding (25,34).
In the second series of experiments, we determined whether mutation of Leu48 could alter receptor interaction with ER chaperone proteins. It has been well demonstrated that terminally misfolded GPCRs display strong and extended interactions with ER resident chaperones such as calnexin (31,35,36). The GFP-tagged α2B-AR and its mutant L48A were expressed in HEK293T cells and immunoprecipitated by anti-GFP antibodies. The amount of calnexin in the GFP-immunoprecipitates was then analyzed by western blotting. Calnexin was identified in the GFP-immunoprecipitates from cells expressing α2B-AR and its mutant L48A but not in immunoprecipitates from cells expressing GFP alone, confirming the specificity of the interaction between the receptor and calnexin (Figure 6A). However, no major differences were found in the amount of calnexin pulled down with α2B-AR or its mutant L48A (Figure 6A), suggesting the L48A mutant does not have a major folding defect and its ER retention is unlikely caused by misfolding.
We previously demonstrated that α2B-AR constitutively forms dimers in the ER and that dimerization of α2B-AR plays a crucial role in modulating its transport from the ER to the cell surface (26). To test if the detrimental effect of Leu48 mutation to Ala on α2B-AR export trafficking was due to an inability of the mutant receptor to dimerize in the ER, we determined whether GFP-tagged L48A could form heterodimers with HA-tagged α2B-AR by co-immunoprecipitation when co-expressed in HEK293T cells. Similar amounts of HA-α2B-AR were detected in the anti-GFP immunoprecipitates from cells transfected with GFP-tagged α2B-AR and its mutant L48A (Figure 6B). In contrast, HA-α2B-AR was undetectable in the anti-GFP immunoprecipitates from cells expressing GFP (Figure 6B). To further ensure specific interaction between α2B-AR and its mutant L48A, the cells were separately transfected with α2B-AR or L48A, mixed and immunoprecipitated with anti-GFP antibodies. There was no detectable HA-α2B-AR in the immunoprecipitates from the mixture (data not shown). These data indicate that α2B-AR forms heterodimers with L48A when co-expressed in the same cell populations.
Our preceding data have strongly demonstrated that the Leu48 residue plays a crucial role in α2B-AR export from the ER. We wondered how many other GPCRs harbor such a Leu residue in their ICL1s. Searching the GPCR database we found that an isolated Leu residue corresponding to Leu48 in α2B-AR is remarkably conserved in the ICL1s among Class A GPCRs (Figure 7). In family A GPCRs, 84.7% of the receptors in human and 82.7% in all species contain a Leu residue in the ICL1s (data not shown). The residue Met has the second highest incidence of 9.7% in the total family A GPCRs whereas the remaining amino acids all have incidences of less than 3%. The highly conserved Leu is localized to the center of the ICL1s and is in the exact same position relative to the most highly conserved residues Asn in the first transmembrane domain (Figure 7) and Asp in the second transmembrane domain (data not shown).
We then sought to determine if the Leu residues were also required for the transport from the ER to the cell surface of β2-AR, AT1R and α1B-AR. Based on the alignment, the isolated Leu corresponding to Leu48 in α2B-AR is located at positions 64, 59 and 78 in β2-AR, AT1R and α1B-AR, respectively (Figure 8A). Mutation of each of these Leu residues in the ICL1s of the three receptors produced a significant reduction in the cell-surface expression as measured by intact-cell ligand binding (Figure 8B). However, the magnitude of the reduction upon mutation was variable. Similar to the effect of mutating Leu48 on α2B-AR transport, mutation of Leu64 abolished β2-AR expression at the cell surface (Figure 8B) and completely arrested the receptor inside the cell (Figure 8C). However, mutation of Leu59 in AT1R and Leu78 in α1B-AR displayed about 60% reduction in their cell-surface expression (Figure 8B). Both the AT1R mutant L59A and the α1B-AR mutant L78A were partially arrested inside the cell (Figure 8C). Similar results were obtained when receptor expression at the cell surface was measured by flow cytometry following staining with anti-HA antibodies in cells transfected with HA-tagged wild-type or mutated receptors (Figure 8B). These data further exclude the possibility that differential effects of mutating single Leu residue on the cell-surface expression of different receptors was because of the influence on receptor binding to different ligands.
