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Originally published In Press as doi:10.1074/jbc.M304504200 on August 25, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45978-45986, November 14, 2003
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A Novel Endocytic Recycling Signal That Distinguishes the Membrane Trafficking of Naturally Occurring Opioid Receptors*

Michael Tanowitz{ddagger} and Mark von Zastrow

From the Departments of Psychiatry and Cellular & Molecular Pharmacology, University of California, San Francisco, California 94143-2140

Received for publication, April 30, 2003 , and in revised form, August 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{delta} and µ opioid receptors are homologous G protein-coupled receptors that are differentially sorted between divergent degradative and recycling membrane pathways following agonist-induced endocytosis. Whereas {delta} opioid receptors are selectively sorted to lysosomes, µ opioid receptors recycle rapidly to the plasma membrane by a process that has been proposed to occur via bulk membrane flow. We have observed that µ opioid receptors do not recycle by default and have defined a specific sequence present in the cytoplasmic tail of the cloned µ opioid receptor that is both necessary and sufficient for rapid recycling of internalized receptors. This sequence is completely distinct from a sequence shown previously to be required for recycling of the {beta}2 adrenergic receptor yet is functionally interchangeable when tested in chimeric mutant receptors. These results indicate that signal-dependent recycling is a more common property of G protein-coupled receptors than previously appreciated and demonstrate that such a modular recycling signal distinguishes the regulation of homologous receptors that are naturally co-expressed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs)1 comprise a large superfamily of signal-transducing receptors that play important physiologic roles and represent prominent drug targets. Many GPCRs are regulated by ligand-induced endocytosis (1-3). This process can mediate diverse functional consequences, which depend in large part on the specific membrane trafficking pathway followed by endocytosed receptors. For example, recycling of internalized receptors to the plasma membrane typically promotes the recovery of cellular responsiveness to agonist as characterized extensively in studies of functional resensitization of the {beta}2 adrenergic catecholamine receptor (B2AR) (2, 4). Trafficking of internalized receptors to lysosomes typically has an opposite effect, promoting proteolytic degradation that is characterized by down-regulation of the total number of receptors present in cells and a prolonged attenuation of cellular responsiveness to agonist (5). The ability of endocytic membrane trafficking to mediate these functionally opposite effects on receptor regulation underscores fundamental questions about the mechanisms that control the specificity of receptor trafficking in the endocytic pathway.

Trafficking of GPCRs to lysosomes requires a specific membrane sorting event involving ubiquitin-dependent or -independent interactions of internalized receptors with cytoplasmic sorting proteins (6-9). In contrast, recycling of receptors to the cell surface has been proposed to represent a "default" pathway that occurs by bulk membrane flow. The idea that GPCRs can recycle by default is consistent with previous studies of constitutively recycling nutrient receptors (10), which traverse a membrane pathway similar to recycling GPCRs, and with the enhanced recycling of various receptors observed after experimental manipulations that disrupt mechanisms of endosomal retention or lysosomal trafficking (8, 11-14). The generalization that all GPCR recycling occurs by default has been challenged by studies of the B2AR where a specific sequence present in the cytoplasmic tail is required for efficient recycling of receptors, suggesting that certain GPCRs do not recycle by default but do so in a signal-dependent manner (15-17).

The sequence required for rapid recycling of the B2AR is not conserved in most GPCRs and binds to cytoplasmic proteins that appear to interact with few other family members, raising basic questions regarding the generality and physiological relevance of this principle of receptor regulation. First, is signal-dependent recycling unique to the B2AR or restricted to a limited subset of GPCRs that possess highly similar cytoplasmic tails? Second, if signal-dependent recycling is a more general phenomenon, can structurally distinct cytoplasmic sequences (which presumably bind to distinct proteins) mediate similar functions as specific recycling signals? Third, while signal-dependent recycling of the B2AR is evident from study of mutant receptors that do not occur in natural tissues, does signal-dependent recycling distinguish the regulation of physiologically relevant GPCRs that are naturally expressed?

We have addressed these questions by focusing on the murine µ and {delta} opioid receptors (MOR and DOR, respectively), homologous G protein-coupled neuropeptide receptors that are natively co-expressed in several cell types (18-20) yet differ significantly in membrane trafficking properties (21-23). Studies of cultured cells indicate that both MOR and DOR are rapidly endocytosed via clathrin-coated pits and colocalize in early endosomes within several minutes after agonist-induced activation. DOR is subsequently down-regulated by endocytic sorting to lysosomes, whereas MOR efficiently recycles to the plasma membrane and is rapidly resensitized (24-26). Endocytic trafficking of DOR does not require covalent modification of receptors with ubiquitin (7) in contrast to certain other GPCRs (6, 27). A candidate lysosomal sorting protein binds strongly to the DOR tail but much less strongly to MOR in vitro and apparently not at all in vivo, suggesting that DOR is sorted to lysosomes via a noncovalent interaction and MOR recycles by default (8). On the other hand, MOR undergoes proteolytic down-regulation under some conditions (28-30), suggesting that this GPCR may be capable of lysosomal trafficking and that additional machinery may modulate its endocytic sorting.

