Cell Surface Expression of GluR5 Kainate Receptors Is Regulated by an Endoplasmic Reticulum Retention Signal
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309585200
ISSN1083-351X
AutoresZhao Ren, Nathan J. Riley, Leigh A. Needleman, James M. Sanders, Geoffrey T. Swanson, John Marshall,
Tópico(s)Receptor Mechanisms and Signaling
ResumoKainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5–2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5–2b and demonstrate that alternative splicing is an important mechanism regulating KAR function. Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5–2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5–2b and demonstrate that alternative splicing is an important mechanism regulating KAR function. Kainate, NMDA, 1The abbreviations used are: NMDAN-methyl-d-aspartateAMPAα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidERendoplasmic reticulumHEKhuman embryonic kidneyKARkainate receptorendo Hendoglycosidase HDMEMDulbecco's modified Eagle's mediumGFPgreen fluorescent proteinNHSN-hydroxysulfosuccinimideFITCfluorescein isothiocyanateTBSTris-buffered salinePBSphosphate-buffered salinePNGase Fpeptide-N-glycosidase FPKCprotein kinase CPKAprotein kinase AFACflow-assisted cytometry. and AMPA receptors mediate glutamatergic excitatory neurotransmission in the mammalian central nervous system (1Seeburg P.H. Trends Neurosci. 1993; 16: 359-365Abstract Full Text PDF PubMed Scopus (815) Google Scholar, 2Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3668) Google Scholar). The intracellular trafficking and surface delivery of multimeric transmembrane proteins, such as kainate receptors, are likely to be tightly controlled processes requiring proper folding and assembly of constituent subunits, so that only fully assembled, functional receptors can be expressed on the plasma membrane. The endoplasmic reticulum (ER) is the primary checkpoint for these complex events (3Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (447) Google Scholar). Recently, we demonstrated that an arginine-rich ER retention signal controls the ER egress and surface expression of KA2 kainate receptor subunits (4Ren Z. Riley N.J. Garcia E.P. Sanders J.M. Swanson G.T. Marshall J. J. Neurosci. 2003; 23: 6608-6616Crossref PubMed Google Scholar). N-methyl-d-aspartate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid endoplasmic reticulum human embryonic kidney kainate receptor endoglycosidase H Dulbecco's modified Eagle's medium green fluorescent protein N-hydroxysulfosuccinimide fluorescein isothiocyanate Tris-buffered saline phosphate-buffered saline peptide-N-glycosidase F protein kinase C protein kinase A flow-assisted cytometry. Alternative splicing and RNA editing of ionotropic glutamate receptors have been shown to play important roles in receptor assembly and trafficking (5Okabe S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar, 6Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 7Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 8Greger I.H. Khatri L. Ziff E.B. Neuron. 2002; 34: 759-772Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). In particular, an RXR (where R is arginine and X a large neutral or positively charged residue) motif was found in the alternatively spliced C1 cassette in the C terminus of NMDA receptor NR1 subunit, mediating the intracellular retention of most C1-containing NR1 isoforms (6Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 7Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar). Furthermore, this RXR-mediated ER retention was regulated by protein kinase C (PKC) phosphorylation (6Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar) and a type I PDZ (postsynaptic density-95/Disc large/zona occludens-1)-binding motif in the alternate C2′ splice cassette (6Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 7Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar). In addition, arginine 607 (Arg-607) at a Q/R (glutamine/arginine) mRNA editing site has been reported to determine the ER retention of GluR2 AMPA receptor subunits in cultured neurons (8Greger I.H. Khatri L. Ziff E.B. Neuron. 