We then determined whether mutation of Leu64 influenced β2-AR dimerization as measured by bioluminescence resonance energy transfer (BRET) as well as the effect of inhibiting receptor transport by expressing the dominant negative mutants of the Ras-like small GTPases Sar1 and Rab1 on receptor dimerization. Similar to wild-type β2-AR, the β2-AR mutant L64A formed heterodimers with wild-type β2-AR (Figure 8D). These data further indicate that mutation of the conserved Leu residues in the first intracellular loops did not interfere with receptor dimerization per se.
Sar1 GTPase plays an essential role in the formation of COPII-coated transport vesicles on the ER membrane, which exclusively mediate protein export from the ER (37,38). Rab1 GTPase modulates protein transport specifically from the ER to the Golgi (39,40). Our previous studies have demonstrated that expression of Sar1H79G, Rab1S25N and Rab1N124I inhibited β2-AR transport from the ER to the cell surface (41–43). Transient expression of Sar1H79G, Rab1S25N and Rab1N124I did not significantly alter dimerization of β2-AR and its mutant L64A (Figure 8D). These data indicate that dimerization of β2-AR occurs constitutively in the ER and is similar for both wild-type β2-AR and mutant L64A.
To confirm the disturbance in cell-surface targeting of the receptors as observed by intact-cell ligand binding and subcellular localization, functional analysis of cAMP production by β2-AR and ERK1/2 activation by AT1R and α1B-AR was then performed. cAMP production in response to ISO stimulation was markedly attenuated in cells expressing the β2-AR mutant L64A, compared to cells expressing wild-type β2-AR (Figure 9A). ERK1/2 activation was attenuated by approximately 50% in cells expressing the AT1R mutant L59A and the α1B-AR mutant L78A as compared to cells expressing their individual wild-type receptors (Figure 9B and C). These data indicate that, similar to α2B-AR, an isolated Leu residue within the ICL1s of β2-AR, AT1R and α1B-AR is also required for efficient receptor cell-surface expression. These results suggest that the Leu residues in the ICL1s may perform a general role in the trafficking of many GPCRs.
The molecular mechanism underlying export from the ER and subsequent transport to the cell surface of the GPCR superfamily remains largely unknown. Studies to define the specific motifs required for GPCR export have been mainly focused on the C- and N-termini of the receptors (25–31). In this study we investigated the role of the ICL1 in the cell-surface transport of GPCRs and identified a single Leu residue, which is essential for the export of α2B-AR, β2-AR, AT1R and α1B-AR from the ER. This isolated Leu is extremely conserved among the family A GPCRs and thus, may provide a common mechanism directing ER export of newly synthesized GPCRs.
We first demonstrated an obligatory role for the ICL1 in α2B-AR trafficking to the cell surface. The cell-surface expression of the α2B-AR mutant lacking the ICL1 was markedly reduced as measured by intact-cell ligand binding, which quantitatively measures receptor expression at the cell surface. The significant reduction of receptor expression at the cell surface was strongly supported by direct visualization of intracellular localization of the mutated receptors and receptor-mediated signal propagation as measured by ERK1/2 activation. Any significant disruption in total receptor expression upon deletion of the ICL1 was ruled out by flow cytometry and western blot. These data indicate that the ICL1 contains structural features important for α2B-AR export to the cell surface.
An alanine-scanning mutagenesis approach was then utilized to identify amino acid residues within the ICL1 required for α2B-AR transport. Mutation of the Leu48 residue in the middle of the ICL1 almost abolished α2B-AR cell-surface expression, whereas mutation of other residues within the ICL1 had minimal to no effect on receptor cell-surface expression. Krause et al. reported that mutation of the Leu62 residue in vasopressin 2 receptor (V2R) resulted in the ER retention of the receptor (27). The function of Leu62 in V2R export from the ER is proposed to be mediated by modulating receptor folding through a long-range hydrophobic interaction with the C-terminal Leu339 residue. The Leu62 residue in V2R is aligned to the Val42 residue in α2B-AR, which is likely located at the border of the first transmembrane domain and the ICL1 and mutation of V42 attenuates α2B-AR expression at the cell surface by 61% (34). The α2B-AR mutant L48A extensively colocalized with the ER marker DsRed2-ER in subcellular colocalization studies. The mutant L48A also completely overlapped with the ER resident protein calnexin in subcellular fractionation. Therefore, we concluded that the single residue Leu48 modulates α2B-AR export at the level of the ER.