The present studies indicate that MOR does indeed have a significant capacity to undergo endocytic sorting to lysosomes and define a 17-amino acid sequence present in the cytoplasmic tail of MOR that specifically promotes sorting of receptors into a rapid recycling pathway. The necessary and sufficient activity of this "MOR-derived endocytic recycling sequence" (MRS) establishes this sequence as a bona fide endocytic sorting signal. The MRS, although structurally distinct from the sorting signal identified previously in the B2AR, is functionally interchangeable with this sequence. These results demonstrate that endocytic recycling signals can distinguish the regulation of physiologically relevant GPCRs that are naturally co-expressed and indicate that such endocytic "recycling signals" are not unique to a single GPCR. Furthermore they suggest that a considerable variety of such signals may exist in the GPCR superfamily.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Constructs—Amino-terminally FLAG-tagged versions of the cloned murine DOR-1 (31), murine MOR-1 (32), and the human B2AR (33) were described previously (3). MOR truncations and point mutations were generated by polymerase chain reaction (Vent polymerase, New England Biolabs) using pcDNA3-SFMOR as template. MOR7 was constructed by polymerase chain reaction of MOR12T where the downstream primer introduced the carboxyl-terminal 5 residues of SFMOR in-frame to the 3' end of the coding region followed by a stop codon. DOR/MOR fusions were constructed by insertion of a synthetic linker-adapter (Operon Technologies) encoding the desired sequence corresponding to segments of the MOR tail followed by a stop codon into an existing SrfI site present in the sequence encoding the carboxyl-terminal cytoplasmic domain of DOR. The truncated B2AR and B2AR/MOR chimera were constructed by inserting synthetic linkers encoding a stop signal or the carboxyl-terminal 17 residues of MOR followed by a stop signal, respectively, into an EcoRV site present in the sequence encoding the B2AR cytoplasmic tail. All mutated cDNAs were cloned into pcDNA3 (Invitrogen), sequences were verified by dideoxynucleotide sequencing (University of California San Francisco Genetics Core Facility), and functionality was confirmed in transfected human embryonic kidney (HEK)293 cells.

Cell Culture and Transfection—HEK293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (University of California San Francisco Cell Culture Facility). Cells grown in 6-cm culture dishes were transfected with 5 µg of plasmid DNA encoding the indicated receptor using calcium phosphate co-precipitation according to standard methods (34). For studies of receptor trafficking in transiently transfected cells, cells were transfected as above and plated onto glass coverslips 24 h after transfection, and experiments were conducted 24-48 h thereafter. Stably transfected cells expressing epitope-tagged receptors were generated by selection for neomycin resistance using 500 µg/ml G418 (Geneticin, Invitrogen). Colonies representing individual neoR clones were isolated, and clones were selected for further study based on receptor expression levels (assessed by fluorescence flow cytometry and Western blot of cell lysates using M1 anti-FLAG antibody). Radioligand binding assay using [3H]diprenorphine (Amersham Biosciences) as tracer indicated that this expression level corresponds to 0.5-1pmol of receptor/mg of membrane protein.

Fluorescence Microscopy—Endocytic trafficking of receptors initially labeled in the plasma membrane was visualized by fluorescence microscopy using a previously described "antibody feeding" method (15). In brief, transiently transfected 293 cells were grown on glass coverslips (Corning), and surface receptors were specifically labeled by incubating intact cells with M1 anti-FLAG antibody (2.0 µg/ml, Sigma). Surface-labeled cells were subsequently incubated (at 37 °C for 30 min) in the presence of the appropriate opioid (10 µM DADLE) or adrenergic (10 µM isoproterenol) agonist to drive agonist-induced internalization. Following this incubation, cells were either fixed immediately or were washed twice in Dulbecco's modified Eagle's medium and subsequently incubated (37 °C for 45 min) in the presence of an excess concentration of the appropriate opioid (10 µM naloxone) or adrenergic (10 µM alprenolol) antagonist to prevent receptor activation by any residual agonist and thereby prevent subsequent agonist-induced endocytosis. Cells were fixed in 4% paraformaldehyde freshly prepared in phosphate-buffered saline (PBS, pH 7.4) for 15 min and then quenched with three washes of Tris-buffered saline (pH 7.5) supplemented with 1 mM CaCl2. Specimens were permeabilized with 0.1% Triton X-100 (Sigma) in a blocking solution (3% dry milk in Tris-buffered saline plus 1 mM CaCl2) and incubated with fluorescein isothiocyanate-conjugated donkey anti-mouse secondary antibody (1:500 dilution, Jackson Immunoresearch) for 30 min to detect antibody-labeled receptors. Epifluorescence microscopy was performed using an inverted microscope (Nikon Diaphot) equipped with a Nikon 60xNA1.4 objective and standard interference filter sets (Omega Optical). Images were collected using a 12-bit cooled charge-coupled device camera (Princeton Instruments) interfaced to a Macintosh computer running IPLab Spectrum software (Scanalytics). For colocalization studies, receptors were detected using a rabbit anti-FLAG antibody (Sigma) followed by incubation with fluorescein isothiocyanate-conjugated donkey anti-rabbit secondary antibody (1:500 dilution, Jackson Immunoresearch). Lysosomes were localized using LAMP1 monoclonal antibody obtained from the Developmental Studies Hybridoma Data Bank followed by incubation with Cy3-conjugated donkey anti-mouse secondary antibody. Confocal fluorescence microscopy was performed using a Zeiss LSM510 microscope fitted with a Zeiss 63xNA1.4 objective operated in single photon mode with standard filter sets and standard (one Airy disc) pinhole.

