Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor*

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, α-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu5b receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu5b receptor and α-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of α-actinin-1 with mGlu5b receptor modulated the cell surface expression of the receptor. This was dependent on the binding of α-actinin-1 to the actin cytoskeleton. In addition, the α-actinin-1/mGlu5b receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an α-actinin-1-dependent mGlu5b receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

Glutamate and aspartate are the major excitatory neurotransmitters in the mammalian central nervous system (1,2). These excitatory amino acids act on glutamate receptors and play an important role in many physiological functions, including learning, memory, and development (3). Glutamate receptors are widely distributed in the central nervous system and include three subtypes of ionotropic glutamate receptors (␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, NMDA, 3 and kainate receptors) and a family of G protein-cou-pled metabotropic glutamate (mGlu) receptors that act through different second messenger pathways. Eight members of the mGlu receptor family have been identified and categorized into three subgroups on the basis of their sequence homology, agonist selectivity and signal transduction pathway. Group I contains the mGlu 1 and mGlu 5 receptor subtypes, which are coupled to phospholipase C in transfected cells and have quisqualic acid as their most potent agonist (4). The mGlu 5 receptor is expressed in two splice variants, mGlu 5a and mGlu 5b , which differ in that mGlu 5b has a 33-amino acid insert in the intracellular C-terminal domain. Interestingly, both subtypes of mGlu 5 are heavily expressed in striatum with the consideration that mGlu 5b might be considered as an "adult" variant and mGlu 5a is more a "neonatal" variant (5).
The actin-based cytoskeleton is connected to the plasma membrane via a lattice-like network of actin-binding proteins that form the membrane skeleton or membrane-associated cytoskeleton (6). The major structural component of the membrane skeleton is spectrin (also referred to as fodrin in nonerythroid cells), a flexible rod-shaped molecule composed of homologous, but non-identical ␣and ␤-subunits. Other actinbinding proteins, like filamin A and ␣-actinin, also participate in the maintenance of this membrane-associated cytoskeleton and are essential for the anchoring of transmembrane proteins. 〈 major F-actin cross-linking protein (7), present in both muscle and non-muscle cells, is ␣-actinin. There are four ␣-actinin genes, two non-skeletal muscle isoforms, ␣-actinin-1 and -4, and two skeletal muscle isoforms, ␣-actinin-2 and -3 (8). All of them share a general structure, which can be divided into three functionally distinct domains: the N terminus containing two calponin homology domains that bind to actin filaments (9), a central region composed of four spectrin-like motifs (10), which acts as a switchboard for interactions with multiple proteins, and the C terminus, which contains EF-hand domains responsible for Ca 2ϩ binding (11) and terminates in a PDZ domain-binding sequence, ESDL (12) (for review see Refs. 13 and 14). Members of the ␣-actinin family, namely ␣-actinin-1, -2, and -4, are abundantly represented in postsynaptic density (PSD) excitatory synapses (15,16), where it is believed they regulate postsynaptic actin dynamics and spine morphology (17). Recently, the spatial expression of ␣-actinin-2 in the rat central nervous system has been analyzed. The highest levels of the protein are found in the striatum, cortex, and hippocampus, where ␣-actinin-2 interacts with both the NMDA subtype of glutamate receptor (18,19) and the adenosine A 2A receptor (20). Also, ␣-actinin-1 showed a high expression level in neurons of striatum, whereas the cerebellum and other subcortical structures showed only weak labeling (21).
In the present study we carried out a GAL-4-based yeast twohybrid screen to identify mGlu 5b partners in adult brain. Using a C-terminal tail region of the receptor as bait we identified ␣-actinin-1 and -4 as novel binding partners of the mGlu 5b receptor. We focus on the characterization of ␣-actinin-1-mGlu 5b interaction, because both proteins are heavily expressed in the same adult brain area, the striatum. This interaction might have relevant physiological consequences, because we demonstrate, in the present work, that ␣-actinin-1 controls the cell surface expression and functioning of mGlu 5b receptor.