2002; 34: 759-772Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The GluR5 kainate receptor subunit also undergoes alternative splicing and RNA editing. Alternative splicing of the N-terminal region of the rat GluR5 mRNA yields two different isoforms, GluR5–1 and GluR5–2, where GluR5–1 contains an extra 15 amino acids compared with GluR5–2 (9Bettler B. Boulter J. Hermans-Borgmeyer I. O'Shea-Greenfield A. Deneris E.S. Moll C. Borgmeyer U. Hollmann M. Heinemann S. Neuron. 1990; 5: 583-595Abstract Full Text PDF PubMed Scopus (512) Google Scholar). In addition, GluR5–2 has three splice variants in the C terminus, named GluR5–2a, GluR5–2b, and GluR5–2c (9Bettler B. Boulter J. Hermans-Borgmeyer I. O'Shea-Greenfield A. Deneris E.S. Moll C. Borgmeyer U. Hollmann M. Heinemann S. Neuron. 1990; 5: 583-595Abstract Full Text PDF PubMed Scopus (512) Google Scholar, 10Sommer B. Burnashev N. Verdoorn T.A. Keinanen K. Sakmann B. Seeburg P.H. EMBO J. 1992; 11: 1651-1656Crossref PubMed Scopus (275) Google Scholar). The shortest isoform, GluR5–2a, results from the introduction of a stop codon after 2 amino acids in the alternatively spliced cassette (10Sommer B. Burnashev N. Verdoorn T.A. Keinanen K. Sakmann B. Seeburg P.H. EMBO J. 1992; 11: 1651-1656Crossref PubMed Scopus (275) Google Scholar), whereas the longest variant, GluR5–2c, is generated by an in-frame insertion of nucleotides, coding for an additional 29 amino acids, into the GluR5–2b sequence (10Sommer B. Burnashev N. Verdoorn T.A. Keinanen K. Sakmann B. Seeburg P.H. EMBO J. 1992; 11: 1651-1656Crossref PubMed Scopus (275) Google Scholar). GluR5–2b and GluR5–2c share a C-terminal consensus type I PDZ-binding domain that is absent in GluR5–2a. Although RNA editing of GluR5 is known to modulate its single-channel conductance, calcium permeability, and rectification properties (10Sommer B. Burnashev N. Verdoorn T.A. Keinanen K. Sakmann B. Seeburg P.H. EMBO J. 1992; 11: 1651-1656Crossref PubMed Scopus (275) Google Scholar, 11Swanson G.T. Feldmeyer D. Kaneda M. Cull-Candy S.G. J. Physiol. (Lond.). 1996; 492: 129-142Crossref Scopus (158) Google Scholar), the functional significance of these splice variants remains to be defined. In the present study, we investigated the intracellular trafficking and surface expression of the GluR5 splice variants, GluR5–2a and GluR5–2b. We now report the identification of a novel ER retention signal present in the alternatively spliced C-terminal domain of GluR5–2b. This signal consists of a critical Arg-896 and surrounding residues, disruption of which promotes ER exit and surface delivery of GluR5–2b receptors in both heterologous cells and hippocampal neurons. In addition, a pair of positively charged residues in the C-terminal region, Arg-900 and Lys-901, were found to regulate ER export of the receptors. Together, our results demonstrate the functional significance of GluR5 alternative splicing and identify ER retention as an important mechanism regulating kainate receptor trafficking and surface expression. Cell Culture—Human embryonic kidney 293 (HEK293) cells, normal rat kidney (NRK) cells, and COS7 cells (ATCC, Manassas, VA) were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 50 units/ml penicillin and streptomycin (37 °C, 5% CO2). For immunocytochemistry experiments, cells were plated onto poly-d-lysine-coated glass coverslips in the same growth medium. Primary cultures of hippocampal neurons were obtained from E18 rat embryos. Briefly, hippocampi were dissected from E18 Sprague-Dawley rats and dissociated with trypsin. Dissociated cells were then plated onto poly-l-lysine (Sigma)-coated glass coverslips at a density of 7500 cells/cm2 in