Quantative Analysis of Receptor Recycling Using Fluorescence Ratio Microscopy—To quantify the extent of recycling observed following agonist removal in individual cells, a modified version of the antibody feeding method was devised. Transiently transfected cells grown on glass coverslips were incubated with Alexa488-conjugated M1 anti-FLAG antibody (prepared by standard methods using Alexa-fluor 488 N-hydroxysuccinimide ester, Molecular Probes) to selectively label FLAG-tagged receptors present in the plasma membrane at the beginning of the experiment. Then cells were incubated (at 37 °C for 30 min) in the presence of 10 µM DADLE or 10 µM isoproterenol to drive internalization as above. At the end of this incubation cells were quickly washed three times in PBS lacking Ca2+ or Mg2+ and supplemented with 0.04% EDTA to dissociate FLAG antibody bound to residual surface receptors remaining in the plasma membrane, thereby leaving antibody bound only to the internalized pool of receptors. EDTA-stripped cells were then incubated (at 37 °C for 45 min) in the presence of 10 µM naloxone or 10 µM alprenolol to prevent subsequent receptor activation and to allow recycling to occur, and then cells were fixed with 4% paraformaldehyde, PBS under non-permeabilizing conditions, quenched with Tris-buffered saline with 3% bovine serum albumin (but no Triton X-100), and incubated with Cy3-conjugated donkey anti-mouse secondary antibody to detect recycled, antibody-labeled receptors. For experiments examining the effect of latrunculin B, immediately following the EDTA stripping step cells were incubated on ice for 1 h in medium containing antagonist and 10 µg/ml latrunculin B followed by a further 45-min incubation at 37 °C to allow recycling of internalized receptors. In each experiment, and for each receptor construct examined, two parallel control coverslips were included, one in which cells were fixed after a 30-min incubation in the absence of agonist and without an EDTA stripping step (100% surface receptor control) and one in which cells were fixed immediately after the EDTA-mediated stripping step (0% recycled control). Cells were examined by epifluorescence microscopy using appropriate filter sets to selectively detect Alexa488 or Cy3, and staining intensities of each fluor in individual cells were integrated using IPLab Image software. This analysis indicated that the efficiency of the EDTA strip (reduction of Cy3 staining intensity in the 0% recycled control relative to the 100% surface receptor control) was >95%, consistent with previous measures using fluorescence flow cytometry. The percentage of receptors recycled in individual cells following agonist washout was then calculated from the red/green ratios determined from the control conditions according to the following formula: (E - Z)/(C - Z) x 100, where E = the mean ratio for the experimental coverslip, Z = the mean ratio for the zero surface control, and C = the mean ratio for the 100% surface control. 20-30 cells/construct/condition were analyzed at random in this manner for each experiment, and average values reported under "Results" represent mean recycling percentages derived from three to five independent experiments.

Fluorescence Flow Cytometry—Internalization and recycling of epitope-tagged receptors were estimated using fluorescence flow cytometry of stably transfected cells to measure changes in the relative amount of FLAG-tagged receptors present in the plasma membrane after surface labeling with Alexa488-conjugated M1 antibody as described previously (16). Fluorescence flow cytometry was performed using a FACScan instrument (BD Biosciences). 20,000 cells were collected for each sample. Triplicate samples were analyzed for each condition in each experiment. The mean fluorescence values for each experiment (n = 5 experiments) were averaged, and the S.E. was calculated across all experiments.

Surface Biotinylation and Assays of Receptor Proteolysis—A previously described cell surface biotinylation method was used to specifically detect FLAG-tagged receptors present in the plasma membrane and to measure their proteolysis (3). Briefly, stably transfected 293 cells expressing the indicated FLAG-tagged receptors were grown in 10-cm dishes, washed twice with ice-cold PBS, and incubated with 300 µg/ml sulfo-N-hydroxysuccinimide-biotin (Pierce) in PBS for 30 min at 4 °C to biotinylate surface proteins. Unreacted biotin was quenched and removed with three washes of ice-cold Tris-buffered saline. Biotinylated cells were then transferred to prewarmed medium containing a 10 µM concentration of the appropriate agonist for the indicated times, and then cells were immediately chilled on ice and lysed in extraction buffer (1.0% (v/v) Triton X-100, 10 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride). Extracts were clarified by centrifugation (12,000 x g for 20 min), and then biotinylated proteins were isolated from cell extracts by immobilization on streptavidin-conjugated Sepharose beads (Amersham Biosciences) and washing five times in PBS containing 0.5% Triton X-100 to remove nonspecifically bound material. Washed beads were eluted with SDS sample buffer, and eluted proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed for FLAG-tagged receptor by immunoblotting using M1 antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham Biosciences), and SuperSignal detection reagent (Pierce). Band intensities were quantified by densitometry of films exposed in the linear range using a range of lysates run in parallel as reference, imaged using a charge-coupled device camera, and analyzed using FluorChem Version 2.0 software (AlphaInnotech Corp.).

GST Fusion Protein Binding Assay—Clarified HEK293 cell lysates were incubated for 4 h with 5 µg of the indicated GST-receptor tail fusion protein, which was prebound to glutathione-Sepharose (Amersham Biosciences). Beads were washed five times with extraction buffer, and bound proteins were eluted with SDS sample buffer. Eluted proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-EBP50 antibody (supplied by Dr. Anthony Bretscher, Cornell University) or mouse anti-NSF antibody (supplied by Dr. Sidney Whiteheart, University of Kentucky).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In the course of characterizing a series of stably transfected cell lines expressing truncated mutant opioid receptors, we noticed that truncation of 17 residues from the cytoplasmic tail of MOR1 created a functional mutant receptor (MOR17T) with significantly reduced stability after agonist-induced endocytosis. A FLAG-tagged version of MOR17T, labeled by surface biotinylation (15) in stably transfected HEK293 cells, was extensively proteolyzed within 2 h after addition of the peptide agonist DAMGO to the culture medium. In contrast, little proteolysis of wild type MOR was observed even after 4 h of agonist exposure (Fig. 1, A and B). The MOR17T mutant receptor colocalized extensively with the late endosome/lysosome marker LAMP1 within 60 min after agonist addition to the culture medium, whereas wild type MOR was localized primarily in LAMP1-negative endocytic vesicles under these conditions (Fig. 1C). Together these results indicate that the MOR17T truncated mutant receptor traffics to lysosomes at a significantly increased rate relative to wild type MOR, and they suggest that the distal cytoplasmic tail of MOR contains a structural element that inhibits lysosomal trafficking of receptors.