Yeast Two-hybrid System-Yeast two-hybrid screening was performed as described previously (20). Briefly, a bait strain was created by transforming pHybLex-LmGlu 5b into Saccharomyces cerevisiae strain L40 as described in the manufacturer's instructions (Hybrid Hunter, Invitrogen). The bait strain was co-transformed with an adult mouse brain cDNA library constructed in the Gal4-activating domain vector pPC86 (Invitrogen), and transformants were plated onto minimal yeast media lacking histidine, tryptophan, uracil, and lysine, containing 300 mg/ml Zeocin (Invitrogen) and 5 mM 3-aminotriazole. Plates were incubated at 30°C for 5 days, and yeast colonies that grew on histidine-deficient media were re-streaked onto fresh selective plates and assayed for ␤-galactosidase activity as per the manufacturer's instructions. Prey plasmids were isolated from yeast and electroporated into Escherichia coli XL-1Blue electrocompetent cells (Stratagene). The 5Ј-end of each clone was sequenced using a vector primer. To confirm the interaction in yeast, purified prey plasmids were re-transformed with the pHybLex-LmGlu 5b and pHybLex-SmGlu 5b baits and with the bait empty bait vector pHybLex/Zeo and tested for growth on selective plates and ␤-galactosidase activity.
For liquid ␤-galactosidase assays 1.5 ml of each culture, grown for 48 h at 30°C, was spun, and the pellet was re-suspended in 200 l of Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , pH 7.0). A small amount of glass beads (425-600 m, Sigma) was added, and the mixture was sonicated for 5-10 min. After cell lysis, the samples were spun to pellet the cell debris. 100 l of supernatant was transferred to a new microcentrifuge tube, and 700 l of Z buffer containing ␤-mercaptoethanol (27 l/10 ml) was added. 150 l of 2.5 mg/ml ortho-nitrophenyl-␤-galactoside (Sigma) was added to the sample, and the mixture was incubated at 37°C for 3 h. The absorbance was read at 420 nm and referred to the amount of protein present in each sample. For strong enzymatic reactions (i.e. when the color started to appear after a few minutes of incubation), a 1:10 dilution of the yeast lysate was used and the absorbance at 420 nm was multiplied by 10.
Cell Culture, Transfection, and Membrane Preparation-HEK-293 cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, and 10% (v/v) fetal bovine serum at 37°C and in an atmosphere of 5% CO 2 . HEK-293 cells growing in 25-cm 3 dishes or 20-mm coverslips were transiently transfected with 10 g of DNA encoding for the proteins specified in each case by calcium phosphate precipitation (23). The cells were harvested at either 24 or 48 h after transfection.
Neuronal striatal primary cultures were obtained as described previously (24) and plated at a density of 5 ϫ 10 4 cells/cm 2 . Membrane suspensions from rat striatum or from transfected HEK cells were obtained as described previously (25,26).
Immunoprecipitation and Immunocytochemistry-For immunoprecipitation, membranes from transiently transfected HEK cells were solubilized in ice-cold lysis buffer (PBS, pH 7.4, containing 1% (v/v) Nonidet P-40) for 30 min on ice. In the case of rat striatum membranes these were solubilized in 2% SDS in PBS and then diluted with 5 volumes of ice-cold 2% (v/v) Nonidet P-40 in PBS (28). In both cases, the solubilized preparation was then centrifuged at 13,000 ϫ g for 30 min. The supernatant (1 mg/ml) was processed for immunoprecipitation, each step of which was conducted with constant rotation at 0 -4°C. The supernatant was incubated overnight with the indicated antibody. Next 40 l of a suspension of protein G cross-linked to agarose beads was added, and the mixture was incubated overnight. The beads were washed and treated as described above.
For immunocytochemistry, transiently transfected HEK-293 cells, or rat neuronal striatal primary cultures, were fixed in 4% paraformaldehyde for 15 min, and washed with PBS containing 20 mM glycine (buffer A) to quench the remaining free aldehyde groups. Cells were permeabilized with buffer A containing 0.2% Triton X-100 for 5 min. Blocking was performed using buffer A containing 1% bovine serum albumin (buffer B). Cells were labeled for 1 h at room temperature with the indicated primary antibody, washed for 30 min in buffer B, and stained with the corresponding secondary antibodies for another hour. Samples were rinsed and then examined using a confocal microscope (29,30). To test the specificity of the antibodies we omitted or replaced the primary antibodies with buffer B. Under these conditions, no selective labeling was observed.