The function of the Leu48 residue in modulating α2B-AR export from the ER is likely dictated by its specific physiochemical and structural features including overall size of the side chain, spacing between the bulky portion of the side chains and the α-carbon and polarity. Our data, demonstrating that mutation of Leu48 to Ile, Val, Tyr and Trp abolished α2B-AR export from the ER and transport to the cell surface, whereas substitution of Leu48 by Phe preserved α2B-AR transport, reveals several important characteristics of the Leu48 residue, which are required to perform its function in ER export. First, disruption of α2B-AR export by mutation of Leu to Ile, which essentially results in the shifting of a methyl group one carbon closer to the α-carbon, suggests that some bulk at the end of the side chain is required for the proper function of the L48 residue. Furthermore, Phe, Tyr and Trp are aromatic residues, but the sizes of Tyr and Trp residues are larger than that of Phe. The export function of Leu48 can be substituted by Phe, but not Tyr or Trp, suggesting that there is a certain size limitation at the position of 48. Second, the only difference between Leu and Val residues is that Leu has one more CH2 group within its side chain, indicating that a particular length between the bulk at the end of the side chain and the α-carbon plays a crucial role in Leu48 function. Third, Tyr and Trp are polar residues, whereas Phe and Leu are nonpolar residues. The export function of Leu48 can be substituted by Phe, suggesting that the nonpolar property may also be an important factor for Leu function.
The molecular mechanism underlying the function of the single Leu residue in regulating GPCR export from the ER remains unknown. The possibility that the Leu48 residue is involved in proper α2B-AR folding in the ER was ruled out, because the treatment with the chemical chaperone DMSO and the low-temperature culture, conditions that have been well characterized to rescue the export of misfolded GPCRs (11,32,33), did not produce marked influences on the cell-surface expression of the α2B-AR mutant L48A. Furthermore, there was no significant difference in the interaction with the ER chaperone calnexin between α2B-AR and its mutant L48A. Increased interactions with ER chaperones have been reported for a number of misfolded GPCRs and used as a sensor for measuring receptor folding (11,35,36). In addition, we have looked at whether pharmacochaperones could rescue cell-surface expression of the mutant receptor. Our data indicate that the α2B-AR antagonists ARC 239, efaroxan and mirtazeoine were unable to rescue the cell-surface expression of α2B-AR mutant L48A as well as F436A (unpublished observation). These data suggest that the Leu48 residue is unlikely involved in α2B-AR folding in the ER. The possibility that the Leu48 residue regulates α2B-AR dimerization was also excluded. We previously demonstrated that α2B-AR constitutively forms dimers in the ER and that an ER export deficient α2B-AR mutant functions as a dominant negative blocker inhibiting wild-type α2B-AR export from the ER (26). Our current data demonstrated that the L48A mutant dimerized with wild-type α2B-AR, indicating that the Leu48 residue is not involved in dimerization of α2B-AR in the ER. Similar to mutation of Leu48 in α2B-AR, mutation of Leu64 did not alter β2-AR dimerization. These data are also consistent with the notion that the motifs required for GPCR dimerization are likely located in the N-termini and the transmembrane domains (44).
It is possible that the Leu48 residue mediates interactions of α2B-AR with some regulatory proteins and that such interactions are crucial for the receptor export from the ER. Consistent with this possibility, a number of accessory proteins directly interact with the intracellular loops of GPCRs to modulate receptor export from the ER to the cell surface (18–20). It is also possible that the single Leu48 may function as an independent ER export motif mediating receptor export from the ER. The ER export of certain proteins is known to be expedited by the presentation of ER export motifs, which are decoded by the COPII vesicle coat. Interaction between the ER export motifs and COPII components serves to gather cargo molecules in high concentrations at ER exit sites for inclusion into the ER-derived COPII vesicles (45–47). Consistent with the possibility, the di-Phe sequence, an ER export motif required for efficient transport of ERGIC53, can be functionally substituted by a single Tyr or Val (47). Furthermore, a single Val residue was shown to be essential for the transport of stem cell factor to the cell surface (48). To address this issue, we have generated intracellular domains (including the first (44T-N53), the second (W117-P131) and the third intracellular loops (T430-E369) and C-terminus (T430-W453) of α2B-AR as GST-fusion proteins and determined their interaction with GFP-tagged Sec24C. We found that the third intracellular loop, but not other intracellular domains (the first and second intracellular loops and C-terminus) interacted with Sec24C isoform (unpublished observation). Whether the single Leu residue mediates GPCR interaction with other components of the COPII vesicles, thus facilitating receptor recruitment to the transport vesicles, is currently under investigation.