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  		FIG. 1.
A truncated mutant MOR exhibits enhanced agonist-induced trafficking to lysosomes. A, stably transfected HEK293 cells expressing FLAG-tagged MOR or MOR17T, a truncation mutant lacking the last 17 carboxyl-terminal residues, were surface-biotinylated and incubated in the absence or presence of 10 µM DAMGO for the indicated times. The fate of surface-labeled receptors was then ascertained by streptavidin pull-down (PD) followed by anti-FLAG immunoblot (IB). A representative immunoblot is shown. B, quantification of anti-FLAG Western blots from experiments performed as in A. Bars represent the mean recovery of surface-biotinylated receptors (relative to the unstimulated control), and error bars represent the S.E. of data derived from four independent experiments. C, colocalization of MOR and MOR17T relative to LAMP1 visualized by confocal fluorescence microscopy in representative HEK293 cells fixed after a 60-min incubation in the presence of DAMGO (10 µM).

 
To determine whether this structural element is required for efficient recycling of MOR to the plasma membrane after agonist-induced endocytosis, we utilized fluorescence flow cytometry to compare ligand effects on internalization and recovery of surface-accessible MOR and MOR17T mutant receptors. Stably transfected cells expressing FLAG-tagged MOR or MOR17T displayed a similar reduction (~50-60%) in surface receptor immunoreactivity following a 30-min exposure to DAMGO, indicating that truncation of the distal cytoplasmic tail of MOR did not markedly affect agonist-induced endocytosis. However, while the number of immunoreactive MORs present in the plasma membrane recovered almost completely within 45 min after removal of agonist from the culture medium, the MOR17T truncated mutant receptor exhibited significantly reduced surface recovery even when assayed 90 min after agonist washout (Fig. 2A). To investigate this observation further, we developed a ratiometric fluorescence-based assay in which the internalized pool of receptors is specifically labeled with a monoclonal antibody that does not interfere with GPCR recycling (15), and recycling of the internal receptor pool is detected by surface accessibility to a secondary antibody (Fig. 2B). This assay was validated by comparing the behavior of FLAG-tagged MOR with that of the murine DOR, a distinct opioid receptor that endocytoses via a similar early endocytic pathway (35) but does not recycle efficiently to the plasma membrane (3). Consistent with this, little recycling of antibody-labeled DOR was observed 45 min after washout of DADLE (a peptide agonist that induces rapid endocytosis of both DOR and MOR) as indicated by the weak cell staining with the surface-applied secondary antibody (Fig. 2C, top panels). In contrast, under the same conditions a strong signal representing recycled MOR was observed (Fig. 2C, bottom panels). Quantification of these data by ratiometric analysis (see "Experimental Procedures") of numerous transiently transfected cells, which represent a range of receptor expression levels, confirmed that the MOR17T mutant receptor recycled to a much smaller degree (24 ± 6.7%) than wild type MOR (81 ± 8.1%). Furthermore the small amount of MOR17T recycling observed in this assay was closely similar to that of the wild type DOR (20 ± 5.9%) (Fig. 2D). These observations indicate that the MOR tail contains a structural element that is required for efficient recycling of internalized receptors to the plasma membrane.



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  		FIG. 2.
Effect of MOR truncation on recycling of receptors to the plasma membrane after agonist-induced endocytosis. A, stably transfected cells expressing FLAG-tagged MOR or MOR17T were analyzed using fluorescence flow cytometry to monitor ligand-regulated effects on surface receptor number. The peptide agonist DAMGO (10 µM) was added at t = -30 min, agonist washout/antagonist addition was performed at t = 0, and recovery of surface receptor number was subsequently monitored. Data points represent mean surface receptor fluorescence (relative to untreated control) derived from five independent experiments with 20,000 cells analyzed/condition in each experiment. The error bars represent the S.E. calculated across experiments. The right panel indicates fractional recycling calculated from these data as described under "Experimental Procedures." B, schematic of an immunofluorescent assay to specifically detect and quantify recycling of internalized receptors to the plasma membrane. C, representative example of results obtained comparing DOR and MOR recycling using the assay schematized in B. Green signal represents Alexa488 fluorescence (total internalized receptors), and red signal represents Cy3 fluorescence (recycled receptors). D, calculation of the fractional recycling of FLAG-tagged DOR, MOR, and MOR17T expressed in transiently transfected HEK293 cells by quantification of green and red fluorescence signals detected in individual cells. Bars indicate mean percentage of receptor recycling (relative to the total amount of receptors internalized after exposure to agonist for 30 min) that was detected 45 min after agonist washout as determined from the mean of four independent experiments in which each experiment involved ratiometric analysis of 20-30 randomly selected cells for each condition and construct. Error bars represent the S.E. recycling values calculated across experiments. int., internalized; R, red; G, green.

