extfieldsofstudy: Biology Medicine

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Intracellular Trafficking of KA2 Kainate Receptors Mediated by Interactions with Coatomer Protein Complex I (COPI) and 14-3-3 Chaperone Systems*

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and β-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.

Kainate receptors (KARs) 2 play a variety of roles in the mammalian central nervous system that include contributions to postsynaptic neurotransmission at a subset of excitatory synapses and presynaptic modulation of both excitatory and inhibitory transmission (1,2). These diverse physiological roles of KARs require selective assembly, subcellular trafficking, and targeting of these proteins to their functional sites. Elucidating the cellular mechanisms that control these processes is important for understanding the full spectrum of KAR-mediated signaling in the brain.
Oligomerization and intracellular trafficking of KARs are controlled in part through interactions with chaperone proteins that bind to discrete cytoplasmic domains on the receptor proteins themselves. For example, GluR6a KAR subunits contain a forward trafficking determinant in their cytoplasmic tail and therefore are highly expressed as homomeric receptors on the plasma membrane of heterologous cell lines (3), whereas the KA2 subunit has an endoplasmic reticulum (ER) retention/retrieval signal and consequently does not reach the plasma membrane in the absence of other KAR subunits (4). The KA2 ER retention/retrieval signal consists primarily of an arginine-rich domain similar to that characterized on several other ionotropic glutamate receptor subunits, including NR1 (5) and GluR5-2c (6), as well as other signaling proteins such as ATP-sensitive potassium channels (K ATP ) (7) and GABA B receptors (8). Although a number of trafficking motifs in KAR subunits have been identified (9), little is known about how these trafficking signals control localization of receptors and which chaperone proteins are responsible for the modulation of receptor trafficking.
In this project, we tested the hypothesis that the polyarginine trafficking determinant in KA2 KAR subunits functions as a COPI interaction domain and thereby mediates sequestration of these subunits in the ER. We found that COPI did indeed associate with KA2 subunits, and this interaction was reduced upon mutation of the arginine-rich ER retention/retrieval domain. As well, mutations that reduced COPI association concomitantly increased association with endogenous and exogenous 14-3-3 proteins. The strong correlation between plasma membrane expression of KA2 receptors and interactions with COPI and 14-3-3 proteins support our hypothesis that these chaperone systems regulate the subcellular trafficking of KA2 subunits. from Dr. Stephen Heinemann (Salk Institute, La Jolla, CA). Myc-GluR6(Q), myc-GluR5-2b, and myc-GluR5-2c were received from Dr. Christophe Mulle (Université Bordeaux II, France). VSVG-14-3-3 was a gift from Dr. Melanie Darstein. All cDNAs were in pcDNA3 vectors. Other cDNA mutants were generated using the QuikChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA). All mutants were subcloned back into the wild type vector using an EcoRI-XbaI site for myc-KA2 cDNAs. PCRamplified sequences including the site mutations were verified by DNA sequencing.
Cerebellar Lysate Preparation-Cerebella were dissected from 129SvEv wild type mice or KA2 Ϫ/Ϫ mice at 21-25 days old, homogenized in 50 mM Tris-HCl, 0.32 M sucrose, pH 7.4, and centrifuged at 800 ϫ g for 5 min at 4°C. Supernatants were centrifuged again at 180,000 ϫ g for 1 h at 4°C. Pellets were resuspended in lysis buffer consisting of 50 mM Tris, 150 mM NaCl, 0.5% Nonidet P-40, Cell Culture, Transfection, and Protein Preparation-COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 100 g/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum. COS-7 cells were plated in 100-mm dishes at 1 ϫ 10 6 cells and transfected with a total of 5 g of cDNA using FuGENE 6 reagent (Roche Applied Science) following the manufacturer's recommended protocol. 48 h after transfection, cells were washed twice with cold phosphate-buffered saline (PBS) and lysed in 0.5 ml of lysis buffer. Crude protein lysates were obtained after centrifugation at 13,000 ϫ g at 4°C for 20 min. Wild type CHO and mutant CHO(ldlF) (clone 2) cells were a gift from Dr. Richard Anderson (University of Texas Southwestern Medical Center, Dallas, TX) and Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA), and were maintained as described in Guo et al. (16). 48 h after transfection, cells were transferred to 39.5°C for 12 h before biochemical analysis.
Immunolocalization of Receptors and Confocal Microscopy-Transfected COS-7 cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.2% Triton X-100 for 5 min. Cells were incubated with anti-myc antibody (4 g/ml in 10% goat serum) at room temperature for 2 h. Cells were washed 3 times with PBS and subsequently incubated with anti-␤-COP antibody (1:500 in 10% goat serum) at room temperature for 2 h. After 3 washes, cells were incubated with appropriate fluorescence-conjugated secondary antibodies: Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Eugene, OR) at room temperature for 1 h. All images were taken from a inverted LSM 510 Meta confocal microscope at the Optical Imaging Laboratory using a Plan-Apochromat ϫ63/1.4 oil objective lens with excitation wavelength at 480 nm for Alexa Fluor 488 and at 543 nm for Alexa Fluor 594.
Peptide-binding Assay-Two peptides containing the KA2 cytoplasmic arginine-rich trafficking determinant and the associated alanine mutant were synthesized with amide terminal groups (KA2-Rpep, CRKTSRSRRRRRP; KA2-Apep, CRKTSRSAAAAAP). These were linked to Sepharose beads at the initial cysteine residue (21st Century Biochemicals, Malboro, MA). Peptide beads were incubated with COS-7 cell lysate overnight. After five washes in lysis buffer, bound proteins were eluted and separated as described in the immunoblotting section. Band densities in Western blots were normalized to total ␤-COP expression in lysate. Experiments were performed four times.
Cell Surface Biotinylation Assay-COS-7 cells were plated in 6-well plates and transfected with 1 g of cDNA. CHO cells were plated into 100-mm dishes and transfected with 5 g of cDNA. Transfected cells were washed twice with cold PBS and incubated with 0.5 mg/ml EZ-link sulfo-NHS-SS-biotin (21331, Pierce) in PBS, pH 8.0, at 4°C for 30 min with gentle agitation. Non-reacted biotin was quenched using quenching solution (50 mM glycine in PBS, pH 8.0) 3 times and then cells were washed 3 times with cold TBS (0.025 M Tris and 0.15 M NaCl), pH 7.2. Cells were harvested in 0.5 ml of lysis buffer containing protease inhibitors. Lysates were incubated with 50 l of 50% streptavidin-Sepharose high performance beads (17-5113-01, Amersham Biosciences) at 4°C overnight. Beads were washed four times, and proteins were eluted and detected as co-immunoprecipitation experiments. In the experiments with CHO cells, the "total" lane was loaded with lysate equivalent to 1% of the protein loaded on the peptide-linked beads. Biotinylated proteins eluted from the beads (corresponding to subunits located in the plasma membrane) were normalized to total protein expression and reported in the term of percentage. All experiments were performed at least four times.
Enzyme-linked Immunosorbent Assay (ELISA)-COS-7 cells were inoculated at 4.5 ϫ 10 4 cells and transfected with 0.6 g of cDNA using FuGENE 6 reagent in 12-well plates. After 48 h, cells were washed once with cold PBS and incubated with anti-myc antibody (4 g/ml in 10% goat serum) at 4°C for 30 min to detect plasma membrane expression. Cells were then washed twice with cold PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. For measuring total protein expression, cells were first fixed with paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and incubated with anti-myc antibody at room temperature for 2 h. Both groups were washed 3 times with PBS and incubated with HRP-conjugated sheep anti-mouse secondary antibody (1:1000 in 10% goat serum, NA931V, Amersham Biosciences) at room temperature for 1 h. After the third washing with PBS, the HRP substrate o-phenylenediamine dihydrochloride (P9187, Sigma) was added and the color reaction was developed for 1 h. The optical density of 0.2 ml of supernatant was detected by spectrophotometer at 490 nm. All values were an average of four replicates in each experiment; the mean background absorbance of the negative controls (sham-transfected cells) were subtracted from surface and total absorbance values. Data are presented in the form of a ratio of plasma membrane expression to total cellular expression. At least four experiments were performed with each cDNA.
Statistical Analysis-Data were tested with a one-way analysis of variance and the post-hoc Tukey-Kramer multiple comparison test, or a one-tailed paired Student's t test.