The isolated Leu residues we found in the ICL1s are remarkably conserved among the family A GPCRs, implying that this single Leu may provide a common mechanism directing GPCR export from the ER. Indeed, similar to α2B-AR, mutation of the conserved Leu residues in the ICL1s of β2-AR, AT1R and α1B-AR significantly blocked their export from the ER to the cell surface. However, the inhibitory effect of mutating the conserved Leu residues on the transport of distinct GPCRs was variable. Mutation of the Leu residues abolished α2B-AR and β2-AR export to the cell surface, but only partially inhibited AT1R and α1B-AR transport. These data indicate that the Leu residue in the ICL1 plays a more important role in ER export trafficking of α2B-AR and β2-AR than AT1R and α1B-AR. Mutation of this conserved Leu residues in the ICL1 of α2B-AR, β2-AR, AT1R and α1B-AR attenuated the receptor-mediated signaling measured as cAMP production or ERK1/2 activation, paralleling the effects of the mutation on the transport of theses receptors to the cell surface. Therefore, we interpret that the attenuation of receptor-mediated signaling resulting from the mutation of the Leu residues in the ICLs was due at least in part to the inability of the mutated receptors to transport to the cell surface. However, we cannot exclude the possibility that mutation of the Leu residues may also impair coupling of the receptors to the signaling molecules involved in receptor signaling systems. In addition, this Leu residue is surrounded by polar positively charged (i.e. Arg, Lys and His) and uncharged residues (e.g. Ser, Thr and Asn) (Figure 7). Although our mutagenesis studies indicate that these neighboring residues are not individually required for α2B-AR export from the ER, the abundance of these polar/basic residues proximal to the highly conserved Leu residue suggests that the physiochemical environment may be important for the function of the Leu residue in modulating receptor export from the ER.
The identification of structural determinants for GPCR export from the ER and the Golgi has greatly advanced our understanding of GPCR targeting to the cell surface, yet the precise mechanisms of their actions remain to be elucidated (25–30). Recent studies have also demonstrated that the transport of GPCRs beyond their export from the ER is tightly controlled at multiple transport steps along the secretory pathway and the key regulators involved at each of these stages have just begun to be revealed (41–43,49–53). For example, we previously determined the roles of Rab1, Rab2, Rab6 and Sar1 GTPases, which specifically coordinate protein transport between the ER and the Golgi (37–40), in the transport of these receptors (41–43,49,50). We demonstrated that the transport from the ER to the cell surface of AT1R, β-AR and α1-AR is dependent on Rab1, Rab2 and Rab6, whereas the transport of α2B-AR is dependent on Rab2, but independent of Rab1 and Rab6. α 2B-AR differs from most other GPCRs as it has no N-linked glycosylation sites. Glycosylation of the receptors occurs in the ER and during their transport through the Golgi resulting in the formation of mature receptors competent for subsequent targeting to the cell surface. It is possible that the glycosylation signal may dictate selection of the transport pathway for the receptors. Nevertheless, these data indicate that the cell surface targeting of different GPCRs may be mediated through distinct pathways.
As the efficient trafficking of GPCRs and the precise positioning of specific receptors at the cell membrane are critical aspects of integrated responses of the cell to hormones and importantly, defective GPCR transport from the ER to the cell surface is clearly associated with the pathogenesis of a variety of human diseases, such as retinitis pigmentosa, nephrogenic diabetes insipidus and male pseudohermaphroditism (15–17). Further elucidation of the molecular mechanisms underlying the export traffic of GPCRs may provide a foundation for development of therapeutic strategies by designing specific drugs to control GPCR biosynthesis and eventually receptor function.