 
The ratiometric recycling assay was used to define more precisely the region in the MOR tail required for efficient recycling of receptors (Fig. 3A). Truncation of the distal 12 residues from the cytoplasmic tail of MOR (MOR12T mutant receptor) caused an inhibition of receptor recycling similar to that observed in the MOR17T mutant receptor, whereas removal of the distal 5 residues (MOR5T mutant receptor) did not significantly inhibit recycling relative to wild type MOR. Internal deletion of the 7-residue sequence that distinguishes the (non-recycling) MOR12T and (recycling) MOR5T mutant receptors (MOR7 mutant receptor) inhibited recycling to a similar degree as the MOR17T or MOR12T truncations (Fig. 3B). These observations indicate that this 7-residue sequence (LENLEAE) is an essential element required for efficient recycling of MOR following ligand-induced endocytosis.



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  		FIG. 3.
Mutational analysis of the cytoplasmic sequence required for rapid recycling of MOR. A, schematic of MOR mutants analyzed. B, fractional recycling of mutant receptors determined by the ratiometric assay 45 min after agonist washout (four independent experiments, 20-30 cells for each condition in each experiment).

 
We next sought to determine whether the sequence required for efficient recycling of MOR is sufficient to function as an autonomous endocytic sorting signal when transferred to a distinct GPCR. To begin to address this question we fused various MOR-derived tail sequences to the cytoplasmic tail of DOR (Fig. 4A), and then used the ratiometric recycling assay to examine endocytic trafficking in transiently transfected HEK293 cells (Fig. 4B). Fusing the carboxyl-terminal 17 residues derived from the MOR tail to DOR created a chimeric mutant receptor that recycled almost completely to the plasma membrane within 45 min after agonist removal (80 ± 6.7%), an extent of recycling indistinguishable from that of wild type MOR (Fig. 2D). Both the amino-terminal and carboxyl-terminal 12 residues from this sequence were also sufficient to significantly enhance recycling of a chimeric DOR (DOR/MOR12p and DOR/MOR12d) (Fig. 4B). However the "core" LENLEAE sequence, present in all three of these recycling chimeras and essential for efficient recycling of MOR, was not sufficient by itself to promote detectable recycling of the DOR/MOR7 chimeric mutant receptor. Nevertheless alanine substitution of this sequence abrogated the recycling activity of the 17-residue sequence (DOR/MORAx7 mutant receptor). These results suggest that the MOR-derived tail sequence is indeed sufficient to promote rapid recycling of a distinct GPCR when fused to the cytoplasmic tail and that the LENLEAE sequence is an essential (but not fully sufficient) element of this MRS.



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  		FIG. 4.
Recycling activity of the MOR-derived tail sequence when fused to DOR. A, schematic of DOR/MOR tail receptor fusion constructs analyzed. Solid bars denote DOR sequence, and open bars denote MOR-derived sequence fused to the cytoplasmic tail constructed as described under "Experimental Procedures." B, fractional recycling of chimeric mutant receptors assayed in transiently transfected HEK293 cells using the ratiometric assay (four independent experiments, 20-30 cells for each condition in each experiment). C, analysis of ligand effects on DOR and DOR/MOR17 surface receptor number in stably transfected HEK293 cells using fluorescence flow cytometry as in Fig. 2A. Results are compiled from five independent experiments. D, fractional recycling calculated from the flow cytometric data. E, rapid internalization kinetics of DOR and DOR/MOR17 assayed by fluorescence flow cytometry. Bars represent mean fractional internalization (relative to untreated cells) at each time point after addition of a saturating concentration of agonist (10 µM DADLE) to the culture medium. Error bars represent the S.D. from triplicate samples (each derived from 20,000 cells) of a representative experiment.

 
The recycling activity of the transplantable MRS was confirmed in stably transfected cells using the flow cytometric assay. The DOR/MOR17 chimera recycled rapidly and nearly completely to the plasma membrane within 30-45 min after agonist removal in contrast to DOR, which recycled to a much smaller extent even when assayed 90 min after agonist removal (Fig. 4, C and D). Further examination of the time course of DOR and DOR/MOR17 recycling revealed the major effect of adding the MRS was on the extent rather than rate of receptor recycling, suggesting that the MRS increased the fraction of endocytosed receptors that traverse a rapid recycling pathway (Fig. 4C). We noticed that the DOR/MOR17 mutant receptor, in addition to its markedly enhanced recycling relative to DOR after agonist washout, exhibited significantly reduced agonist-induced internalization at the 30-min time point of agonist exposure. This effect could represent the ability of the MRS to promote rapid recycling of receptors even before agonist washout with a corresponding reduction in the net internalization measured (36) or could reflect an additional effect of the MRS as an inhibitor of receptor endocytosis. Thus we compared internalization of the DOR and DOR/MOR17 mutant receptors measured at early time points after agonist addition when initial endocytosis is expected to be the major determinant of net internalization. These receptors were indistinguishable in their net internalization when assayed 2 and 4 min after agonist addition, whereas significantly reduced internalization of the DOR/MOR17 mutant receptor relative to DOR was apparent after 6 min and was further reduced thereafter (Fig. 4E). These observations indicate that the MRS does not detectably inhibit the initial agonist-induced endocytosis of receptors but specifically promotes recycling of internalized receptors. Furthermore they suggest that the MRS is sufficient to promote rapid recycling of receptors even in the continuous presence of agonist.