Immunoprecipitation of KA2 and COPI Proteins-
The COPI chaperone complex was shown recently to be a critical mediator of ER retrieval mediated by polyarginine signals in a subset of integral membrane proteins (11,12,14). The KA2 kainate receptor subunit is retained efficiently in the ER through a mechanism primarily mediated by a polyarginine signal in the cytoplasmic carboxyl-terminal domain. To determine whether COPI proteins subserve a retention/retrieval function for the KA2 kainate receptor subunit, we first tested for a biochemical interaction between KA2 subunits and members of the COPI complex in transfected COS-7 cells. NH 2 -terminal myc-tagged recombinant rat KA2, GluR5-2b, GluR5-2c, or GluR6a cDNAs were transfected into COS-7 cells before immunoprecipitation of cell lysates with an anti-␤-COP antibody; KA2 or other subunit protein was then detected in Western immunoblots using an anti-myc antibody. We found that homomeric myc-KA2 receptors co-precipitated with endogenous ␤-COP in either COS-7 cells (IP lane, Fig. 1A) or in transfected HEK293 cells (data not shown). The PC lane in Fig. 1 showed the nonspecific binding of protein to the beads used for immunoprecipitation. In contrast to KA2, myc-GluR5-2b, myc-GluR5-2c, and myc-GluR6a subunits did not co-immunoprecipitate with ␤-COP (Fig. 1A), demonstrating that the interaction is relatively specific to KA2 subunits. We also tried showing the background binding of proteins to beads lacking antibody. Anti-␤-COP antibody immunoprecipitated KA2 subunits but not GluR5-2b, GluR5-2c, or GluR6a subunits. Blots were stripped and re-probed with an anti-␤-COP antibody (right panel ) to verify precipitation of ␤-COP proteins. B, ␤-COP was weakly detected after immunoprecipitation with anti-myc antibody (left panel). Blots were stripped and re-probed with an anti-myc antibody (right panel) to demonstrate precipitation of myc-KA2 receptors. C, cerebellar lysate prepared from 129SvEv or KA2 Ϫ/Ϫ mice was used for immunoprecipitation with anti-KA2 antibody. ␤-COP protein co-precipitated with KA2 subunits in wild type cerebellar lysate but not lysate from KA2 Ϫ/Ϫ cerebella. The right panel demonstrates the presence of KA2 protein in the immunoprecipitate from wild type mice. D, immunoprecipitation of ␤-COP from cerebellar lysate from 129SvEv mice co-precipitated KA2 (left blot) but not GluR6/7 (middle blot) subunits. E, immunoprecipitation of GluR6/7 subunits from cerebellar lysates did not co-precipitate ␤-COP protein (left blot), but did pull down GluR6/7 and KA2 protein (middle and right blots). F, co-localization of ␤-COPI (green) with myc-KA2 subunits (red) in transfected COS-7 cells. COPI staining was most obviously localized to the Golgi apparatus and in small puncta likely representing transport vesicles. Homomeric myc-KA2 receptor staining showed the characteristic reticular pattern of endoplasmic reticulum. An overlay of ␤-COP and KA2 immunofluorescence at higher magnification shows that KA2 subunits were partly co-localized with ␤-COP (arrows). the converse experiment, immunoprecipitation of myc-KA2 and immunoblotting for ␤-COP protein, and observed a very weak but detectable coprecipitation (Fig. 1B). We postulate from these data that the majority of KA2 subunits are resident in the ER and that a relatively small proportion of the receptor subunit transiently associates with COPI during retrieval to the ER.
We next tested if the interaction between KA2 subunits and COPI complex proteins could be detected in the central nervous system. The KA2 subunit is highly expressed in cerebellar granule cells, where they likely form heteromeric receptors with GluR6 subunits and modulate excitatory synaptic transmission at parallel fiber synapses (17). Membrane protein lysates were prepared from homogenized cerebella from 129SvEv mice as well as KA2 Ϫ/Ϫ gene-targeted mice (as a control for nonspecific interactions with the precipitating antibody). KA2 protein was immunoprecipitated from the lysates with an anti-KA2 antibody and ␤-COP was detected in Western blots (Fig. 1C). As shown in Fig. 1, ␤-COP subunit co-immunoprecipitated with KA2 protein from wild type but not KA2 Ϫ/Ϫ cerebella (Fig. 1C, left panels). Blots were stripped and re-probed with anti-KA2 antibody to verify that the immunoprecipitation of KA2 subunit protein was successful (Fig. 1C, right panel), and as expected immunoreactivity was not detected in the samples from KA2 Ϫ/Ϫ mice. A reciprocal immunoprecipitation using an anti-␤-COP antibody also supported a robust association between the KA2 subunit and ␤-COP (Fig. 1D). Because cerebellar KA2 subunits likely assemble with GluR6 subunits as heteromeric receptors, we also tested to see if ␤-COP co-precipitated GluR6 either directly or in association with KA2. As shown in Fig. 1D, GluR6/7 immunoreactivity did not precipitate with ␤-COP. In the reciprocal experiment, we immunoprecipitated GluR6/7 protein and Western blotted for ␤-COP. The vesicle protein was not detected in this assay (Fig. 1E). These results support the interpretation that KA2 subunits exist in at least two distinct pools in cerebellum: those assembled with GluR6 as heteromeric KARs and those associated with the COPI retrograde trafficking pathway fated for retrieval to the ER.
Subcellular Co-localization of KA2 and ␤-COP-Next, we determined if the subcellular localizations of KA2 subunits and COPI proteins were consistent with their interaction in our biochemical assays using confocal immunofluorescence staining of transfected COS-7 cells. Myc-KA2 subunits were detected with an anti-myc antibody (red in Fig. 1F ) and ␤-COP was stained with an anti-␤-COP antibody (green in Fig. 1F ). In addition to COPI vesicle staining, anti-␤-COP antibody strongly labels the Golgi apparatus, which is manifested as the intense green labeling in the image. The vast majority of KA2 receptor subunits are located in the ER and are unlikely to be actively associated with COPI, accounting for very weak ␤-COP bands when the KA2 subunit is the target of immunoprecipitation. As shown in the last row of images in Fig. 1F, the discrete vesicle-like structures (punctate dots) positively stained for both KA2 and ␤-COP likely represent COPI vesicles containing KA2 as cargo protein.
Elimination of COPI Function Promotes Plasma Expression of KA2 Receptors-If COPI association plays a central role in limiting forward trafficking of KA2 subunits beyond the cis-Golgi, homomeric KA2 receptors should exhibit higher plasma membrane expression when COPI functionality is disrupted. CHO(ldlF) cells contain a temperaturesensitive (ts) mutation in one subunit of the complex, -COP (16). At a nonpermissive temperature (39.5°C), -COP is degraded and the cells lose the capability of forming COPI vesicle coats. We compared the plasma membrane expression of homomeric myc-KA2 receptors expressed in wild type CHO cells and CHO(ldlF) cells using a cell surface biotinylation assay ( Fig. 2A). Cell surface proteins were labeled with biotin, purified on streptavidin columns, and eluted proteins were immunoblotted with anti-myc antibody. We included lanes with 1% of the input myc-KA2 protein to determine the relative amount of KA2 expressed at the plasma membrane, and detected actin immunoreactivity to verify equivalent protein loading ( Fig. 2A). The relative plasma membrane to total protein expression of myc-KA2 receptors in CHO(ldlF) cells increased significantly by 3.6-fold (n ϭ 4, p Ͻ 0.05) after a 12-h incubation at 39.5°C compared with CHO(ldlF) cells kept at 34°C for the same period of time (Fig. 2B). Transfected CHO cells kept at either 37 or 39.5°C expressed the same relative level of myc-KA2 on the plasma membrane as CHO(ldlF) cells maintained at the permissive temperature. This data support the hypothesis that COPI-mediated retention/ retrieval prevents plasma membrane expression KA2 receptors.