FRET Experiments Analyzed by Fluorometry-Forty-eight hours after transfection, cells were rapidly washed twice in PBS, detached, and re-suspended in the same buffer. To control the number of cells, the protein concentration of the samples was determined using a Bradford assay kit (Bio-Rad) using bovine serum albumin dilutions as standards. Cell suspension (20 g of protein) was distributed in duplicate into 96-well microplates (black plates with a transparent bottom). Plates were read in a Fluostar Optima Fluorometer equipped with a high energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 400 nm (393-403 nm), and 10 nm bandwidth emission filters corresponding to a 506 -515 nm filter (Ch 1) and a 527-536 nm filter (Ch 2). Gain settings were identical for all experiments to keep the relative contribution of the fluorophores to the detection channels constant for spectral un-mixing. Quantitation of FRET was performed as described previously (31). The contribution of each fluorophore to both detection channels was calculated from the readings obtained by expressing each GFP variant separately. The spectral signatures of the different receptors fused to either GFP 2 or YFP did not significantly vary from the determined spectral signatures of the fluorescent proteins alone. Linear un-mixing was performed according to Zim-mGlu 5b Receptor ␣-Actinin-1 Interaction APRIL 20, 2007 • VOLUME 282 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12145 mermann et al. (32) and was used to determine the fluorescence emitted by each of the fluorophores.
Biotinylation of Cell Surface Proteins-Cell surface proteins were biotinylated as described previously (33,34). Briefly, HEK-293 cells transiently transfected with the mGlu 5b receptor in the absence, or presence, of ␣-actinin-1-YFP constructs were washed three times in borate buffer (10 mM H 3 BO 3 , pH 8.8; 150 mM NaCl) and then incubated with 50 g/ml Sulfo-NHS-LC-Biotin (Pierce) in borate buffer for 5 min at room temperature. Cells were washed three times in borate buffer and again incubated with 50 g/ml Sulfo-NHS-LC-Biotin in borate buffer for 10 min at room temperature, and then 13 mM NH 4 Cl was added for 5 min to quench the remaining biotin. Cells were washed in Tris-buffered saline, disrupted with three 10-s strokes in a Polytron, and centrifuged at 14,000 ϫ g for 30 min. The pellet was solubilized in ice-cold lysis buffer (see above) for 30 min and centrifuged at 14,000 ϫ g for 20 min. The supernatant was incubated with 80 l of streptavidin-agarose beads (Sigma) for 1 h with constant rotation at 4°C. The beads were washed and treated as described above and processed for immunoblotting.
Extracellular Signal-regulated Kinase Assay-Before stimulation with quisqualic acid transiently transfected HEK-293 cells were serum-starved for 16 h by replacing the usual culture medium for normal Dulbecco's modified Eagle's medium without glutamine and fetal bovine serum but containing 2 mM sodium pyruvate and 1 unit/ml glutamate-pyruvate transaminase (Roche Applied Science) to eliminate glutamate from the medium. After stimulation, cells were washed with ice-cold PBS and scraped into 1 ml of lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH 7.6, 45 mM ␤-glycerophosphate, 50 mM NaF, and 1 mM NaVO 4 in the presence of a protease inhibitor mixture (Sigma). Lysed cells were centrifuged for 20 min at 14,000 rpm at 4°C, and equal protein concentrations were resolved on 10% SDS-PAGE, blotted onto Immobilon-P membrane, and incubated with rabbit anti-ERK1/2 (1/40,000) or mouse anti-phospho-ERK1/2 (1/2,500). Quantitative analysis of detected bands was performed by using densitometric scanning (35).

RESULTS
Yeast Two-hybrid Screening-To identify intracellular proteins interacting with the mGlu 5b receptor, a region containing 178 amino acids of the C-terminal tail of the receptor (amino acids 828 -1006) were fused in-frame with LexA in the pHybLexA/Zeo vector (LmGlu 5b , Fig. 1A) and used to screen a mouse brain cDNA library using the yeast two-hybrid system. Of the seven clones, from the 1 ϫ 10 6 total transfor-FIGURE 1. ␣-Actinin-1 interacts with mGlu 5b receptor in the yeast twohybrid system. A, schematic representation of the pHybLex-LmGlu 5b (LmGlu 5b ) fusion protein containing amino acids 828 -1006 and pHybLex-SmGlu 5b (SmGlu 5b ) fusion protein containing amino acids 828 -932 of the C-terminal tail mGlu 5b receptor. Quantitation of the interaction of ␣-actinin-1 