Rat α2B-AR in vector pcDNA3, human β2-AR in vector pBC, rat AT1R in vector pCDM8 and human α1B-AR tagged with green fluorescent protein (GFP) at its C-terminus were kindly provided by Dr Stephen M. Lanier, Dr John D. Hildebrandt (Medical University of South Carolina, Charleston, SC), Kenneth E. Bernstein and Kenneth P. Minneman (Emory University, Atlanta, GA), respectively. Antibodies against GFP, phospho-ERK1/2 and calnexin and anti-HA antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ERK antibodies detecting total ERK1/2 expression were from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against GM130 and p230 were from Transduction Laboratories (San Diego, CA). Antibodies against α-tubulin, isoproterenol (ISO), phenylephrine (PE), UK14304, rauwolscine, phentolamine and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, MO). Human angiotensin II (Ang II) was purchased from Calbiochem (Gibbstown, NJ). [3H]-RX821002 (specific activity = 41 Ci/mmol), [3H]-CGP12177 (51 Ci/mmol), [7-methoxy –3H]-prazosin (70 Ci/mmol) and [125I]-[Sar1, Ile8]-angiotensin II ([125I]-Ang II) (2200 Ci/mmol) were purchased from PerkinElmer (Waltham, MA). Alexa Fluor 594-labeled secondary antibodies and 4, 6-diamidino-2-phenylindole were from Molecular Probes, Inc. (Eugene, OR). The ER marker pDsRed2-ER was from BD Biosciences (Palo Alto, LA). All other materials were obtained as described elsewhere (25,41).
α2B-AR, β2-AR and AT1R tagged with GFP at their C-termini were generated as described previously (41). α2B-AR tagged with three HA at its N-terminal was purchased from UMR cDNA Resource Center (Rolla, MO). The GFP and HA epitopes have been used to label GPCRs resulting in receptors with similar characteristics to the wild-type receptors (54–56). The construct α2B-ARΔICL1 (in which the ICL1 of α2B-AR was truncated) and receptor mutants were generated using the QuikChange site-directed deletion mutagenesis kit (Stratagene, La Jolla, CA). The sequence of each construct used in this study was verified by restriction mapping and nucleotide sequence analysis (Louisiana State University Health Sciences Center DNA Sequence Core).
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin, and 100 units/mL streptomycin and transiently transfected by using Lipofectamine 2000 reagent as described previously (25,41). Transfection efficiency for both 6-well and 100-mm dish formats was estimated to be greater than 70% based on the GFP fluorescence.
Cell-surface expression of α2B-AR, β2-AR, AT1R and α1B-AR in HEK293T cells was measured by ligand binding of intact live cells using [3H]-RX821002, [3H]-CGP12177, [125I]-Ang II and [7-methoxy –3H]-prazosin, respectively, as described (42,43,49,56–58). HEK293T cells cultured on 6-well dishes were transiently transfected with 1.0 μg of plasmids. After 6 h, the cells were split into 12-well dishes pre-coated with poly-L-lysine at a density of 5 × 105 cells/well. The cells were serum-starved 24 h post-transfection. The cells were incubated 48 h post-transfection with DMEM plus [3H]-RX821002, [3H]-CGP12177 or [7Methoxyn –3H]-prazosin at a concentration of 20 nM in a total of 400 μL for 90 min at room temperature to measure the cell-surface expression of α2B-AR, β2-AR and α1B-AR, respectively. The binding was terminated and excess radioligand eliminated by washing the cells twice with ice-cold DMEM. All of the retained radioligand was then extracted by digesting the cells in 1M NaOH for 2 h at room temperature. The liquid phase was collected and suspended in 5 ml of Ecoscint A scintillation fluid (National Diagnostics Inc., Atlanta, GA). The amount of radioactivity retained was measured by liquid scintillation spectrometry. For measurement of AT1R expression at the cell surface, HEK293T cells were incubated with 400 μL DMEM containing 125I-Ang II at a concentration of 20 pM for 2 h. After washing the cells twice with 1 mL DMEM, the bound ligand was extracted by mild acid treatment (2 × 5 min with 0.5 mL buffer containing 50 mM glycine and 125 mM NaCl, pH 3). The radioactivity was counted in a γ-counter. The nonspecific binding of α2B-AR, β2-AR, α1B-AR and AT1R was determined in the presence of rauwolscine (10 μM), alprenolol (20 μM) and phentolamine (10 μM) and nonradioactive Ang II (10 μM), respectively. For chemical rescue, HEK293T cells were incubated with DMSO at a concentration of 2% in a total of 2 mL of DMEM without fetal bovine serum for 24 h. For low-temperature rescue, the cells were cultured at 30°C for 24 h. Cell-surface expression of α2B-AR was then measured by ligand binding of intact live cells using [3H]-RX821002 as described earlier. All radioligand binding assays were performed in triplicate.