Neurobasal medium (Invitrogen) supplemented with Glutamax and B27. Arabinofuranosyl-cytosine (5 μm) was added 3 days after plating, and cells were fed twice weekly thereafter. Molecular Biology—The pMLSV N1/N4 expression vector for human Tac was purchased from ATCC. The Tac coding sequence was then amplified by PCR and subcloned into a HindIII-XbaI site in pcDNA3. To generate the Tac-GluR5 and Tac-GluR6 chimeras, we first amplified the N-terminal and transmembrane sequences of Tac, as well as the C termini of GluR5/2a, GluR5–2b, and GluR6 by PCR, then ligated both fragments into a HindIII-XbaI site in pcDNA3 vector after digestion. Deletion mutants of Tac-G5/2b were made by PCR amplification of the desired sequences and subsequent subcloning into the HindIII-XbaI site in pcDNA3 expression vector. Tac-G5/2b and Tac-G6 point mutations were also made by PCR. GluR5–2a-pRK5 and GluR5–2b-pBluescript SK(–) were generous gifts from Dr. Peter Seeburg (Max Planck Institute for Medical Research, Heidelberg, Germany) and Dr. Steve Heinemann (Salk Institute, La Jolla, CA), respectively. To make GFP-GluR5–2b in pRK5 expression vector, we first replaced a C-terminal BamHI-EcoRI fragment in GluR5–2a-pRK5 with the corresponding piece from GluR5–2b amplified by PCR. The GFP coding sequence was then amplified by PCR from pEGFP-C2 vector (BD Biosciences, San Jose, CA) and inserted after residue 36 (counted from the initiator methionine) of GluR5–2b in pRK5, using an XhoI site created by QuikChange (Stratagene, La Jolla, CA). The pDsRed2-ER vector was purchased from BD Biosciences. Myc-tagged PICK1 (protein interacting with C kinase) and GRIP1a (glutamate receptor interacting protein) in pRK5 vector were gifts from Dr. Richard Huganir (Johns Hopkins University, Baltimore, MD), and PSD-95-pGW1 (postsynaptic density-95) was a gift from Dr. Morgan Sheng (Massachusetts Institute of Technology, Cambridge, MA). All the mutagenesis and PCR-derived sequences were sequenced for verification. Transfection—HEK293, COS7, and NRK cells were transfected using FuGENE 6 transfection reagent (Roche Molecular Biochemicals), following the manufacturer's protocol for transient transfection of adherent cells. Cultured hippocampal neurons (DIV7) were transfected with the LipofectAMINE 2000 transfection reagent (Invitrogen), using the protocol from the manufacturer. Surface and intracellular protein expressions were analyzed 48 h after transfection. Antibodies—Monoclonal anti-Tac (Covance, Princeton, NJ) was used at 1:800 or 1:2000 for immunostaining of heterologous cells or hippocampal neurons, and 1:4000 for flow cytometry. Polyclonal anti-Tac (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:200 for staining and 1:300 for Western blots. Monoclonal anti-TGN38 (Calbiochem, San Diego, CA), anti-mannosidase II (Covance), and polyclonal anti-giantin (Covance) were used at 1:1000 for immunofluorescence of heterologous cells. Polyclonal anti-calnexin (Stressgen, Victoria, British Columbia, Canada) and anti-GFP (Santa Cruz Biotechnology) were used at 1:200 for immunostaining. Monoclonal anti-autofluorescent protein antibody (Qbiogene, Carlsbad, CA) was used at 1:200 for immunofluorescence and 1:500 for flow cytometry. Monoclonal anti-GFP (B-2) antibody (Santa Cruz Biotechnology) was used at 1:300 for Western blots. Polyclonal anti-GluR5 (Upstate Biotechnology, Inc., Lake Placid, NY) was used at 1:100 for immunofluorescence and 1:1000 for Western blots. Monoclonal anti-PSD-95 (Upstate Biotechnology, Inc.) and anti-Myc (Santa Cruz Biotechnology) were used at 1:100,000 and 1:250, respectively, for Western blots. All secondary antibodies conjugated to fluorescein (FITC), phycoerythrin, or rhodamine red-X (Jackson Immunoresearch, West Grove, PA) were used at 1:200 for immunostaining. All secondary antibodies conjugated to horseradish peroxidase (BD Biosciences) were used at 1:10,000 for Western blot. Protein