isoform with mGlu 5b receptor fusion proteins was determined using a liquid ␤-galactosidase assay as described under "Experimental Procedures" (inset panel in A). Data are mean Ϯ S.E. values of three replicates. pHyb, pHybLex (Invitrogen); TM7, seven transmembrane domains. B, schematic representation of the interacting region of ␣-actinin-1. The interacting region of ␣-actinin-1 with the C-Terminal tail mGlu 5b receptor comprises amino acids 369 -892 of ␣-actinin-1. CH, calponin homology domain; SPEC, spectrin-like motif; EFH, EF-hand domain. C, the regions containing transmembrane 7 (TM7) and C-terminal tail of hmGlu 5b (accession code: D28539) and hmGlu 5a (accession code: D28538) are aligned. Dashed lines indicate the region of deletions in the hmGlu 5a (32 amino acids) receptor variant. The putative ␣-actinin-1 binding motif is underlined in black (amino acids 932-1006). The two boxed regions represent the Ca 2ϩ /calmodulin binding motifs. Also illustrated are motifs required for Homer and PDZ domain interactions. . mGlu 5b receptor was detected using a polyclonal antibody against the mGlu 5a/b receptor (1/1,000), and the GST fusion proteins with a polyclonal antibody were used against GST (1/2,000). The primary bound antibody was detected using a HRP-conjugated goat anti-rabbit antibody (1/60,000). The immunoreactive bands were visualized by chemiluminescence. mGlu 5b Receptor ␣-Actinin-1 Interaction mants screened that were found to grow onto nutritional-deficient plates and activated the ␤-galactosidase assay, three were identified as different isoforms of the actin binding protein ␣-actinin, one clone for ␣-actinin-1, and another two for ␣-actinin-4. The isolated ␣-actinin-1 clone comprises amino acids 369 -892 that include part of the spectrin-like motif and the Ca 2ϩ binding domain (Fig. 1B). To determine the region of the C-terminal domain of the mGlu 5b receptor that interacted with ␣-actinin-1, another LexA fusion protein missing the last 74 amino acids of the former LmGlu 5b was constructed (SmGlu 5b , Fig. 1A) and tested for its ability to bind ␣-actinin-1. This shorter fusion protein could not interact with ␣-actinin-1 as tested using a liquid ␤-galactosidase assay (Fig. 1A, inset panel), thus mapping the interacting domain to within amino acids 932-1006 of mGlu 5b receptor. This region is common in both mGlu 5a and mGlu 5b receptor isoforms and close to the described Ca 2ϩ /calmodulin binding motifs (36) (Fig. 1C).
To assess the physiological relevance of the ␣-actinin-1/ mGlu 5b receptor interaction, co-immunoprecipitation experi- . Cells were processed for immunoprecipitation (see "Experimental Procedures") using a monoclonal anti-GFP antibody (2 g/ml). The crude extracts (Crude) and immunoprecipitates (IP: anti-GFP) were analyzed by SDS-PAGE and immunoblotted using a polyclonal antibody against mGlu 5a/b receptor (1/1,000) and a monoclonal anti-GFP antibody (1/2,000). The primary bound antibody was detected using a HRP-conjugated goat anti-rabbit antibody (1/60,000) or HRP-conjugated rabbit anti-mouse antibody (1/6,000). The immunoreactive bands were visualized by chemiluminescence. mGlu 5b Receptor ␣-Actinin-1 Interaction ments on adult rat striatal homogenates and double immunolabeling on primary cultures of rat striatum neurons were performed. Using soluble extracts from adult rat striatum, which had been shown by Western blotting to contain both ␣-actinin and the mGlu 5a/b receptor (Fig. 6A), the anti-␣-actinin antibody could co-immunoprecipitate a band ϳ130 kDa, which was detected using an anti-mGlu 5a/b receptor antibody (Fig. 6A, upper panel, lane 3). This band did not appear when an irrelevant rabbit IgG was used for immunoprecipitation (Fig.  6A, upper panel, lane 1), showing that the reaction was specific and that the detected band might correspond mainly to mGlu 5b receptor variant, because this is the major adult form expressed in striatum (5). Interestingly, when the same blot was reacted with an antibody against NR1 subunit of the NMDA-type glutamate receptor, a band of 130 kDa corresponding to this NMDA subunit was detected in the immunoprecipitate with the anti-␣-actinin antibody (Fig. 6A, lower panel, lane 3), as expected (18,19). Also, a similar faint band was observed in the immunoprecipitate with the anti-mGlu 5a/b antibody (Fig. 6A,  lower panel, lane 2), suggesting that NMDA receptor might be somehow physically associated to the mGlu 5a/b receptor in rat striatum.