Dependence of COPI Association on the KA2 Trafficking Determinant-We hypothesized that COPI proteins interacted with KA2 subunits at the arginine-rich domain in the intracellular carboxyl terminus. To determine whether the critical amino acid residues responsible for COPI association site correlated with those required for ER sequestration, we examined COPI association with truncated and alanine-substituted KA2 subunits generated by site-directed mutagenesis of the receptor cDNA. The cytoplasmic tail of KA2 contains 155 amino acids; we generated a mutant subunit truncated at residue 827 (827-stop, with our numbering beginning at the initial methionine), which eliminated the bulk of the COOH terminus, and 856-stop, which terminated the cytoplasmic tail prior to the polyarginine domain (Fig. 3A). Truncated myc-KA2(827-stop) and myc-KA2(856-stop) subunits expressed in COS-7 cells exhibited significantly decreased association with ␤-COP in immunoprecipitation experiments (Fig. 3B). Quantification of the opti-FIGURE 2. Plasma membrane expression of homomeric myc-KA2 receptors increases upon disruption of the COPI pathway. A, myc-KA2 was expressed in either CHO or CHO(ldlF) cells grown at 37 or 34°C, respectively. Two days after transfection, one group from each type of transfected cell was shifted to 39.5°C for 12 h, whereas the other groups were kept at the control temperatures. Cell surface proteins were then labeled with biotin. Biotin-labeled plasma membrane proteins were purified on streptavidin columns and immunoblotted with anti-myc antibody (middle panel, PM myc-KA2). 1% of the total membrane protein loaded on the streptavidin beads was immunoblotted to normalize protein expression (top panel, total myc-KA2). The lower panel shows the actin loading controls. Incubation of CHO(ldlF) cells at the nonpermissive temperature (39.5°C), resulting in the inactivation of COPI function, increased the relative level of plasma membrane KA2 subunits (while concurrently reducing total KA2 expression). B, band density from A was measured by densitometry. After 12 h incubation at 39.5°C, homomeric myc-KA2 receptors expressed in CHO(ldlF) cells had higher relative plasma membrane expression to total protein expression than cells incubated at 34°C, representing a 3.6-fold increase. Data represent mean Ϯ S.E. (n ϭ 4, *, p Ͻ 0.05).
These data indicated that the majority of ␤-COP association with KA2 subunits depended on domains downstream from the 856-stop site, consistent with the proposal that the polyarginine trafficking domain comprised a putative site for interaction. In addition to the polyarginine site, a pair of COOH-terminal leucine residues were shown to influence cell surface expression (4) and therefore also were considered as a candidate interaction site. Alanine substitution of all five arginines, in the mutant construct myc-KA2(R862-6A), or both cytoplasmic trafficking determinants, in myc-KA2(R862-6A,L908 -9V), markedly decreased the association of KA2 subunits with ␤-COP (by 59 Ϯ 11 and 67 Ϯ 6%, n ϭ 6 and 3, respectively, p Ͻ 0.01) to a level similar to that observed for the truncated KA2 subunits (Fig. 3, B and C). To further define the COPI binding site, we tested the interaction of ␤-COPI with two KA2 mutants in which the polyarginine determinant was substituted with two sets of three alanines: myc-KA2(R862-4A) and myc-KA2(R864 -6A). Association of ␤-COP protein with either of these mutants was not reduced from that of wild type KA2 subunits (Fig. 3, B and C), suggesting that all five arginine residues (or a different subset of residues) in KA2 are necessary for interaction with the chaperone system. All blots were stripped and re-probed with an anti-␤-COP antibody to verify the efficiency of COPI immunoprecipitation (Fig. 3B).
␤-COP is one of seven proteins that comprise the COPI chaperone system; to further validate the association between KA2 subunits and the COPI complex we tested for association between the receptor subunit and ␣-COP, another component of COPI vesicle coats. Immuno-precipitation of KA2-transfected cells with ␣-COPI antibody resulted in co-precipitation of KA2 protein (Fig. 4A). As with ␤-COP, the truncation of receptor (myc-KA2(856-stop)) and mutation of the polyarginine motif (myc-KA2(R862-6A)) markedly reduced association with ␣-COP (by 88 Ϯ 4 and 66 Ϯ 15%, n ϭ 3, respectively, p Ͻ 0.01) (Fig. 4,  A and B). These results further support our hypothesis that the argininerich domain plays a role in COPI association.  Interaction between ␤-COP and Peptides Containing the KA2 Trafficking Determinant-We next tested if the association inferred from our immunoprecipitations likely resulted from a direct interaction between COPI proteins and the polyarginine domain in the KA2 subunit. This was tested by comparing the binding of ␤-COP protein in COS-7 cell lysates to two short peptides, KA2-Rpep and KA2-Apep, which were conjugated to Sepharose beads (Fig. 5A). KA2-Rpep contained the KA2 cytoplasmic arginine-rich trafficking determinant, whereas KA2-Apep had the five alanine substitutions used in previous immunoprecipitation experiments. The Lysate lane in the Fig. 5 shows expression of the ␤-COP protein in the COS-7 lysate, and the NS lane shows the nonspecific interaction between lysate and Sepharose beads. We found that ␤-COP was readily detected in Western blots after elution from KA2-Rpep beads. In contrast, the protein was only weakly detected after elution from the KA2-Apep peptide (Fig. 5A). To quantitate this result, we normalized the band densities of ␤-COP staining in the peptide lanes to the density of the lysate lane (each experiment was run with all four groups in parallel). We found that alanine substitution in the peptide reduced the interaction with ␤-COP by 90 Ϯ 3% (n ϭ 4, p Ͻ 0.01) (Fig. 5B). This result strongly supports the hypothesis that COPI proteins directly interact with the KA2 receptor at the argininerich sequence in the COOH terminus.
COPI Association Is Reduced in Heteromeric GluR6a/KA2 Receptors-Co-assembly of GluR6a with KA2 subunits leads to efficient plasma membrane expression of heteromeric GluR6a/KA2 receptors, suggesting that the interaction between COPI and KA2 subunits and consequent ER retention is occluded in the presence of GluR6a subunits. To test this hypothesis, COS-7 cells were co-transfected with myc-KA2 and GFP-GluR6a cDNAs, and cell lysate was immunoprecipitated with an anti-␤-COP antibody. As predicted, we found a reduction in co-precipitated myc-KA2 (by 64 Ϯ 6%, n ϭ 5, p Ͻ 0.01, Fig. 6, A and  B). GFP-GluR6a was not detectable in the immunoprecipitated complex of ␤-COP and myc-KA2 (Fig. 6A), suggesting that heteromeric GFP-GluR6a/myc-KA2 receptors did not interact with COPI to a significant degree. It therefore is likely that myc-KA2 detected after immunoprecipitation in these experiments represented a pool of unassembled or homomeric subunits that were not associated with GFP-GluR6a subunits.
Inhibition of COPI association with KA2 by GluR6a subunits could occur through masking of the polyarginine retention motif similar to the mechanism proposed for K ϩ channels (7) and GABA B receptors (8), although our previous data suggested that this was not the case for ER retention of KA2 subunits (3). To test if COPI association with KA2 was masked by the GluR6a COOH terminal tail, we co-transfected myc-KA2 with GFP-GluR6a(850-stop) cDNA, a truncated mutant that lacks most of the GluR6a cytoplasmic tail. Previously, we showed that heteromeric GFP-GluR6a(850-stop)/myc-KA2 receptors were expressed at the plasma membrane (3), suggesting that retention signals in KA2 were suppressed despite the absence of the forward trafficking motif in GluR6a. We found that heteromeric assembly of myc-KA2 with GFP-GluR6a(850-stop) subunits decreased myc-KA2-␤COP interaction by 72 Ϯ 9% (n ϭ 5, p Ͻ 0.01) (Fig. 6, A and B). These data support our earlier hypothesis that the retention signal in KA2 subunits were not masked by the GluR6a cytoplasmic tail, but rather became unavailable for COPI association as a result of conformational changes in the KA2 subunit induced by heteromeric assembly.