Preparation and Immunoblotting—Forty-eight hours after transfection, HEK cells grown in 60-mm culture dishes were washed once with cold TBS and homogenized in 1 ml of lysis buffer (1% Triton X-100 in TBS with 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 2 mm EDTA, pH 8.0). The samples were then solubilized for 1 h at 4 °C and cleared by centrifugation at 14,000 × g for 30 min. Aliquots of the supernatants were boiled for 5 min in 1× sample buffer and resolved on 8% or 10% SDS-PAGE. Gels were then Western blotted, immunostained, and visualized with Supersignal West Pico chemiluminescent substrate (Pierce). Biotinylation of Cell Surface Protein—Transfected HEK cells, grown in poly-d-lysine-coated 60-mm culture dishes, were washed three times with ice-cold PBS and incubated 15 min with 1.0 mg/ml EZ-link™ sulfo-N-hydroxysulfosuccinimide (NHS)-S-S-biotin (Pierce) in cold PBS, pH 8.0, with gentle agitation at 4 °C. Cells were washed once and incubated with a quenching buffer (192 mm glycine, 25 mm Tris in PBS) for 10 min. Next, cells were rinsed twice in cold PBS, collected, homogenized in lysis buffer, and centrifuged. Supernatants were then incubated with 50 μl of 50% slurry of streptavidin-Sepharose beads (Amersham Biosciences) for 2 h at 4 °C. Beads were pelleted by brief centrifugation, and aliquots of the supernatant were taken to represent the unbound, intracellular pool. Beads were then washed three times with lysis buffer, and biotinylated proteins were eluted by boiling in 1× sample buffer for 5 min, separated by SDS-PAGE, and immunoblotted as above. Immunofluorescence—For selective labeling of surface receptors, transfected live COS7 cells were incubated with appropriate primary antibodies (monoclonal anti-Tac or anti-autofluorescent protein) diluted in DMEM supplemented with 5% fetal bovine serum for 1 h at 4 °C. The cells were then washed with cold PBS and fixed with 4% paraformaldehyde and 4% sucrose on ice for 20 min. After fixation, cells were washed with PBS and labeled with fluorescence-conjugated secondary antibodies. Next, cells were permeabilized at room temperature with 0.2% Triton X-100 for 5 min. Intracellular expression was then determined by sequentially incubating with a second set of primary (polyclonal anti-Tac or anti-GFP) and secondary antibodies. A slightly modified protocol was used for staining transfected hippocampal neurons. Briefly, for surface labeling, cultured live neurons were incubated with anti-Tac diluted in PBS with 5% normal goat serum for 30 min at room temperature, fixed, and incubated with a rhodamine-conjugated anti-mouse secondary antibody. To detect intracellular expression, neurons were then permeabilized and sequentially incubated with anti-Tac and FITC-conjugated anti-mouse secondary antibody diluted in PBS with 10% normal goat serum. Both surface and intracellular expression were visualized on a Axioskop fluorescence microscope (Zeiss, Thornwood, NY) coupled to a CCD camera (Hamamatsu, Hamamatsu City, Japan) and analyzed by OpenLab imaging software (Improvision, Coventry, United Kingdom). Colocalization images were obtained with the permeabilized staining protocol, visualized with a Zeiss LSM410 confocal microscope (Zeiss), and analyzed with Renaissance imaging software (Microcosm, Columbia, MD). Flow-assisted Cytometry—Transfected live HEK293 cells grown in 6-well tissue culture plates were incubated with monoclonal anti-Tac or anti-Myc antibodies diluted in DMEM supplemented with 5% serum for 1hat4 °C. Cells were then washed with PBS and incubated with FITC- or phycoerythrin-conjugated anti-mouse secondary antibodies in PBS for 1 h at 4 °C. After extensive washing, cells were detached from the plates with 500 μl of PBS plus 5 mm EDTA, and transferred to 12 × 75-mm polystyrene test tubes (VWR Scientific, South Plainfield, NJ). Finally, 500 μl of 4% paraformaldehyde in PBS was added to fix the