The distribution of ␣-actinin and the mGlu 5a/b receptor in primary rat striatal neurons was also analyzed using confocal microscopy analysis, and a similar punctate distribution and some degree of co-distribution for both proteins were found (Fig. 6B). Co-distribution occurred mainly at specific aggregates in dendrites (Fig. 6B, arrows in inset panel). Interestingly, the single labels for mGlu 5a/b or for ␣-actinin give the same pattern as seen in the double co-staining (i.e. simultaneous detection of mGlu 5 plus ␣-actinin), suggesting that the co-immunodetection is indeed specific (data not shown). These observations are consistent with the concept that ␣-actinin and mGlu 5a/b receptor associate in striatal neurons.
␣-Actinin-1 Promotes Cell Surface Expression of mGlu 5b Receptor-To gain insight into the physiological consequences of the ␣-actinin-1/mGlu 5b receptor interaction, the effect of ␣-actinin-1 on the mGlu 5b receptor cell surface expression was studied. To this end we isolated mGlu 5b receptors present in the plasma membrane by cell surface protein biotinylation, using a membrane impermeant biotin ester, followed by streptavidin-agarose affinity precipitation of the membrane proteins. The results showed that the amount of mGlu 5b receptor present at the cell surface is increased when mGlu 5b receptor and ␣-actinin-1 are co-expressed, compared with the properties present when mGlu 5b receptor is expressed alone (Fig.  7A, Cell Surface, upper panel, lanes 6 versus 5). Quantitation of FIGURE 5. FRET efficiency of the mGlu 5b -GFP 2 and ␣-actinin-YFP pair by sensitized emission in living cells. HEK-293 cells were transiently transfected with the plasmids encoding the mGlu 5b -GFP 2 (donor) and the ␣-actinin-1-YFP constructs (acceptor) using a ratio of donor to acceptor DNA of 1:2. The ␣-actinin-1-YFP constructs used in the co-transfection were the same used in Fig. 3. The plasmid encoding the construct GFP 2 -YFP was transfected and used as a positive control. Fluorescence readings were performed 48 h post transfection as described under "Experimental Procedures." Linear unmixing of the emission signals was applied to the data (see "Experimental Procedures"), and the results are shown as the sensitized emission of the acceptor when the cells were excited at 400 nm. Data are the mean Ϯ S.D. of five to nine independent experiments performed in triplicate. Data of the different transfection groups were analyzed by one-way analysis of variance followed by Newman-Keuls post-hoc comparisons. **, p Ͻ 0.05 or ***, p Ͻ 0.001 versus the mGlu 5b -GFP 2 and YFP co-transfected cells (negative control). FIGURE 6. In vivo interaction of mGlu 5 receptor and ␣-actinin in rat striatum. A, solubilized extracts from rat striatum (see "Experimental Procedures") were subjected to immunoprecipitation analysis using nonspecific rabbit IgG (lane 1), rabbit anti-mGlu 5a/b receptor antibody (2 g/ml) (lane 2), and rabbit anti-␣-actinin (2 g/ml) (lane 3). Extracts (Crude) and/or immunoprecipitates (IP) were analyzed by SDS-PAGE and immunoblotted using a polyclonal antibody against mGlu 5a/b receptor (1/1000), a monoclonal antibody against NR1 (1/1000), and a polyclonal antibody against ␣-actinin (1/1000). The primary bound antibody against NR1 was detected using a HRP-conjugated rabbit anti-mouse antibody (1/6000) and the anti-mGlu 5a/b receptor antibody was detected using a HRP-conjugated anti-rabbit IgG TrueBlot TM (1/2000) to avoid IgG cross-reactivity. The immunoreactive bands were visualized by chemiluminescence. B, primary cultures of rat striatum neurons (DIV 14 -21) were cultured and processed for immunocytochemistry (see "Experimental Procedures") using a polyclonal antibody against mGlu 5a/b receptor (1/200) and a monoclonal anti-␣-actinin antibody (1/100) followed by Texas red-conjugated goat anti-rabbit (1/2000) and AlexaFluor488-conjugated goat antimouse IgG (1/1000). Cells were analyzed by double immunofluorescence with a confocal microscope. Superimposition of images (merge) reveals codistribution in yellow (arrow in the inset panel). Scale bar: 10 m. mGlu 5b Receptor ␣-Actinin-1 Interaction the increase of membrane bound/localized mGlu 5b receptor indicated that the levels of surface receptor had risen by up to 4-fold in the ␣-actinin-1 co-transfected cells (Fig. 7B). Under similar conditions, when the mGlu 5b receptor was co-transfected with ␣-actinin-1 mutants lacking the actin binding domain, i.e. ␣-actinin-1-(358 -892)-YFP, ␣-actinin-1-(746 -892)-YFP, and ␣-actinin-1-(816 -892)-YFP, there was a reduction in plasma membrane mGlu 5b receptor expression when compared with cells transfected with the mGlu 5b receptor alone (Fig. 7A, Cell Surface, upper panel, lanes 7-9 versus lane  5). Interestingly, when the streptavidin isolates were reacted with the anti-GFP antibody to detect ␣-actinin-1 constructs, it became apparent that ␣-actinin-1 could be observed in the streptavidin isolates from the cells that were co-transfected with the mGlu 5b receptor (Fig. 7, Cell Surface, middle panel, lane 6), suggesting that ␣-actinin-1 might be associated with the cell surface mGlu 5b receptor. These results are in agreement with the marked overlap observed in the distribution of these two proteins found at the plasma membrane level (Fig. 3). Because no calnexin could be detected in the streptavidin isolates, it was clear that the biotin ester had not penetrated the cell membrane (Fig. 7, Cell Surface, lower panel). Because ␣-actinin-1 mutants lacking the domain responsible for the interaction with actin (calponin homology domain) inhibit receptor cell surface expression, these results suggest that the ␣-actinin-1-mediated-mGlu 5b receptor plasma membrane expression requires the actin cytoskeleton.