For measurement of total receptor expression, HEK293T cells were transiently transfected with 500 ng of GFP-tagged receptors for 36 h. The cells were collected, washed twice with PBS and resuspended at a density of 8 × 106 cells/mL. Total GFP fluorescence was then measured on a flow cytometer (BD Biosciences FASCalibur) as described previously (38). For measurement of receptor expression at the cell surface, HEK293T cells, transfected with HA-tagged receptor, were suspended in PBS containing 1% FCS at a density of 4 × 106 cells/mL and incubated with high affinity anti-HA-fluorescein (3F10) at a final concentration of 2 μg/mL for 30 min at 4°C. After washing twice with 0.5 mL of PBS/1% FCS, the cells were resuspended and the fluorescence was analyzed on a flow cytometer (Dickinson FACSCalibur) as described (41).
For fluorescence microscopic analysis of receptor subcellular localization, HEK293T cells were grown on coverslips pre-coated with poly-L-lysine in 6-well plates and transfected with 500 ng of GFP-tagged receptors. For colocalization of GFP-tagged receptors with the ER marker DsRed2-ER, HEK293T cells grown on coverslips were transfected with 500 ng of GFP-tagged receptors and 300 ng of pDsRed2-ER. The cells were fixed with 4% paraformaldehyde–4% sucrose mixture in PBS for 15 min and stained with 4, 6-diamidino-2-phenylindole for 5 min. For colocalization of GFP-tagged receptors with the cis-Golgi marker GM130 or the TGN marker p230, HEK293T cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 min, and blocked with 5% normal donkey serum for 1 h. The cells were then incubated with antibodies against GM130 or p230 at a dilution of 1:50 for 1 h. After washing with PBS (3 × 5 min), the cells were incubated with Alexa Fluor 594-labeled secondary antibody (1:2000 dilution) for 1 h at room temperature. The coverslips were mounted, and fluorescence was detected with a Leica DMRA2 epifluorescent microscope as described previously (41). Images were deconvolved using SlideBook software and the nearest neighbor deconvolution algorithm (Intelligent Imaging Innovations, Denver, CO).
HEK293T cells were cultured on 6-well dishes and transfected with 1.0 μg of plasmids. The cells were starved for at least 3 h after 36 to 48 h post-transfection, and then stimulated with individual receptor agonists. The stimulation was terminated by addition of 1 × SDS gel loading buffer (25,41). After solubilization of the cells, 20 μL of sample was separated by 12% SDS-PAGE. Activation of ERK1/2 was determined by measuring the level of ERK1/2 phosphorylation with phospho-specific ERK1/2 antibodies. The blots were then stripped and reprobed with total ERK1/2 antibodies to confirm equal loading of the gel.
HEK293T cells cultured in 100-mm dishes were transfected with 6 μg of the GFP-tagged α2B-AR mutant L48A. The cells from three dishes were collected 24 h later in ice-cold buffer A containing 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA. The cells were spun at 300 ×g for 5 min and resuspended in 1 mL of homogenization buffer [85% buffer A and 15% buffer B containing 10 mM Tris, pH7.4, 5 mM KCl, 1 mM EDTA, 128 mM NaCl]. The cells were then homogenized by passing through a 26 gage needle 12 times followed by centrifuge at 1, 000 × g for 10 min at 4°C. The supernatant was then loaded on the top of pre-formed 0–26% OptiPrep gradient (Axis-Shield Techniques, Dundee, Scotland) (59). Discontinuous gradients were made manually using a syringe following the manufacturer’s instructions and the gradient was then stored in a refrigerator overnight to produce a continuous gradient. The samples were then centrifuged in a Beckman SW41 rotor at 100000 × g for 3 h at 4°C. Sequential 800 μL fractions were collected from the top of the gradient. Each fraction of 20 μL was mixed with 2 × SDS gel loading buffer and separated on 10% SDS-PAGE.