cells. Surface expression was quantified using a FACScalibur cell sorter (BD Biosciences). Background fluorescence was determined using mock-transfected HEK293 cells incubated with corresponding primary and secondary antibodies. Mean fluorescence intensity was acquired and plotted with Prism4.0 software (GraphPad, San Diego, CA). In Vitro Deglycosylation—Transfected HEK cells grown in 35-mm dishes were washed with ice-cold TBS and homogenized in 1 ml lysis buffer (0.1% SDS, 5% deoxycholate and 1% Nonidet P-40 in TBS, plus 2 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml chemostatin, and 1 μg/ml leupeptin, pH 8.0). After brief sonication and centrifugation (14,000 rpm for 20 min at 4 °C), the supernatants were collected. 6 μl of Denature Buffer (New England Biolabs, Beverly, MA) was added to 54 μl of cell lysate. The samples were then boiled for 10 min, divided into thirds, and treated with 500 units of peptide-N-glycosidase F (PNGase F; New England Biolabs), 500 units of endoglycosidase H (endo H; New England Biolabs), or no enzyme for 5 h at 37 °C. After incubation, samples were boiled for 5 min in 1× sample buffer, resolved on 10% SDS-PAGE, and immunoblotted as described. Kinase Activation Experiments—HEK cells transfected with Tac-G5/2b were treated with (a) 100 nm phorbol 12-myristate 13-acetate in Me2SO for 30 min, washed with fresh media, and allowed to recover at 37 °C for 3 h; (b) 50 μm forskolin together with 10 μm phosphodiesterase inhibitor, isobutylmethylxanthine, in Me2SO for 2 h; or (c) Me2SO only. Cells were then examined for surface expression as by flow-assisted cytometry (FAC) analysis. Transfected COS cells were treated similarly; surface and intracellular expression of the receptors were then examined by live immunostaining as described. All experiments were done in a blind manner. Electrophysiology—HEK293 cells were plated on glass coverslips coated with 100 μg/ml collagen and poly-d-lysine. The following day, cells were transfected with plasmids containing the wild type or mutant GFP-GluR5–2b (0.2 μg/coverslip) using FuGENE 6 transfection reagent according to the instructions from the manufacturer. Whole-cell patch clamp recordings were made with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) 2–3 days after transfection. The external bath solution contained 150 mm NaCl, 2.8 mm KCl, 10 mm glucose, 2 mm CaCl2, 1.0 mm MgCl2, and 10 mm HEPES (pH was adjusted to 7.3 with NaOH). The internal solution was composed of 110 mm CsF, 30 mm CsCl, 4 mm NaCl, 0.5 mm CaCl2, 10 mm HEPES, and 5 mm EGTA (adjusted to pH 7.3 with CsOH). For rapid agonist application, cells were lifted from the coverslip into a laminar solution stream exiting a three-barrel glass pipette (Vitro Dynamics, Rockaway, NJ) pulled to an internal barrel diameter of ∼80 μm and mounted on a piezo-ceramic bimorph. The solution stream was rapidly moved across the transfected cells by applying voltage to the bimorph with a stimulation-isolation unit triggered by a digital signal from pClamp 8 software. Data were acquired and analyzed using pClamp 8 software (Axon Instruments, Foster City, CA) and Origin 6.0 (OriginLab Corp., Northampton, MA). GluR5–2b C-terminal Domain Contains an Intracellular Retention Signal—The distinct C-terminal sequences of GluR5 splice variants (Fig. 1A) are known to be responsible for their differential interactions with intracellular scaffolding molecules (12Hirbec H. Francis J.C. Lauri S.E. Braithwaite S.P. Coussen F. Mulle C. Dev K.K. Coutinho V. Meyer G. Isaac J.T. Collingridge G.L. Henley J.M. Neuron. 