To examine which residues within the LENLEAE sequence are critical for its recycling activity we generated several additional chimeric receptors containing individual amino acid mutations in this portion of the MRS (Fig. 5A) and examined effects on recycling after agonist washout (Fig. 5B). Alanine substitution of the three glutamic acid residues or of the single asparagine residue had no detectable effect on the MRS recycling activity. Mutation of the leucine residues, either together or individually, abrogated the recycling activity and resulted in chimeric receptors that recycled to an extent similar to that of wild type DOR. To test whether these leucine residues are also essential for the MRS activity in its native context, we generated a full-length mutant MOR in which only these residues were mutated. This mutation blocked recycling to a similar extent as truncation of the entire MRS, resulting in a mutant MOR that was indistinguishable from DOR in its failure to recycle efficiently. Furthermore mutation of these leucine residues resulted in substantially enhanced agonist-induced proteolysis of receptors labeled by surface biotinylation (Fig. 5C), confirming that these residues are critical to the functional activity of the MRS in specifying recycling rather than degradative membrane trafficking of the full-length MOR.



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  		FIG. 5.
Identification of two critical leucine residues in the MRS. A, schematic of constructs analyzed. Solid bars denote DOR sequence, and open bars denote MOR sequence. Bold letters indicate point mutations of individual residues in the MOR-derived tail sequence. B, fractional recycling of the indicated mutant receptors determined by ratiometric assay 45 min after agonist washout (four independent experiments, 20-30 cells for each condition in each experiment). C, effect of the MOR2xL-A (MOR-LA) mutation on agonist-induced proteolysis of surface-biotinylated receptors analyzed as in Fig. 1A. A representative immunoblot is shown from three independent experiments. PD, pull-down; IB, immunoblot.

 
To further investigate how fully the MRS distinguishes the endocytic trafficking properties of these physiologically relevant opioid receptors, we next examined how strongly the MRS affects lysosomal trafficking when fused to DOR. This is a more stringent test of endocytic sorting activity than the measurement of recycling measured after agonist washout because it requires receptors to recycle efficiently during multiple rounds of endocytosis and in the continuous presence of agonist. As observed previously, surface-labeled DOR was almost completely proteolyzed within 2 h after addition of agonist to the culture medium, consistent with its postendocytic sorting to lysosomes. In contrast, ligand-induced proteolysis of the DOR/MOR17 chimera was significantly inhibited as indicated by the greatly increased recovery of surface-biotinylated receptors following agonist-induced endocytosis (Fig. 6A). Quantification of these results from multiple experiments confirmed this conclusion and indicated that the MRS is sufficient to inhibit proteolysis of internalized DOR (Fig. 6B) to a similar degree as it inhibits proteolytic targeting of MOR (Fig. 1B). The sorting activity of the MRS was further evaluated by examining the localization of endocytosed receptors relative to LAMP1. Whereas wild type DOR colocalized extensively with LAMP1-positive endocytic structures 60 min after addition of agonist to the culture medium as shown previously (7), little colocalization of internalized DOR/MOR17 with LAMP1 was observed under these conditions (Fig. 6C). These results show the MRS is sufficient both to promote rapid recycling of opioid receptors and to strongly inhibit their postendocytic trafficking to lysosomes. The MRS thus functions as a major determinant distinguishing the endocytic trafficking of physiologically relevant GPCR subtypes.



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  		FIG. 6.
The MRS is sufficient to prevent lysosomal trafficking of DOR. A, agonist-induced proteolysis of surface-biotinylated DOR and DOR/MOR17 assayed in stably transfected HEK293 cells as in Fig. 1A. B, quantification of anti-FLAG immunoblots from experiments performed as in A. Error bars represent the S.E. of data derived from four independent experiments. C, colocalization of DOR and DOR/MOR17 with the lysosomal marker LAMP1 observed in representative HEK293 cells fixed after a 60-min incubation in the presence of DADLE (10 µM). C, control; PD, pull-down; IB, immunoblot.

 
The B2AR is a member of a distinct GPCR subfamily that also contains a sequence in the cytoplasmic tail that is necessary for its rapid recycling (15). This sequence functions as a ligand to engage in a PDZ domain-mediated interaction with NHERF/EBP50, an ezrin-binding protein that promotes recycling of B2AR (15, 16, 37). A distinct interaction of this sequence with NSF, a protein required for vesicle fusion, is also proposed to mediate B2AR recycling (17). The opioid MRS sequence is completely distinct from the B2AR recycling signal and conforms to no known PDZ ligand consensus sequence, which in the vast majority of cases requires a carboxyl-terminal hydrophobic residue. Nevertheless the opioid-derived MRS could, in principle, mediate binding of MOR to NHERF/EBP50 via a novel PDZ-mediated binding interaction or a different domain of EBP50 or to NSF. To test this possibility we conducted an in vitro binding assay by incubating GST fusion proteins encoding the MOR carboxyl-terminal tail with HEK293 cell lysate. The MOR carboxyl-terminal tail did not detectably interact with either NHERF/EBP50 or NSF. In contrast, we easily detected binding of both proteins to the B2AR carboxyl-terminal tail (Fig. 7C). The B2AR and MOR-derived recycling signals, therefore, appear to be molecularly distinct both at the level of primary amino acid sequence and with respect to protein-protein interactions. Both sequences, however, are sufficient to promote efficient recycling when fused to DOR (16). Thus it seemed possible that these otherwise diverse sequences may represent functionally interchangeable endocytic trafficking signals. To test this we examined the effect of introducing the opioid-derived MRS into the cytoplasmic tail of a non-recycling mutant B2AR created by deleting its native recycling signal (15). Indeed the opioid-derived MRS was fully sufficient to restore efficient recycling when fused to this recycling-defective mutant catecholamine receptor as indicated both by visual assay of receptor redistribution to the plasma membrane (Fig. 7A) and by quantitative assay of recycling after agonist washout (Fig. 7B). Recycling of the B2AR is sensitive to the actin-depolymerizing agent latrunculin B presumably through a disruption of the cortical actin cytoskeleton and consequently ezrin and associated proteins such as NHERF/EBP50 (15). We were therefore interested in whether MOR recycling would be similarly affected. We tested the sensitivity of B2AR and MOR to latrunculin B in parallel experiments using the ratiometric recycling assay. B2AR recycling was dramatically inhibited by 10 µg/ml latrunculin B, whereas there was no significant effect of this compound on recycling of MOR (Fig. 7D). This result suggests that recycling of B2AR and MOR proceed through different downstream mechanisms.