DISCUSSION
The intracellular chaperone systems that control assembly and entry of kainate receptors into the secretory pathway are largely unknown. In this study, we elucidated the cellular mechanism for retention of homomeric KA2 kainate receptors in the ER. These receptors do not form functional glutamate receptors when expressed on the plasma membrane, and therefore retention of these subunits contributes to the assembly of appropriately functional heteromeric channels when other KAR subunits are expressed. Endogenous KA2 subunits in the cerebellum, where they are expressed at high levels in granule cells, and exogenous KA2 in transfected cells both associated with COPI proteins in immunoprecipitation assays. The association was dependent upon the arginine-rich determinant in the cytoplasmic tail of KA2 and results in efficient sequestration of KA2 subunits in the ER. We also found that KA2 subunits immunoprecipitated with a variety of endogenous 14-3-3 proteins. As well, association with the 14-3-3 isoform was increased by mutations of the arginine-rich COPI interaction site, suggesting that 14-3-3 proteins might play a role in the KA2 biosynthetic pathway.
These changes in COPI and 14-3-3 association were correlated with altered plasma membrane expression of KA2 receptors. In summary, our results identify the first set of proteins associated with critical trafficking determinants in kainate receptor subunits.
Regulation of ER Localization of Homomeric Kainate Receptor Subunits by COPI-The major function of COPI in the intracellular trafficking of proteins is thought to be retrieval of ER-resident proteins from the Golgi back to the ER (13). Membrane proteins containing A, interactions of endogenous 14-3-3 and exogenous VSVG-14-3-3 with GluR5-2b receptors were tested utilizing anti-14-3-3 and anti-VSVG antibodies, respectively. Western blotting with an anti-myc antibody showed that myc-GluR5-2b receptors weakly co-precipitated with endogenous 14-3-3 and exogenous VSVG-14-3-3. Top blots were stripped and re-probed with anti-14-3-3 and anti-VSVG antibodies for endogenous 14-3-3 and exogenous 14-3-3, respectively. Abbreviations: L, lysate; PC, preclear background. B, GluR5-2c receptors did not show detectable association with endogenous 14-3-3 or exogenous VSVG-14-3-3. C, similar to GluR5-2b receptor experiments, GluR6a receptors showed weak association with exogenous VSVG-14-3-3. arginine-based trafficking motifs, such as K ATP channels and GABA B receptors, exhibit an association with COPI inversely correlated with their level of plasma membrane expression (12,18). We also found that either the disruption of COPI functionality, in the CHO(ldlF) cells, or a reduction in KA2-COPI association by mutagenesis was similarly correlated with increased cell surface expression (by 3-8-fold). We note that despite this significant increase, the large majority of KA2 protein remained in intracellular compartments (predominantly the ER). This suggests other mechanisms exist that limit the forward progress of homomeric KA2 receptors through the biosynthetic pathway. Among the possibilities are inefficient folding or oligomerization processes. Indeed, our antibody-based detection assays would not differentiate between KA2 proteins in monomeric, dimeric, or tetrameric assemblies, and therefore much of the total KA2 protein we detect in these assays could be in immature forms. Finally, GluR5-2b and GluR5-2c receptors, both of which are largely retained in the ER via argininebased determinants (6,19), did not associate with COPI at a detectable level, suggesting that other retention/retrieval pathways exist for these receptors.
The Arginine-rich Motif as a COPI Interaction Domain in Homomeric KA2 Receptors-The classically defined COPI binding motif consists of a cytoplasmic dilysine motif K(X)KXX (20), but more recent research has demonstrated that the protein complex also mediates argininebased retention/retrieval mechanisms in a variety of proteins that include Iip35, KCNK3 channels, and K ATP channels (21,22). Importantly, Yuan and colleagues (12) demonstrated that this interaction occurred through a direct binding of the RKR sequence of K ATP channels by COPI proteins, rather than an association through intermediary proteins. We found that the string of arginines comprising residues 862-866 in the cytoplasmic tail of KA2 are critical for intracellular retention and immunoprecipitation with COPI vesicle coat proteins. As well, this determinant controlled interaction between ␤-COP protein and immobilized KA2-Rpep peptide. Thus, the robust interaction between COPI and immobilized peptide and myc-KA2 receptors was significantly reduced upon alanine substitution of all five of the constituent arginines in the trafficking determinants (by 90 Ϯ 3 and 59 Ϯ 11%, respectively). In contrast, alteration of either the first or last three arginines did not decrease COPI association with myc-KA2 mutants compared with wild type KA2 subunit. COPI association also was unaffected by added mutation of a dileucine motif known to play a role in plasma membrane expression of KA2 receptors (4). Interestingly, the residual COPI association remaining after COOH-terminal truncation or mutation of the arginine trafficking determinant, and the nearly complete absence of binding to the KA2-Apep peptide, suggest that additional COPI association domains exist within the KA2 subunit. These could be located within the COOH-terminal juxtamembrane domain or in the short intracellular loops between membrane domains. Indeed, the first intracellular loop in the KA2 subunits contains a putative argininebased retention/retrieval signal, RAR, that could represent the site of residual COPI association. It will be of interest to determine whether these residues also promote intracellular retention of KA2 subunits. We thus conclude that COPI vesicle coat proteins are important mediators of the efficient intracellular sequestration of homomeric KA2 proteins via an interaction with the polyarginine determinant, given the similar reduction in COPI ␣ and ␤ subunit association upon mutation of KA2 at Arg 862 -Arg 866 , the direct interaction of ␤-COP protein with the KA2-Rpep peptide, the correlation with plasma membrane expression, and the co-localization of KA2 subunits and ␤-COP in vesicular structures.
Inactivation of COPI Binding to KA2 by Heteromeric Assembly of KARs-In neurons, KA2 subunits are assembled with other KAR subunits to form defined sets of heteromeric receptors that likely are differentially targeted to various membrane domains. We predicted that co-assembly of KA2 with other subunits would relieve COPI-mediated retention, because heteromeric KARs are trafficked to plasma membrane (in heterologous cell lines) and pre-and postsynaptic sites of action (in neurons). This was indeed the case; co-expression of KA2 with GluR6a subunits reduced association of ␤-COP with KA2 subunits by 64 Ϯ 6%, consistent with the robust plasma membrane expression of heteromeric GluR6a/KA2 receptors. It is likely that the residual interaction between COPI and KA2 occurred with subunits that had not formed heteromeric assemblies with GluR6a, because the latter subunit was not detectable in the immunoprecipitates isolated with the anti-␤-COP antibody. Thus, the arginine-based retention/retrieval in KA2 becomes occluded upon heteromeric assembly of receptors.
The mechanism of occlusion of the KA2 trafficking determinant upon assembly with GluR6a subunits is obscure. Similar motifs in K ATP channels, the major histocompatibility protein MHC II, N-methyl-Dasparatate receptors, and GABA B receptors are thought to be sterically masked by heterotypic cytoplasmic domains in other subunits or associated proteins (22). More recently, Gassmann and colleagues (23) proposed that inactivation of the GABA B1 subunit RSRR retention signal upon heteromeric assembly with GABA B2 subunits occurred because the determinant was removed from an "active zone" rather than by being masked through coiled-coiled domain interactions. Similarly, we previously reasoned that steric masking of the KA2 signal by cytoplasmic domains of co-assembled kainate receptor subunits (e.g. GluR6a) did not underlie inactivation of the KA2 arginine determinant because GluR6a subunits with truncated COOH-terminal domains assembled with KA2 subunits and promoted forward trafficking to the plasma membrane (3). Our current results further support this interpretation. Heteromeric assembly of KA2 with truncated GluR6a(850-stop) subunits, which contains only 9 amino acids in the COOH terminus, markedly decreased COPI binding by 72 Ϯ 9%, demonstrating that the occlusion of COPI binding and the resulting release of the heteromeric KAR into the secretory pathway likely did not occur through direct masking by the COOH terminus of GluR6a subunits. These observations are most consistent with our hypothesis that structural alterations within the KA2 subunit itself upon assembly with appropriate partner subunits into hetero-oligomers are responsible for occlusion of COPI binding and inactivation of the retention/retrieval signal.
Suppression of Arginine-based ER Retention/Retrieval Motif via Other Chaperone Proteins-Inactivation of COPI-dependent arginine-based ER retention/retrieval signals in a number of integral membrane proteins results from competitive binding to cytoplasmic domains by other chaperone proteins, which permits egress from the ER into the secretory pathway. In particular, interactions with the 14-3-3 protein family were shown to counteract the COPI associations through both phosphorylation-dependent and phosphorylation-independent mechanisms (21,22). Several novel binding sites of 14-3-3 proteins overlap with arginine-based ER retention/retrieval motif (11,12,18). It has been proposed that the association to 14-3-3 proteins competes and prevents COPI binding, leading to the release of proteins from the ER retrieval pathway (11,12), although this hypothesis was challenged recently (18). 14-3-3 proteins are a widely expressed family of chaperone proteins that subserve a variety of cellular functions including cell cycle and growth control, signal transduction, and apoptosis (24). Their participation in subcellular trafficking pathways and modulation of the signaling function of ion channels has only recently begun to be elucidated.
Our interest in the potential relevance of 14-3-3 proteins to KA2 subunit trafficking was initiated by low-stringency analysis of proteinprotein interaction domains in the KA2 cytoplasmic tail, which identified a putative 14-3-3 binding site overlapping the arginine-based trafficking determinant (25). Consistent with this prediction, we found that KA2 subunits interacted with multiple 14-3-3 isoforms including those endogenous to the COS-7 heterologous cell line. However, mutagenesis of the polyarginine domain unexpectedly increased 14-3-3 binding, effectively eliminating the possibility that a putative association with the KA2 subunit occurs in a directly competitive fashion with COPI (at least at this particular site). The domain in KA2 that mediates association with 14-3-3 proteins has yet to be elucidated. We found that all mutations to the KA2 COOH terminus that increased 14-3-3 binding correlated with a higher relative expression on the plasma membrane, even if the mutations did not significantly effect COPI interactions. For example, mutation of the dileucine motif (Leu 908 -Leu 909 ), which increases surface expression and 14-3-3 association, does not significantly effect COPI binding; this site is a putative endocytic signal and therefore it is possible that reduction in the rate of endocytosis increases the pool of KA2 subunits available for interaction with 14-3-3 proteins. Because the interaction between KA2, 14-3-3, and COPI is correlative in our study, it also is possible that 14-3-3 association with the KA2(R862-6A) and other mutant receptors increased because of greater availability in subcellular compartments not readily accessed in the presence of COPI-mediated retention/retrieval. Thus, the precise role of the association with 14-3-3 proteins in KA2 subunit trafficking remains unclear.
Interestingly, proteomic analysis of proteins associated with GluR6a subunits from mouse brain identified the 14-3-3␥ isoform as an interacting chaperone protein, suggesting that this interaction is relevant to kainate receptor trafficking in the central nervous system (26). We did not observe robust interactions between 14-3-3 and GluR6a (or GluR5-2b and GluR5-2c), in contrast to KA2 subunit, and therefore postulate that different 14-3-3 isoforms could be involved in heteromeric kainate receptor biosynthesis through interactions with distinct subunits.
In conclusion, COPI vesicle coat association with the arginine-rich trafficking determinant in the KA2 COOH terminus leads to the ER localization of these receptors. This ER retention process is inactivated by conformational changes following heteromeric assembly with GluR6 subunits and is inversely correlated with interactions between KA2 and 14-3-3 chaperone proteins. The COPI pathway therefore constitutes an important early pathway for cellular control of KAR subunits and their availability for assembly into functional receptors.