2003; 37: 625-638Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), however their roles in receptor trafficking remain unexplored. To facilitate the search for potential trafficking signals in these alternatively spliced C termini and to avoid the complication of assembly with native subunits in neurons, we constructed chimeric receptors consisting of the N-terminal and transmembrane domains of the human interleukin-2 receptor α subunit (Tac), and the C terminus of either GluR5–2a or GluR5–2b (designated Tac-G5/2a and Tac-G5/2b, respectively) (Fig. 1A). The expression profiles of the Tac-G5 chimeras and Tac alone were then examined in HEK cells with a biotinylation method using sulfo-NHS-S-S-biotin. Because this modified biotin is membrane-impermeable, it only binds to surface proteins, which can then be separated from the intracellular pool by conjugation with streptavidin beads and visualized by Western blotting. Using this method, we observed strong surface expression of Tac and Tac-G5/2a (Fig. 1B), whereas Tac-G5/2b signal could not be detected on the plasma membrane, despite its robust intracellular expression (Fig. 1B). Furthermore, the intracellular Tac was present as both a high molecular mass (55 kDa) and a low molecular mass (45 kDa) form (Fig. 1B), but only the high molecular mass species was detected in the biotinylated surface fraction (Fig. 1B). This is consistent with previous studies demonstrating that Tac is synthesized as a 45-kDa immature form in the ER and further processed upon transport into the Golgi apparatus, resulting in a shift of molecular mass (13Leonard W.J. Depper J.M. Robb R.J. Waldmann T.A. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6957-6961Crossref PubMed Scopus (169) Google Scholar, 14Bonifacino J.S. Suzuki C.K. Klausner R.D. Science. 1990; 247: 79-82Crossref PubMed Scopus (162) Google Scholar). The same pattern was also observed for Tac-G5/2a (Fig. 1B), whereas Tac-G5/2b could only be detected as a low molecular mass species (Fig. 1B). The lack of a mature form for Tac-G5/2b suggests that it is unable to exit the early secretory compartments that include ER and ER-Golgi intermediate compartment (ERGIC). To quantify receptor surface expression, we performed a FAC analysis on HEK293 cells transfected with Tac or Tac-G5 receptors. Cells expressing Tac or Tac-G5/2a had 70-fold higher surface fluorescence compared with that of Tac-G5/2b (Fig. 1C). In all cases, total protein abundance of different receptors was comparable, as revealed by Western blot analyses of whole cell extracts (data not shown). To confirm the intracellular retention of Tac-G5/2b, we employed a live immunofluorescence technique to visualize surface receptors. In agreement with the biotinylation and FAC data, Tac-G5/2b was not found on the plasma membrane, but rather retained intracellularly in a mesh network-like intracellular compartment reminiscent of ER morphology, with most of the staining concentrated in the perinuclear region (Fig. 1D). In contrast, Tac and Tac-G5/2a were readily detectable on the cell surface (Fig. 1D). Finally, to determine whether the intracellular retention of Tac-G5/2b receptor occurs in neurons, we transfected cultured hippocampal neurons with the Tac-G5 chimeras and analyzed their surface expression. As in the HEK and COS cells, Tac-G5/2b was retained inside the neurons, whereas both Tac and Tac-GluR5/2a were efficiently delivered to the neuronal surface (Fig. 1E). Together these data indicate that the C-terminal domain of GluR5–2b contains signal(s) sufficient to confer intracellular retention of Tac receptors in both heterologous cells and neurons. Tac-G5/2b Receptors Are Retained in the ER—The two major intracellular organelles involved in protein biogenesis and trafficking are the ER and Golgi apparatus, both of which have distinctive morphology and unique molecular markers. To determine the precise intracellular location of the retained Tac-G5/2b receptor, we compared its distribution profile with those of the ER and Golgi markers. When expressed in COS7 or NRK cells, Tac-G5/2b colocalized extensively with the ER markers, calnexin and DsRed-ER (Fig. 2A), while showing no overlap with the Golgi proteins, giantin, mannosidase II, and TGN-38 (Fig. 2B). Furthermore, in agreement with the earlier observation that Tac-G5/2b failed to reach the Golgi apparatus for post-translational processing (Fig. 1B), the receptor remained sensitive to endo H (Fig. 2C), an enzyme that preferentially hydrolyzes the high mannose N-glycans present on immature proteins in the ER (15Trimble R.B. Maley F. Anal. Biochem. 