For dimerization studies, HEK293T cells cultured on 100-mm dishes were transfected with 2 μg of HA-tagged α2B-AR together with 2 μg of the pEGFP-N1 vector or GFP-tagged wild-type or mutated α2B-AR in the pEGFP-N1 vector for 28 h. The cells were washed twice with PBS, harvested and lysed with 500 μL of lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and Complete Mini protease inhibitor cocktail. After gentle rotation for 1 h, samples were centrifuged for 15 min at 14000 × g and the supernatant was incubated with 50 μL of protein G Sepharose for 1 h at 4°C to remove nonspecific bound proteins. Samples were then incubated with 3 μg of anti-GFP antibodies overnight at 4°C with gentle rotation, followed by an incubation with 50 μL of protein G Sepharose 4B beads for 5 h. Resin was collected by centrifugation and washed three times with 500 μL of lysis buffer without SDS. Immunoprecipitated receptors were eluted with 100 μL of 1× SDS gel loading buffer, separated by 8% SDS-PAGE and visualized by immunoblotting using anti-HA and GFP antibodies (26).
For co-immunoprecipitation of α2B-AR with the ER chaperone calnexin, HEK293T cells cultured on 100-mm dishes were transfected with 4 μg of the pEGFP-N1 vector or GFP-tagged wild-type or mutated α2B-AR in the pEGFP-N1 vector for 24 h and subjected to immunoprecipitation using anti-GFP antibodies as described earlier. From each sample, 30 μL was then separated by SDS-PAGE to probe for calnexin in the GFP-immunoprecipitates by immunoblotting. In parallel, each sample was further diluted five times with 1 ×SDS gel loading buffer, separated by SDS-PAGE and probed with anti-GFP antibodies to determine the amount of the receptor in the immunoprecipitates (31).
BRET was performed as previously described (51,52). Briefly, 48 h after transfection with GFP10-, and Rluc-tagged wild-type and L64A β2-AR fusion proteins, HEK293 cells were washed twice with PBS, resuspended in PBS + (0.1 % glucose, 5 μg/mL leupeptin, 10 μg/mL benzamidine and 5 μL/mL trypsin inhibitor) and distributed into 96 well microplates (white Optiplate; PerkinElmer Life and Analytical Sciences). BRET2 was performed using coelenterazine 400a (Cedarlane) at a final concentration of 5 μM. Signals were collected on a Packard Fusion instrument (PerkinElmer Life and Analytical Sciences) using either 410/80-nm filter (luciferase) or 515/30-nm filter (GFP10). BRET background was determined under conditions where resonance energy transfer between RLuc and GFP could not occur. This was accomplished by expressing CD8-RLuc together with GFP-tagged receptors as a negative control. This background BRET was subtracted to yield net BRET.
cAMP concentrations were measured by using cAMP enzymeimmunoassay system (Biotrak, Amersham Pharmacia Biotech, Piscataway, NJ) as described previously (34,60). HEK293T cells were cultured in 100-mm dishes and transfected with 3 μg of β2-AR or its mutants tagged with GFP. After 6 h, the cells were split into 12-well plates and cultured for 12 h. The cells were then starved for 24 h and then incubated with isobutyl-methylxanthine (0.1 mM) for 30 min before stimulation with ISO at a concentration of 10 μM for 5 min at room temperature. The reactions were stopped by aspirating the medium and the cells were lysed using 200 μL of dodecyltrimethylammonium (2.5%). One hundred microliter of cell lysate was transferred into microtitre plates and incubated with anti-cAMP antiserum, followed by the incubation with cAMP-peroxidase conjugate. After washing and addition of substrate, peroxidase activity was measured by spectrometry.
Western blot analysis of protein expression was carried out as previously described (25,41). Samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The signal was detected using ECL Plus (PerkinElmer Life Sciences) and a Fuji Film luminescent image analyzer (LAS-1000 Plus) and quantitated using the Image Gauge program (Version 3.4).
Differences were evaluated using Student’s t test, and p < 0.05 was considered as statistically significant. Data are expressed as the mean ± S.E.
This work was supported by National Institutes of Health grants R01GM076167 (to G.W.) and P20RR018766 (PI: Daniel R. Kapusta, Ph.D.) and by grants from the Canadian Institutes of Health Research (to T.E.H). Trainee support included a Louisiana Board of Regents graduate fellowship (to M.T.D.), an American Heart Association, Southeast Affiliate postdoctoral fellowship (to C.D.) and a scholarship from the Fonds de la Recherche en Santé du Québec (to M.R.). T.E.H. holds a Chercheur National Award from the Fonds de la Recherche en Santé du Québec. We are grateful to Drs Stephen M. Lanier, John D. Hildebrandt, Kenneth E. Bernstein and Kenneth P. Minneman for sharing reagents.