Functional Implications of the mGlu 5b Receptor-␣-Actinin-1 Interaction-Recently, we have described that mGlu 5b receptor can signal through the extracellular signal-regulated MAPK cascade (35). On the other hand, it has been shown that ␣-actinin isoforms interact with the MEK activator MEKK1 (38) or the extracellular signal-regulated kinase, ERK (39). To test the functional consequences of ␣-actinin-1/mGlu 5b receptor interaction we studied the activation of the MAPK pathway by the mGlu 5b receptor in HEK cells transiently expressing the mGlu 5b receptor in the absence, or presence, of ␣-actinin-1 (receptor densities were controlled by immunoblotting, data not shown). Treatment with quisqualic acid (100 M) did not induce ERK1/2 phosphorylation in cells transfected with ␣-actinin-1 alone. However, in cells transfected with the mGlu 5b receptor alone quisqualic acid did induce a significant ERK1/2 phosphorylation, as expected (35). Interestingly, when cells were transiently transfected with both ␣-actinin-1 and the mGlu 5b receptor a synergistic potentiation of ERK1/2 phosphorylation after receptor activation was observed (Fig. 8).

DISCUSSION
In this study, we have identified an interaction between the mGlu 5b receptor and ␣-actinin-1 and have shown that this interaction can regulate cell surface expression and function of the receptor. A yeast two-hybrid screen was initially used to identify a novel interaction between the heptaspanning membrane mGlu 5b receptor and the actin cross-linking protein ␣-actinin-1. This interaction was subsequently confirmed by means of pull-down experiments using GST and ␣-actinin-1-GST fusion constructs, and by co-distribution and co-immunoprecipitation experiments in transfected HEK-293 cells. Moreover, co-distribution of both proteins in rat striatum primary cultures and the ability of anti-␣-actinin antibodies to immunoprecipitate mGlu 5 receptor from rat striatum homogenates suggest that the interaction is physiologically relevant.