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  		FIG. 7.
The opioid-derived recycling signal is functionally interchangeable with that from the B2AR, yet they are molecularly distinct. A, visualization of endocytic recycling effects of the MRS when fused to B2ART, a mutant B2AR in which the native recycling signal has been eliminated. B, fractional recycling of receptors observed 45 min after agonist washout using the ratiometric assay (four independent experiments, 20-30 cells for each condition in each experiment). C, comparison of the abilities of the B2AR and MOR carboxyl-terminal tails to bind NHERF/EBP50 and NSF, two proteins implicated in recycling of the B2AR. GST fusion proteins encoding the indicated receptor tail were incubated with 293 cell lysate followed by Western blotting to detect associated NHERF/EBP50 or NSF. The figure shows a representative experiment in which the blot was first probed for NHERF/EBP50 and then stripped and reprobed for NSF. The bottom panel shows a Ponceau stain of this blot to demonstrate equal loading of fusion proteins. GST-B2AR-Ala and GST-MOR12 are tail mutations that abrogate recycling of full-length receptors. D, comparison of the effects of actin depolymerization on recycling of the B2AR and MOR. Cells were incubated with or without 10 µg/ml latrunculin B prior to analysis by ratiometric recycling as described under "Experimental Procedures." Iso, isoproterenol; Alp, alprenolol.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies of signaling receptors have indicated that sorting of receptors between degradative and recycling pathways is mediated by signals that direct the trafficking of receptors to lysosomes (6, 38-40). When these signals are absent or interfered with, receptors are thought to then recycle by default (8, 11). Our results indicate that efficient recycling of opioid receptors to the plasma membrane does not occur by default but requires a specific sequence that is conserved in the MOR tail across species but is absent in DOR. This MRS is both necessary for efficient recycling of MOR and sufficient to promote rapid recycling of DOR and therefore constitutes a fully functional endocytic recycling signal. Previous studies indicate that covalent modification with ubiquitin is not necessary for trafficking of DOR to lysosomes (7) and that a non-covalent protein interaction modulates lysosomal sorting of DOR (8). Because this protein displays significantly stronger binding to DOR than MOR, it was previously proposed that MOR recycles by default. However, this relatively simple hypothesis is not consistent with the present results demonstrating that MOR has a latent capacity to traffic to lysosomes. In contrast to other membrane proteins that recycle in a signal-independent manner and in which recycling signals are thought not to exist (41), the differential endocytic membrane trafficking of opioid neuropeptide receptors appears to be determined primarily by receptor-specific differences in sorting of receptors into a rapid recycling pathway.

Functionally the MRS is reminiscent of a cytoplasmic sequence shown previously to be required for efficient recycling of the B2AR (15, 17). Indeed the present studies indicate that the recycling signals derived from these distinct GPCRs are functionally interchangeable. Thus MOR represents a new example of a GPCR whose normal recycling to the plasma membrane requires the activity of a modular recycling signal. The recycling activity of the B2AR tail sequence was identified by comparison of wild type receptors with mutant versions that do not occur in nature (15, 17). In contrast, the MRS distinguishes the endocytic trafficking of MOR from that of DOR, two naturally occurring physiologically important GPCRs that are natively co-expressed in tissues such as brain and spinal cord (18-20).

Remarkably the opioid-derived MRS is completely different in sequence from the recycling signal present in the B2AR and fails to detectably bind proteins implicated in B2AR recycling. Moreover B2AR and MOR recycling have different sensitivities to actin depolymerization, suggesting that the opioid-derived MRS functions by a distinct biochemical mechanism. Nevertheless these molecularly distinct trafficking signals are functionally quite similar. This observation suggests that there may exist a considerable diversity of modular endocytic recycling signals in the GPCR superfamily and raises interesting questions regarding the physiological role of this apparent complexity. Possibilities include roles in different downstream effector pathways, regulation of resensitization kinetics, and specific membrane targeting pathways in differentiated cells. We presently favor the idea that signal-mediated recycling may be important to a large number of mammalian GPCRs where the endocytic pathway is thought to mediate multiple effects on receptor signaling and regulation and where similar receptors differ greatly in the structure of their carboxyl-terminal cytoplasmic domains. There are a number of studies in the literature that are consistent with this idea. For example, the carboxyl-terminal cytoplasmic tail of the endothelin type A receptor influences receptor internalization in ways that have been reported to represent an increase in receptor recycling (42). In addition, studies of the human lutropin receptor have identified a sequence that, although not required for recycling of the natural receptor, is sufficient to promote recycling of a rat/human lutropin receptor chimera (43, 44). Furthermore recycling of the {kappa} opioid receptor can be promoted via a tail-dependent protein interaction previously implicated in trafficking of the B2AR (45). Thus, signal-mediated receptor recycling may emerge as a widespread mechanism for regulating GPCR postendocytic sorting.