s2fieldsofstudy: Biology Medicine

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Immune Restoration Disease in HIV Patient

We describe a severely immunosuppressed HIV-1–positive man in whom immune restoration disease associated with pulmonary infection caused by Mycobacterium microti developed after antiretroviral treatment. The diagnosis was made by using convenient spoligotyping techniques, but invasive investigations were required to exclude a tumor.

D uring the first few months of highly active antiretroviral treatment, immune restoration may be complicated by clinical events in which either previously subclinical infections are found or preexisting partially treated opportunistic infections deteriorate. This condition, termed immune restoration disease (IRD), is thought to be caused by the improvement in the host's immune response to pathogens. We report what we believe is the first recorded case of IRD with confirmed Mycobacterium microti infection. M. microti is an unusual infection associated with small rodents. Novel genetic techniques have confirmed it as a human pathogen, but its true incidence remains unclear.

Case Report
A 33-year-old man was admitted to the hospital in 2002 with a 3-month history of intermittent hemoptysis. He had previously visited hematology outpatient clinics for many years for hemophilia A (3%-5% factor VIII) and HIV-1 had been diagnosed in 1996 (a stored sample from 1989 retrospectively tested HIV-1-antibody positive). CD4 cell counts had been recorded as 434 × 10 6 /L in 1996 and 260 × 10 6 /L in 1998. After this time, he failed to attend any further clinic appointments.
In addition to worsening hemoptysis, he had minor weight loss but no night sweats. He appeared moderately unhealthy, with oral thrush and mild splenomegaly. A chest radiograph showed left upper lobe shadowing (Figure 1, panel A), sputum smears contained numerous acid-fast bacilli, and sputum was sent for culture, speciation, and sensitivity testing. Blood tests showed a CD4 cell count of 6 ×10 6 /L and HIV viral load of 272,000 (log 5.4) copies/mL. His hemoglobin was 12.1 g/dL, leukocyte count 4.9 × 10 9 /L, and platelets 367 × 10 9 /L. Renal and liver function test results were normal, apart from a γ-glutamyltransferase level of 200 U/L (normal <35 U/L). An ultrasound scan of his abdomen showed a diffuse increase in liver echogenicity and an enlarged (13.6-cm) spleen. He was naturally immune to hepatitis B, and tests for hepatitis C antibody and viremia were negative.
He was given broad-spectrum antimycobacterial therapy (rifampin, isoniazid, pyrazinamide, ethambutol, and azithromycin) as treatment for both M. tuberculosis complex (MTB complex) and opportunistic mycobacteria. He improved throughout a 2-week hospital stay and highly active antiretroviral therapy (HAART) was begun. It consisted of zidovudine and lamivudine and an increased dose of efavirenz (800 mg daily) because of concomitant rifampicin therapy, together with cotrimoxazole for prophylaxis against Pneumocystis jirovecii.
At a 3-week follow-up appointment, the patient had continued to improve. No further hemoptysis had occurred, and he had gained 2 kg body weight. After that, by telephone, he complained of occasional fevers and malaise but declined to be seen. Nine weeks after his initial visit, he came to the ward and with symptoms of breathlessness, nausea, and diarrhea. He had fevers spiking to >38°C, and newly palpable lymph nodes were evident in the right axilla and over the left parotid gland. A chest radiograph showed increased shadowing (Figure 1, panel B) and a computed tomographic (CT) scan of the chest showed a 6×3-cm solid mass in the upper left chest with visible air bronchograms and minor volume loss, reported as "almost certainly solid neoplasm" (Figure 2). A bronchoscopy, before which the patient received factor VIII, showed an area of irregular nodular tissue in the posterior segment of the left upper lobe. His CD4 count was 26 × 10 6 /L.
While we awaited histologic examination results, the patient's medication was withdrawn, and he improved within days. The lymph nodes reduced in size, and the spiking temperature resolved. Eight weeks after admission, the results of IS6110 polymerase chain reaction (PCR) testing on the slow-growing, positive mycobacterial culture confirmed the presence of MTB complex. The bronchial biopsy specimen showed necrotizing granulomatous bronchitis; acid-fast bacilli were not visible, and this specimen did not grow any mycobacteria.
The Mycobacterium species was not growing well in the laboratory, but M. microti was identified in the culture with spoligotyping methods (1,2). On extension of culture, the isolate was confirmed as M. microti phenotypically and found to be sensitive in vitro to rifampin, isoniazid, ethambutol, and clarithromycin but resistant to pyrazinamide.
The antimycobacterial drugs were reintroduced without problems 4 weeks later, as was HAART after an additional 8 weeks. Thirty months after restarting antimycobacterial therapy, the patient remained well with a total weight gain of 19 kg, complete radiologic resolution of the pulmonary mass, undetectable HIV viral load (<50 copies/mL), and a progressive rise in his CD4 count to 249 × 10 6 /L.

Conclusions
M. microti is a slow-growing member of the MTB complex. It has most commonly been described in association with small rodents (2,3). Its role as a pathogen in humans has been proposed by a handful of case reports involving both immunocompetent and immunodeficient persons (4)(5)(6). Because of the slow growth of the bacterium, confirmatory diagnosis is difficult, and cultures may be wrongly discarded as negative after a routine 8-to 12-week culture period. In such cases several commercial tests can identify the organism to the level of the MTB complex. The members of the tuberculosis complex are not reliably distinguished on biochemical grounds. Restriction fragment length polymorphism, DNA fingerprinting, is a time-consuming, slow, and expensive method of distinguishing members. Spoligotyping (spacer oligotyping) is a PCR-based test that can be quickly performed, even on the small amounts of DNA in poorly growing cultures. Strains differ in the size of spacer regions that intersperse direct repeat regions. These polymorphisms create differences in the PCR products produced, a process that allows strain identification (1,7). Other PCR-based tests are being developed that may have advantages over spoligotyping for identifying some members of the complex (8). Culture remains the standard and allows drug sensitivity testing. We suggest that such cultures be extended for up to 6 months if acid-fast bacilli are noted in specimens from a patient with consistent pulmonary pathologic findings.
Severe immunosuppression associated with HIV-1 can allow coinfections to remain subclinical. After HAART is introduced, IRD can lead to the development of opportunistic infections with atypical and severe clinical signs and symptoms. In this case, necrotizing granulomatous inflammation occurred without isolation of mycobacteria, which is atypical in an immunodeficient HIV patient. These clinical manifestations have been described in many diseases, including MTB and other opportunistic mycobacterial infections (9), herpesvirus infections, and hepatitis B and C (10). Similar granulomatous endobrochial lesions have been described during immune restoration with M. avium complex in HIV-infected patients (11). IRD has been described as occurring in 30% to 40% of those with very low CD4 counts when they begin HAART, developing within the first few months of treatment as the CD4 count rises. IRD is a diagnosis of exclusion: other processes such as drug fever, resistance, treatment failure, or other infections must be excluded. IRD may be a life-threatening condition, and clinical interventions, such as stopping medications, steroids, or both, may be indicated. An associated increase in markers of immune activation occurs, which seems to vary depending on the pathogen involved (12,13) .
In MTB, the major risk factor for IRD is beginning HAART within 2 months of antituberculosis treatment. This fact has led many experts to recommend delaying HAART for 2 months, a strategy that may also reduce the incidence of adverse events. However, a recent retrospective review suggested that delaying HAART would, particularly in those with CD4 cell counts<100 cells × 10 6 /L, lead to significant increase in risk for death and new opportunistic infections (14). IRD should always be considered along with treatment failure if a patient's clinical condition deteriorates after HAART is introduced.