␣-Actinin-1 is a rod-shaped molecule composed of two 100-kDa anti-parallel monomers, linking actin filaments in a parallel way (Fig. 9). In the present work the mGlu 5b receptor interacting region of ␣-actinin-1 was mapped within the last 76 amino acids of the molecule. Interestingly, for ␣-actinin-2, one of the two skeletal muscle isoforms of ␣-actinin that is also expressed in brain (18,19), this domain is involved in the interaction with the Z repeats of titin in skeletal muscle (40,41). Furthermore, the same 76 residues of ␣-actinin-2 have been . Cell surface labeling was performed as described under "Experimental Procedures." Crude extracts and biotinylated proteins were subsequently analyzed by SDS-PAGE and immunoblotted using a rabbit anti-mGlu 5 receptor antibody (1/1,000), a rabbit anti-␣-actinin antibody (1/2,000), and a mouse anti-calnexin antibody (1/250). The primary bound antibody was detected using a HRP-conjugated goat anti-rabbit antibody (1/60,000) or HRP-conjugated rabbit anti-mouse antibody (1/6,000). The immunoreactive bands were visualized by chemiluminescence. B, quantification of cell surface receptor. The intensities of the immunoreactive bands on x-ray film corresponding to crude extracts and biotinylated protein were measured by densitometric scanning. Cell surface receptor values were normalized using the total amount of receptor in the crude extract for each sample. The results are presented as means Ϯ S.E. of three independent experiments. mGlu 5b Receptor ␣-Actinin-1 Interaction shown to interact with ZASP (Z band alternately spliced PDZcontaining protein), another sarcomere Z disk protein (8,42,43). In the central nervous system, this region in the ␣-actinin-4 interacts with the PDZ (PSD-95, Dgl, Z0 -1) domain of densin-180 and with Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII), forming a ternary complex stabilized by multiple interactions (12,37,44). Also, for ␣-actinin-4 the same region is involved in the interaction with densin-180, a transmembrane protein that is tightly associated with the postsynaptic density in central nervous system neurons and that is postulated to function as a synaptic adhesion molecule (12). The PDZ domain of densin-180 contributes to its binding to ␣-actinin-4 (12). Furthermore, the C-terminal region of ␣-actinin-2 (amino acids 819 -894), and the highly related proteins ␣-actinin-1 and ␣-actinin-4 interact with CaMKII (37). Apart from these interactions, the ␣-actinin family members also interact with cell surface receptors such as the Kv1-type potassium channel (45), the ATP-gated ion channel P2X 7 (46), and the glutamate NMDA receptor (47). ␣-Actinin binds to the NMDA receptor NR1 and NR2B subunit C termini at the C0 region, where it competes with calmodulin, which also binds NMDA receptors at the same site (48,49). Displacement of ␣-actinin from the C0 region by calmodulin has been implicated in calcium-dependent inactivation of NMDA receptor-mediated whole cell currents (50). It has also been postulated that under resting cellular conditions ␣-actinin is bound to the NMDA receptor. This interaction predominantly decreases single channel closed time, resulting in an increased open probability (P open ). When the intracellular calcium concentration increases during neuronal excitation, calmodulin binds to, and ␣-actinin dissociates from, the receptor, causing an increase in mean channel closed time, a decrease in mean channel open time, and an overall reduction in P open (51). It has also been suggested that the association of ␣-actinin with NMDA receptors may contribute to the NR2 subunit-selective modulation of this receptor, by localizing inactive CAMKII to the NMDA receptor (44,52). In this context, it is interesting to note that an NMDA/␣-actinin interaction has been reported in rat striatum (18,19) where the mGlu 5a/b receptor is also expressed. Furthermore, activation of mGlu 5a/b receptors results in a pronounced potentiation of NMDA responses in several brain regions (53,54), including the striatum (55), suggesting that mGlu/NMDA receptor interactions are of widespread significance. Indeed, cross-talk between type I mGlu and NMDA receptors has also been demonstrated in different types of central nervous system neurons, including cultured cortical neurons (56), cultured striatal neurons (57), and hippocampal CA3 pyramidal cells (58). The one or more mechanisms by which activation of mGlu 5a/b receptor modulates NMDA receptor function are not well understood, and different hypotheses to explain the enhancement of NMDA currents by type I mGlu receptors have been proposed. For example, receptor-mediated phosphorylation of the NMDA receptor NR2A/B subunits by protein kinases, such as protein kinase C (56,58), increases the open probability of the channel. Interestingly, NMDA receptor-mediated responses in layer V pyramidal neurons of the rat prefrontal cortex were facilitated by purinergic P2 receptor activation. The mechanisms underlying this facilitation implicated the activation of type I mGlu receptors, FIGURE 8. Stimulation of ERK1/2 activity by mGlu 5b receptor. Serumstarved HEK cells expressing mGlu 5b receptor in the absence or presence of ␣-actinin were stimulated with quisqualic acid (100 M) for 5 min. The phosphorylation of ERK1/2 was determined by immunoblotting (see "Experimental Procedures"). Bottom, representative ERK assay; top, the intensities of the immunoreactive bands on x-ray film corresponding to ERK1/2 and phospho ERK1/2 protein were measured by densitometric scanning. All values of phosphorylated ERK1/2 were normalized using ERK1/2 and expressed as means Ϯ S.E. (in relative densitometric scanning (RDS) obtained in non-treated transfected cells) of three independent experiments. mGlu 5b Receptor ␣-Actinin-1 Interaction namely mGlu 1 and mGlu 5a/b receptors, via the G q /phospholipase C/inositol 1,4,5-trisphosphate/Ca 2ϩ /CAMKII transduction pathway (59).