1984; 141: 515-522Crossref PubMed Scopus (200) Google Scholar). In contrast, Tac and Tac-G5/2a were resistant to endo H (Fig. 2C), indicating that these receptors had been processed in the Golgi, a result consistent with their strong surface expression (Fig. 1, B–E). To ensure that endo H-mediated deglycosylation was selective for Tac-G5/2b, we used PNGase F, a less selective glycosidase that cleaves most asparagine-bound glycans (16Tarentino A.L. Gomez C.M. Plummer Jr., T.H. Biochemistry. 1985; 24: 4665-4671Crossref PubMed Scopus (919) Google Scholar). Indeed, all the receptors exhibited increased electrophoretic mobility, indicating PNGase F sensitivity (Fig. 2C). These experiments provide both immunocytochemical and biochemical evidence for the selective ER retention of Tac-G5/2b receptors. Arg-896 Is Critical for the ER Retention of Tac-G5/2b—The C terminus of GluR5–2b contains a number of arginine- and lysine-rich sequences, but no regions that match previously identified ER retention signals. To map the residue(s) conferring ER retention of Tac-G5/2b, we first made a series of deletional mutations (Fig. 3A), based on the positions of these arginine- and lysine-rich sequences, and examined their effects on ER retention. Deletion of the last 10 amino acids (Tac-G5/2bΔ10), but not the last 6 residues (Tac-G5/2bΔ6), effectively released Tac-G5/2b from ER retention in both COS7 cells (Fig. 3B) and hippocampal neurons (Fig. 3C). In contrast to Tac-G5/2b and Tac-G5/2bΔ6, Western blot analyses showed that Tac-G5/2bΔ10 was present as both mature and immature forms, providing biochemical evidence for its egress from ER (Fig. 3D). Quantification of surface expression using FAC indicated that the Tac-G5/2bΔ10 mutant had 35-fold greater surface fluorescence than that of Tac-G5/2b (Fig. 3E). The other two deletion mutants, Tac-G5/2bΔ15 and Tac-G5/2bΔ36, also exhibited robust surface expression (Fig. 3, B, C, and E). Together these results suggest that the ER retention signal(s) is likely embedded in a short sequence, LTCHQRRTQ, in the distal portion of GluR5–2b C terminus. To determine the precise molecular composition of the ER retention signal, each of the 9 residues (LTCHQRRTQ) (Fig. 4A, top panel) was replaced with an alanine. The mutants were then expressed in HEK cells and examined for surface expression using FAC analysis. A robust surface signal was only detected for the R896A mutant (Tac-G5/2b-R896A), whereas other mutations either had no effect (i.e. L891A, T892A, Q895A, T898A, and Q899A) or showed an insignificant increase (C893A, H894A, and R897A) (Fig. 4A, middle panel). Accordingly, no mature form could be detected for any other receptor except the R896A mutant (Fig. 4A, bottom panel). Furthermore, when expressed in COS cells and hippocampal neurons, Tac-G5/2b-R896A showed strong surface expression (Fig. 4B). These data indicate that Arg-896 is essential for the ER retention of the Tac-G5/2b, whereas other residues do not have such a dominant effect. Consistent with this idea, mutating multiple residues surrounding Arg-896 (i.e. CHQRR/AAAAA) did not further enhance surface expression. In addition, replacement of Arg-896 with a lysine residue (Tac-G5/2b-R896K) also resulted in robust surface expression, both in heterologous cells (Fig. 4, A and C) and hippocampal neurons (Fig. 4C). Surface expression of the R896A mutant (Fig. 4A), although robust, was significantly lower than that of the deletion mutants Δ10, Δ15, and Δ36 when tested in para
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