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Alix Facilitates the Interaction between c-Cbl and Platelet-derived Growth Factor β-Receptor and Thereby Modulates Receptor Down-regulation*

Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) β-receptor (PDGFRβ) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRβ removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRβ. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRβ. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRβ. Our data suggest that Alix inhibits down-regulation of PDGFRβ by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.

Ligand binding induces dimerization and autophosphorylation of the receptors. The phosphorylated tyrosine residues have key roles in coupling to downstream signaling pathways as they mediate interaction with Src homology 2 domain (SH2)-containing molecules.
Alix (ALG-2-interacting protein X; also referred to as AIP1 or HP95) is a protein that was identified as a binding partner for ALG-2 (apoptosis-linked gene 2) (2). Apoptosis induced by various stimuli requires ALG-2, and several studies have indicated a role for Alix in regulation of cell death (3)(4)(5)(6). Alix possesses an N-terminal Bro1 domain and a C-terminal region rich in proline and tyrosine residues, suggesting an adaptor/scaffold function. Recently, Alix has been implicated in receptor endocytosis and trafficking (7)(8)(9). Work by Schmidt et al. (8) demonstrates that Alix antagonizes epidermal growth factor receptor (EGFR) down-regulation by the c-Cbl⅐CIN85 complex, resulting in reduced ubiquitination and internalization (8). The same group also showed that Alix interacts with and is phosphorylated by Src family kinases and that this phosphorylation reduces the interaction between Alix and EGFR, CIN85, or Pyk2, thereby releasing the inhibitory function of Alix on endocytosis (9). Another role that has emerged for Alix is in the organization of endosomes and formation of multivesicular bodies required for transportation of proteins from the early endosome to the lysosome (10,11). Furthermore, it has been demonstrated that Alix interacts with several cytoskeletal proteins, e.g. actin and tubulin, and with tyrosine kinases such as FAK and Pyk2, thereby negatively regulating cell adhesion (12). Overexpression of Alix in NIH3T3 cells promotes flat cell morphology and reduced proliferation (13), and Alix overexpression in HeLa cells leads to cell cycle arrest in G 1 phase (14). In summary, Alix has been shown to be involved in numerous cellular events, such as modulation of cellular architecture, apoptosis, growth, and endocytosis; however, the precise function of Alix is not well understood.
In the present report, we show that Alix interacts constitutively with PDGFR␤. It modulates the stability of the E3 ubiquitin ligase c-Cbl, thus affecting receptor ubiquitination and down-regulation.

EXPERIMENTAL PROCEDURES
Reagents-Recombinant human PDGF-BB was generously provided by Amgen (Thousand Oaks, CA). The following antibodies were used for immunoprecipitation and immunoblotting: phosphotyrosine (sc-7020) and PDGFR␤ (sc-339) antisera were from Santa Cruz Biotechnology (Santa Cruz, CA); the monoclonal PDGFR␤ antibody recognizing the extracellular domain (GR14L) was from Oncogene Research Products (Cambridge, MA); monoclonal ␤-actin antibody was from Sigma (A5441); the monoclonal HA antibody (12CA5) was from Roche Applied Science; the monoclonal c-Cbl antibody (610442) was from BD Transduction Laboratories; and Ras-GAP antibodies were from Upstate Biotechnology (05-178). An antiserum against Alix was raised by immunizing a rabbit with the synthetic peptide CSYPFPQPPQQSYYPQQ conjugated to keyhole limpet hemocyanin. The antibodies were purified by chromatography over a column of the corresponding peptide immobilized on Sepharose beads. Purified antibodies were stored in 0.15 M NaCl, 20 mM Hepes, pH 7.4, and 50% glycerol at Ϫ70°C. Rabbit antiserum against PLC␥ was generated by immunizing rabbits with a peptide corresponding to the C terminus of bovine PLC␥ (15).
Cell Culture and Transfection-Porcine aortic endothelial (PAE) and 293T cells were cultured in Ham's F-12 and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum and 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. Cells were transfected by Lipofectamine according to the protocol provided by the manufacturer (Invitrogen). For transient transfections, cells were assayed 48 h after transfection. To establish stable transfectants, cells were selected 48 h after transfection by the presence of 400 g/ml zeocin until only resistant cells were left.
Affinity Purification of Tyrosine-phosphorylated Proteins-Subconfluent PAE cells (total 10 9 cells) expressing either PDGFR␣ or PDGFR␤ were serum-starved overnight and then stimulated for 5 min with 100 ng/ml PDGF-BB and lysed in 0.5% Triton, 50 mM NaCl, 10 mM NaF, 30 mM tetrasodium pyrophosphate, 10% glycerol, 1 mM EDTA, 20 mM Tris, 1 mM Pefabloc, 1% Trasylol, and 1 mM sodium orthovanadate, pH 7.4. Extracts were cleared by centrifugation, and tyrosine-phosphorylated proteins were immunoprecipitated with 4G10 antibodies coupled to agarose beads (Upstate Biotechnology) for 2 h. The immunocomplex was washed three times in lysis buffer and then eluted twice with 100 mM phenylphosphate in 10 mM Hepes, pH 7.4, at 4°C. Eluted proteins were precipitated using the Wessel-Flügge procedure (16) and resuspended in 100 l of sample buffer containing 10 mM dithiothreitol. The samples were treated with 25 mM iodoacetamide for 15 min at room temperature before separation by SDS-PAGE and visualization by silver staining using an in-gel digestion compatible protocol (17).
Protein Identification-Excised protein bands were destained (18) and dried with acetonitrile. A solution of porcine sequence grade trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate, 10% acetonitrile was allowed to soak into the gel pieces on ice for 45 min. Approximately 100 ng trypsin/gel band was applied and incubated at 30°C overnight. The digestion was stopped by making the samples 1% with respect to trifluoroacetic acid. After dilution in 0.1% trifluoroacetic acid, samples were concentrated and desalted on a Zip-Tip C18 column (Millipore Corp., Bedford, MA), and the digest mixture was eluted directly onto the matrix-assisted laser desorption/ionization (MALDI) target with 70% acetonitrile, containing about 8 mg/ml matrix ␣-cyano-4-hydroxycinnamic acid. Peptide mass fingerprinting was done by MALDI time-offlight mass spectrometry (MALDI-TOF MS) on a Bruker Ultraflex TOF/TOF (Bruker Daltonics, Bremen, Germany). The instrument was operated in reflector mode and optimized for analytes up to 4000 Da. The spectra were internally calibrated using autolytic trypsin peptides. The peptide mass lists were used to scan protein sequence data banks for protein identities via the ProFound search engine. Significance of matches were judged after taking several parameters into account, such as "z-score," error distribution, number of miscuts, and migration behavior.
Immunoprecipitation and Western Blotting-Subconfluent cells were starved in 0.1% fetal bovine serum overnight and incubated and with 100 ng/ml PDGF-BB for the indicated periods of time. Cells were washed with ice-cold phosphate-buffered saline and lysed (0.5% Triton, 50 mM NaCl, 10 mM NaF, 30 mM tetrasodium pyrophosphate, 10% glycerol, 1 mM EDTA, 20 mM Tris, 1 mM Pefabloc, 1% Trasylol, 1 mM sodium orthovanadate, pH 7.4). Extracts were clarified by centrifugation, and protein concentration was determined by the BCA protein assay system (Pierce). Equal amounts of lysates were incubated with 1 g of antibody as indicated. The immunoprecipitates were collected on protein A-Sepharose beads, washed three times in lysis buffer, and then boiled in SDS sample buffer containing dithiothreitol. Immunoprecipitates or equal amounts of total cell lysates were analyzed by SDS-PAGE. For Western blotting, samples were electrotransferred to polyvinylidene difluoride membranes (Immobilon P), which were blocked in 5% bovine serum albumin in phosphate-buffered saline solution containing 0.1% Tween 20. Primary antibodies were used with the concentrations and buffers recommended by the suppliers and incubated overnight in the cold. After washing, the membranes were incubated with horseradish peroxidase-conjugated antimouse or anti-rabbit IgG antibodies (both from GE Healthcare/ Amersham Biosciences), and proteins were visualized using ECL Western blotting detection systems from Roche Applied Science on a cooled charge-coupled device camera (CCD; Fuji, Minami-Ashigata, Japan). Before reprobing, the membranes were stripped with 0.4 M NaOH for 10 min at room temperature, blocked, and incubated with the corresponding antibody.
Biotinylation of Cell Surface Proteins-Cells were serumstarved overnight and stimulated with 20 ng/ml PDGF-BB for different periods of time at 37°C. Next, cells were washed twice in ice-cold phosphate-buffered saline, pH 7.4, and incubated for 1 h on ice with 0.2 mg/ml sulfo-NHS-SS-biotin (Pierce) in phosphate-buffered saline. To inactivate unbound sulfo-NHS-SSbiotin, cells were treated for 5 min with 50 mM Tris, pH 8.0. Cells were lysed, and biotinylated proteins were precipitated with streptavidin-agarose (Sigma) for 1 h at 4°C. The beads were washed three times in lysis buffer and boiled with SDS sample buffer containing dithiothreitol. Eluted proteins were separated using SDS-PAGE and transferred to Immobilon P membranes. The membranes were blocked with 5% bovine serum albumin in phosphate-buffered saline, and the amount of PDGFR␤ was visualized and quantified by immunoblotting with PDGFR␤ antibodies.