It is important to note that the ␣-actinin domains mediating interactions with NMDA and mGlu 5a/b receptors are different, meaning that simultaneous interaction of ␣-actinin with both receptors could take place. Under this scenario, the close association of NMDA and mGlu 5a/b receptors would facilitate the modulation of NMDA receptor-mediated currents by the mGlu 5 receptor. On the other hand, it is also likely that the actin cytoskeleton, and ␣-actinin in particular, may have a role in the regulation of NMDA receptor function by the mGlu 5a/b receptor in the rat striatum.
The presence of a complex involving the mGlu 5a/b receptor and ␣-actinin suggests that ␣-actinin may mediate the association of the receptor with the actin cytoskeleton. Other studies have identified filamin A, another actin cross-linking protein similar to ␣-actinin, as an intracellular binding partner for other heptaspanning membrane receptors, namely the dopamine D 2 and D 3 receptors (60, 61), the calcium-sensing receptors (CaRs) (62), the metabotropic glutamate receptor 7 (63), the -opioid receptor (64), and the calcitonin receptor (65). Filamin A/D 2 receptor interaction is required for the proper targeting or stabilization of dopamine D 2 receptor at the plasma membrane (61,66) and may contribute to its cell surface clustering (60). On the other hand, the interaction of CaR with filamin A prevents the degradation of the receptor, increasing its total cellular expression and plasma membrane localization, thus facilitating CaR signaling to the MAPK pathway (67). Furthermore, silencing the filamin A gene expression inhibits CaR signaling (68). In the case of the -opioid receptor its interaction with filamin A is required for proper trafficking and regulation of the receptor (64). The calcitonin receptor-filamin A interaction causes an increase in the recycling of the receptor to the cell surface and decreased degradation of the receptor, suggesting an important role for filamin in the endocytic sorting and recycling of the internalized calcitonin receptor (65). In contrast to the well documented interaction of filamin A with several heptaspanning membrane receptors, for ␣-actinin only one previous study has reported an interaction of this actinbinding protein with a G-protein-coupled receptor, namely the adenosine A 2A receptor (20). Here it was shown that the attachment of the A 2A receptor to the actin cytoskeleton through a direct interaction with ␣-actinin-2 is a pre-requisite for its agonist-induced plasma membrane clustering and ␤-arrestin-mediated internalization (20). Although the A 2A receptor was the first G-protein-coupled receptor documented to bind to an ␣-actinin isoform, namely the ␣-actinin-2, here we show that mGlu 5b receptor also interacts with ␣-actinin-1 and that this interaction promotes cell surface expression of the receptor. Interestingly, this ␣-actinin-1-dependent cell surface expression of the receptor is maintained by the actin cytoskeleton, because mutants lacking the calponin homology domain, which renders them unable to bind actin, do not promote cell surface expression of the receptor.
␣-Actinin isoforms also interact with proteins involved in signal transduction, such as the MEK activator MEKK1 or the extracellular signal-regulated kinase, ERK, as mentioned previ-ously (38,39). Also, mGlu 5b receptor can signal through the extracellular signal-regulated MAPK cascade (35). Taking all this evidence together, it seems that ␣-actinin has a dual role as an actin cytoskeleton component and as a scaffolding protein, anchoring receptors to their target signaling molecules and thus ensuring a rapid and efficient signal transduction. A similar hypothesis has been suggested for filamin A, because this protein interacts with MEKs 1/2, p38 kinases (69), and the Rasrelated GTPases, Rac, RhoA, Cdc42, and RalA (70). Also consistent with this double function as a scaffolding and an adaptor protein, the interaction of filamin A increases the coupling efficiency of the dopamine D 2 receptor with adenylate cyclase (60,61) and is a prerequisite required for activation of MAPK signaling by the calcium-sensing receptor (67). Here we demonstrate, as for filamin A, that ␣-actinin-1 promotes mGlu 5 receptor signaling through the extracellular signal-regulated MAPK cascade, suggesting a functional role for the ␣-actinin-1/ mGlu 5b receptor interaction in addition to anchoring the receptor to the actin cytoskeleton.
In summary, a direct interaction between ␣-actinin-1 and mGlu 5b receptor has been identified by using the yeast twohybrid system and confirmed by convergent techniques in transfected HEK-293 cells and in more physiological models such as cultured neurons or rat striatum. Finally, we describe that the ␣-actinin-1-dependent cell surface expression of the receptor depends on the proper ␣-actinin-1 attachment to the actin cytoskeleton, facilitating the receptor coupling to the signal transduction machinery.