A major goal of future study will be to identify cytoplasmic proteins that interact with the MRS and to elucidate the mechanism by which this recycling signal functions. The present studies also raise more general questions regarding how recycling signals function in relation to other machinery controlling the endocytic membrane trafficking of GPCRs. In this regard it is particularly noteworthy that the MRS acts as a functional recycling signal in both opioid and adrenergic receptor chimeras. This is surprising because, in contrast to DOR, the B2AR requires ubiquitylation to traffic to lysosomes (27). Thus the opioid-derived MRS appears to function as a dominant trafficking signal relative to both ubiquitylation-dependent and -independent lysosomal sorting mechanisms. It must be noted that this is likewise true of the B2AR recycling signal, indicating a potential similarity in the underlying mechanisms by which these different receptors recycle. This underscores the important challenge of resolving into common principles the apparent diversity of mechanisms controlling the recycling and lysosomal trafficking of receptors. The present results provide a significant step forward by defining a novel recycling signal involved in sorting functionally relevant GPCRs and by establishing that signal-dependent recycling may be a more general property of GPCR membrane trafficking than previously realized.


   FOOTNOTES
 
* This work was supported by a research grant from the National Institutes of Health (to M. v. Z.) and by a National Institutes of Health individual national research service award (to M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: N-216 Genentech Hall, University of California, 600 16th St., San Francisco, CA 94143-2140. Tel.: 415-476-7855; Fax: 415-504-0169; E-mail: mbt2m{at}itsa.ucsf.edu.

1 The abbreviations used are: GPCR, G protein-coupled receptor; B2AR, {beta}2 adrenergic catecholamine receptor; MOR, µ opioid receptor; DOR, {delta} opioid receptor; MRS, MOR-derived endocytic recycling sequence; DADLE, [D-Ala2,D-Leu5]-enkephalin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; NSF, N-ethylmaleimide-sensitive factor; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; NHERF, Na+/H+ exchanger regulatory factor; EBP50, ezrin-radixin-moesin (ERM)-binding phosphoprotein 50; LAMP1, lysosome-associated membrane protein 1. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Anthony Bretscher for providing EBP50 antibody and Dr. Sidney Whiteheart for anti-NSF.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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The Composition of the {beta}-2 Adrenergic Receptor Oligomer Affects Its Membrane Trafficking after Ligand-Induced Endocytosis
Mol. Pharmacol., January 1, 2005; 67(1): 288 - 297.
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A. Heydorn, B. P. Sondergaard, B. Ersboll, B. Holst, F. C. Nielsen, C. R. Haft, J. Whistler, and T. W. Schwartz
A Library of 7TM Receptor C-terminal Tails: INTERACTIONS WITH THE PROPOSED POST-ENDOCYTIC SORTING PROTEINS ERM-BINDING PHOSPHOPROTEIN 50 (EBP50), N-ETHYLMALEIMIDE-SENSITIVE FACTOR (NSF), SORTING NEXIN 1 (SNX1), AND G PROTEIN-COUPLED RECEPTOR-ASSOCIATED SORTING PROTEIN (GASP)
J. Biol. Chem., December 24, 2004; 279(52): 54291 - 54303.
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W. Liang and P. H. Fishman
Resistance of the Human {beta}1-Adrenergic Receptor to Agonist-induced Ubiquitination: A MECHANISM FOR IMPAIRED RECEPTOR DEGRADATION
J. Biol. Chem., November 5, 2004; 279(45): 46882 - 46889.
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G. A. Vargas and M. von Zastrow
Identification of a Novel Endocytic Recycling Signal in the D1 Dopamine Receptor
J. Biol. Chem., September 3, 2004; 279(36): 37461 - 37469.
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V. C. Dang and J. T. Williams
Chronic Morphine Treatment Reduces Recovery from Opioid Desensitization
J. Neurosci., September 1, 2004; 24(35): 7699 - 7706.
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 EndocrinologyHome page
N. R. Farid and M. W. Szkudlinski
Minireview: Structural and Functional Evolution of the Thyrotropin Receptor
Endocrinology, September 1, 2004; 145(9): 4048 - 4057.
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I. Lecoq, N. Marie, Ph. Jauzac, and S. Allouche
Different Regulation of Human {delta}-Opioid Receptors by SNC-80 [(+)-4-[({alpha}R)-{alpha}-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] and Endogenous Enkephalins
J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 666 - 677.
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M. Corbani, C. Gonindard, and J.-C. Meunier
Ligand-Regulated Internalization of the Opioid Receptor-Like 1: A Confocal Study
Endocrinology, June 1, 2004; 145(6): 2876 - 2885.
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B. M. Cox and A. T. Crowder
Receptor Domains Regulating {micro} Opioid Receptor Uncoupling and Internalization: Relevance to Opioid Tolerance
Mol. Pharmacol., March 1, 2004; 65(3): 492 - 495.
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C. Galet, T. Hirakawa, and M. Ascoli
The Postendocytotic Trafficking of the Human Lutropin Receptor Is Mediated by a Transferable Motif Consisting of the C-Terminal Cysteine and an Upstream Leucine
Mol. Endocrinol., February 1, 2004; 18(2): 434 - 446.
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