RESULTS
Identification of Tyrosine-phosphorylated Proteins after PDGF Stimulation-We were interested in identifying proteins that become tyrosine-phosphorylated after PDGF stimulation and hence are likely to participate in signal transduction downstream of the PDGFRs. To this end we used monoclonal antiphosphotyrosine antibodies (4G10) immobilized on Sepharose beads to immunoprecipitate proteins from PDGF-BB-stimulated PAE cells transfected with either PDGFR␣ or PDGFR␤. This allowed us to investigate the tyrosine-phosphorylated proteins downstream of the two PDGFR isoforms, expressed at comparable levels in the same cellular background. Bound proteins were eluted, separated by SDS-PAGE, and visualized by silver staining (supplemental Fig. 1). Proteins tentatively phosphorylated after PDGF stimulation were excised and identified by mass spectroscopy as indicated in supplemental Fig. 1. Among the proteins identified, Alix was selected for further study in regard to PDGFR␤ signal transduction. Interestingly, we observed that Alix was not significantly phosphorylated by PDGFR␣, indicating that Alix is a preferred substrate for PDGFR␤.
Alix Is Phosphorylated in Response to PDGF and Interacts Constitutively with PDGFR␤-To verify the mass spectroscopy results, we immunoprecipitated Alix from cells stimulated with PDGF-BB and resolved the immunocomplexes by SDS-PAGE followed by transfer to an Immobilon P membrane. A phosphotyrosine immunoblot revealed bands with sizes corresponding to Alix (ϳ95 kDa) and PDGFR␤ (ϳ180 kDa) in the Alix immunoprecipitate (Fig. 1A, left panels). The 95-and 180-kDa phosphoproteins were confirmed to be Alix and PDGFR␤, respectively, by reprobing the membrane with Alix-or PDGFR␤-specific antibodies (Fig. 1A, left panels). Interestingly, we found that Alix interacted with PDGFR␤ in a constitutive manner, although its tyrosine phosphorylation was ligand-dependent. The interaction could also be detected in a reciprocal experiment (Fig. 1A, right panels). Next, we performed a kinetic analysis of Alix phosphorylation. We found that Alix was transiently phosphorylated in response to PDGF-BB (Fig. 1B, top panel) with kinetics similar to the overall receptor activation as determined by anti-phosphotyrosine immunoblotting of WGA-Sepharose-enriched PDGFR␤ (Fig. 1B, bottom panel). Our results suggest that Alix is phosphorylated by PDGFR␤. It is possible that Alix is also phosphorylated indirectly by Src family kinases activated by PDGFR␤; however, such a contribution is likely to be minor, because Alix phosphorylation was only slightly inhibited by the low molecular weight inhibitor SU6656 (data not shown).
Alix Binds to the C-terminal Region of PDGFR␤-To determine the binding region for Alix in PDGFR␤, we decided to analyze different C-terminally truncated mutants of the receptor for their abilities to interact with Alix. By expressing deletion mutants of the receptor in 293T cells, we found a partial loss of interaction when the 75 most C-terminal amino acid residues of PDGFR␤ were deleted and an almost complete loss of binding when the last 98 amino acid residues were deleted ( Fig. 2A). Because Alix, despite its not being significantly phosphorylated by PDGFR␣, was also found to interact with this receptor (data not shown), we compared the amino acid sequence of PDGFR␣ and -␤ in the region important for binding. We observed that the sequence surrounding Tyr-1021 in PDGFR␤ is similar between both receptor isoforms (Fig. 2B). Furthermore, this region is rich in proline residues, which can interact with SH3 domain or WW domain-containing proteins in a constitutive manner. To clarify whether this region is responsible for the interaction with Alix, we synthesized peptides corresponding to the amino acid sequence between residues 1015 and 1030, with the addition of an N-terminal cysteine to facilitate coupling to a solid support (Fig. 2B), with Tyr-1021 either phosphorylated or not phosphorylated. Next we performed in vitro interaction assays using this peptide immobilized on beads followed by immunoblotting for target proteins. We found that Alix interacted with both peptides, independent of phosphorylation, in agreement with the constitutive binding observed in vivo (Fig. 2C). As a control, we analyzed the interaction between these peptides and its known interaction partner PLC␥1. Indeed, we could detect a phosphorylationdependent interaction with PLC␥1 (Fig. 2C). As a negative control we used a peptide containing tyrosine 771 in the PDGFR␤, which is known to bind RasGAP. As seen in Fig. 2C, this peptide, when phosphorylated on tyrosine, could pull down RasGAP but not Alix. Thus, we concluded that Alix can interact with the PDGFR␤ in the region between residues 1015 and 1030 in a receptor phosphorylation-independent manner. However, other regions in the PDGFR␤ may contribute, because there was a weak residual binding also when the last 98 amino acids of the receptor were deleted.

Alix Inhibits PDGF-BB-induced Removal of PDGFR␤ from the Cell Surface-To investigate whether the expression level of
Alix influenced the rate of PDGFR␤ cell surface clearance, we analyzed cells that were engineered to overexpress Alix (Fig.  3A). To measure the amount of receptors on the cell surface, we stimulated cells for different periods of time and biotinylated the cell surface proteins prior to lysis. Biotinylated proteins were purified using streptavidin-agarose, and the levels of PDGFR␤ were then analyzed by immunoblotting (Fig. 3B). As can be seen, the PDGF-BB-induced clearance of PDGFR␤ from the cell surface was reduced in Alix-overexpressing cells as compared with mock-transfected control cells.

Alix Overexpression Enhances Interaction between c-Cbl and PDGFR␤ and Phosphorylation of c-Cbl-Ubiquitination of receptor tyrosine kinases is important for their internalization, and c-Cbl is a major ubiquitin ligase connected with PDGFR␤.
Because we noticed a reduced rate of PDGFR␤ down-regulation from the cell surface in cells overexpressing Alix (Fig. 3B), we investigated the effect of increased Alix levels on c-Cbl phosphorylation and association with the PDGFR␤ after ligand stimulation. We found that elevated Alix levels resulted in increased interaction between c-Cbl and PDGFR␤ (Fig. 4). In addition, we observed that Alix co-immunoprecipitated with c-Cbl. Interestingly, neither the interaction between c-Cbl and Alix nor that between c-Cbl and PDGFR␤ was induced by PDGF stimulation, but it existed in a constitutive manner both in control and Alix-overexpressing cells. Moreover, PDGF-induced c-Cbl phosphorylation was augmented in cells overexpressing Alix compared with control cells (Fig. 4).

Elevated Levels of Alix Lead to Reduced PDGFR␤ Down-regulation but Increased c-Cbl Degradation in
Response to PDGF-BB-Next, we wanted to determine whether the reduced rate of PDGFR␤ removal from the cell surface in cells overexpressing Alix was associated with changes in receptor stability. We therefore stimulated cells with PDGF-BB for different periods of time and determined the amount of PDGFR␤ by immunoblotting. Indeed, ligand-induced PDGFR␤ degradation was reduced in Alixoverexpressing cells compared with mock-transfected cells (Fig. 5). In contrast, we could not observe any effects of Alix overexpression on PDGFR␤ phosphorylation. However, c-Cbl was hyperphosphorylated in cells overexpressing Alix (Figs. 4 and 5). We found that the enhanced c-Cbl phosphorylation in response to PDGF-BB stimulation in cells with elevated levels of Alix correlated with increased c-Cbl degradation (Fig.  5). The enhanced rate of c-Cbl degradation was completely inhibited by MG132, suggesting that the degradation occurred in proteasomes (Fig. 6).
Elevated Alix Expression Decreases Ligand-induced PDGFR␤ Ubiquitination-The observation that increased Alix expression resulted in increased proteasomal degradation of c-Cbl suggested that the level of PDGFR␤ ubiquitination might also be affected. To investigate this possibility, we transfected Alixoverexpressing or mock-transfected cells with ubiquitin fused with an HA epitope and performed immunoprecipitation of PDGFR␤ followed by immunoblotting with HA antibodies. Indeed, we found that the PDGFR␤ was significantly less ubiquitinated in cells overexpressing Alix (Fig. 7). However, after shorter periods (5-15 min) of PDGF-BB stimulation, we could not demonstrate a significant difference in level of PDGFR␤ ubiquitination between control and Alix-overexpressing cells (data not shown). This suggests that the bulk of c-Cbl FIGURE 2. Alix binds to the C-terminal tail of PDGFR␤. A, 293T cells were transiently transfected with PDGFR␤ constructs with progressively longer deletions of the C-terminal tail (CT) and stimulated with PDGF-BB (100 ng/ml) for 5 min. Alix was immunoprecipitated (Ip) and the membrane immunoblotted (Ib) with antibodies recognizing the extracellular domain PDGFR␤. The membrane was then reprobed with Alix antibodies. To verify comparable receptor expression and activation in all transfections, PDGFRs were precipitated with WGA-Sepharose, and the filter was immunoblotted with pTyr and subsequently reprobed for total receptor. WT, wild type. B, the amino acid sequence of PDGFR␤ corresponding to the region between CT98 and CT75 is shown in bold letters. The PDGFR␣ sequence is aligned below. On the right, the position numbers for the C-terminal residues are indicated. In addition, the amino acid sequence for the peptide used for in vitro interaction studies is shown, to which an N-terminal cysteine was added to facilitate coupling to Sulfolink beads. C, a synthetic peptide corresponding to the amino acid sequence surrounding tyrosine 1021 (residues 1015-1030, see panel B) or tyrosine 771 (residue 764 -781), either phosphorylated or not, was used to investigate the interaction among Alix, PLC␥1, and RasGAP in vitro by immunoblotting as indicated.
proteins must first be degraded before the reduction in ubiquitination can be observed. In summary, Alix overexpression stabilizes the PDGFR␤, possibly through promoting enhanced proteasomal degradation of c-Cbl in response to PDGF-BB and thereby reducing c-Cbl-mediated ubiquitination of the PDGFR␤.

DISCUSSION
Down-regulation of receptor tyrosine kinases is essential for proper response to ligand, and disturbances in this process may contribute to transformation (19). It has been shown that monoubiquitination of receptor tyrosine kinases is an important mechanism that regulates receptor internalization and intracellular trafficking (20). c-Cbl is a ubiquitin ligase targeting several receptor tyrosine kinases including the PDGFRs. In this report, we present data on c-Cbl and the multifunctional adaptor protein Alix and on their involvement in PDGFR␤ downregulation. In general, our results are consistent with earlier work with the EGFR (8,9).
We found that Alix forms a constitutive complex with PDGFR␤ and is transiently phosphorylated in response to ligand stimulation. An activation-independent interaction has also been reported between Alix and the EGFR (8). We mapped the binding site of Alix to the C-terminal tail of PDGFR␤. However, contributions from other regions of the receptor in this interaction cannot be excluded. The C-terminal region contains a proline-rich sequence that may mediate the interaction with Alix. However, Alix does not contain any domain that is known to interact with proline-rich regions. Thus, it is possible the interaction between Alix and PDGFR␤ is indirect, through an adaptor protein. In fact, Alix is believed to bind indirectly to the EGFR because the association cannot be observed using recombinant proteins (8). Moreover, the interaction between Alix and PDGFR␤ was not affected by mutation of tyrosines 1009 and 1021 in the C-terminal tail of the receptor to phenylalanine (data not shown).
It was recently shown that overexpression of Alix results in a reduced EGFR internalization (8). We found that overexpression of Alix also reduced the rate of ligand-induced PDGFR␤ removal from the cell surface compared with mock-transfected cells; this reduction was accompanied by a decreased rate of receptor degradation. Moreover, we observed c-Cbl hyperphosphorylation in response to PDGF-BB in Alix-overexpressing cells that was associated with enhanced proteasomal degradation of c-Cbl. These observations are in concurrence with a study from Bao et al. (21) showing that expression of active Src (or overexpression of normal Src) promotes c-Cbl hyperphos- . A, total cell lysate (TCL) was separated and immunoblotted with Alix antibodies to determine Alix overexpression. B, cells were serum-starved overnight and treated with PDGF-BB for the indicated periods of time at 37°C. Cell surface proteins were then biotinylated before lysis. Biotinylated proteins were precipitated with streptavidin-agarose, separated on SDS-PAGE, and transferred onto Immobilon P membranes. The level of receptor was determined by immunoblotting (Ib) with PDGFR␤ antibodies. The intensity of the PDGFR␤ bands was measured and plotted as percent of initial receptor levels. . c-Cbl exists in a constitutive complex with Alix and PDGFR␤, and overexpression of Alix increases c-Cbl phosphorylation. PAE/ PDGFR␤-Alix and PAE/PDGFR␤ cells were serum-starved overnight and treated with 100 ng/ml PDGF-BB for 5 min at 37°C. Lysates were prepared, and c-Cbl was immunoprecipitated (Ip). Proteins were separated by SDS-PAGE, transferred onto Immobilon P membranes, and subjected to immunoblotting (Ib) with pTyr, PDGFR␤, Alix, and c-Cbl antibodies as indicated. PDGFR␤ was precipitated with WGA-Sepharose, and the filter was probed with pTyr antibodies to verify equal receptor stimulation.
phorylation and, as a consequence, its degradation (21). However, the low molecular weight Src inhibitor SU6656 had no significant effect on PDGF-BB-induced c-Cbl phosphorylation in our cell model (data not shown).
Our results suggest that that c-Cbl, Alix, and PDGFR␤ interact constitutively, although our data could not distinguish whether a ternary complex or separate binary complexes are formed. However, elevated Alix expression enhances the interaction between c-Cbl and PDGFR␤. Notably, the constitutive interaction between c-Cbl and PDGFR␤ was also detected in mock-transfected cells and hence is not an artifact of overexpression. The enhanced c-Cbl phosphorylation observed in cells overexpressing Alix may be explained by enhanced interaction between c-Cbl and the receptor, which may allow a more efficient phosphorylation.
Given that c-Cbl is a major ubiquitin ligase downstream of receptor tyrosine kinases (22), we anticipated that an increase in c-Cbl degradation would be reflected in a decreased level of receptor ubiquitination. Indeed, we observed that Alix overexpression correlated with a reduced level of PDGFR␤ ubiquitination. It has been suggested that degradation of hyperphosphorylated c-Cbl allows the EGFR to recycle back to the plasma membrane instead of being sorted for lysosomal degradation (21), potentially because of a lack of sustained ubiquitination (23,24). This is compatible with our results showing that Alix overexpression leads to increased c-Cbl phosphorylation and degradation, as well as to a reduced rate of PDGFR␤ clearance from the cell surface. It remains to be determined whether this FIGURE 5. Alix overexpression inhibits PDGFR␤ degradation and enhances Cbl degradation. A, PAE/PDGFR␤-Alix and PAE/PDGFR␤ cells were serum-starved overnight and treated with 100 ng/ml PDGF-BB for the indicated periods of time at 37°C. Lysates were prepared, and PDGFR␤ was immunoprecipitated (Ip) using WGA-Sepharose and c-Cbl. Proteins were separated by SDS-PAGE, transferred onto Immobilon P membranes, and subjected to immunoblotting (Ib) as indicated. As a loading control, a fraction of total cell lysate (TCL) was subjected to immunoblotting for ␤-actin levels. B, the average intensities from three experiments were measured and plotted Ϯ1 S.D. as indicated. The value for unstimulated cells was used for normalization. a.u., arbitrary units. FIGURE 6. Cbl is degraded via the proteasomal pathway. A, PAE/PDGFR␤-Alix and PAE/PDGFR␤ cells were serum-starved overnight and treated with 25 M MG132 or dimethyl sulfoxide (DMSO) for 4 h followed by stimulation with 100 ng/ml PDGF-BB for the indicated periods of time at 37°C. Lysates were prepared, and c-Cbl was immunoprecipitated (Ip), separated by SDS-PAGE, transferred onto Immobilon P membranes, and immunoblotted (Ib) as indicated. As a loading control a fraction of total cell lysate (TCL) was immunoblotted for ␤-actin levels. B, the intensities of the bands shown in A were measured and plotted as indicated. The value for unstimulated mock cells was used for normalization. a.u., arbitrary units.
is because of decreased internalization or increased recycling. In conclusion, Alix may function as an adaptor/scaffold protein regulating PDGFR␤ ubiquitination and consequently intracellular sorting by modulating the degradation of c-Cbl.