Stargazin and Other Transmembrane AMPA Receptor Regulating Proteins Interact with Synaptic Scaffolding Protein MAGI-2 in Brain

Yeast two-hybrid screening

The eight Ca²⁺ channel γ subunit proteins (γ1–γ8) encoded by the genes Cacng1–Cacng8 share similar predicted topologies, with four transmembrane segments, short cytoplasmic N-terminal leaders, and variable-length cytoplasmic C-terminal tails (Fig. 1A). The more pronounced size and amino acid sequence differences between the C-terminal tails of these proteins compared with the transmembrane segments and extracellular loops suggested that the tail region might be particularly important for distinguishing functions and protein–protein interactions among the otherwise similar γ subunits.

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Figure 1.

Cacng protein segments used for yeast two-hybrid screening. A, Model of Cacng (γ) protein topology, showing a cytoplasmic N terminus, four transmembrane segments, two extracellular loops, and a cytoplasmic C terminus. All eight γ proteins are similar in size except for the variable-length C termini, labeled on the model for each indicated protein. The cytoplasmic tails of all γ proteins except for the smaller γ1 and γ6 were used as baits for yeast two-hybrid screens. The filled arrowhead marks the beginning of each bait, and the open arrowheads mark the ends, which correspond to the C terminus of each protein. B, Alignment of the γ protein C-termini amino acid sequences (bait regions). The sequences are grouped in boxes by similarity with the inferred evolutionary relationships indicated by branching lines at left (Price et al., 2005). Note that the C-terminal tail of γ8 is longer than that of the related γ2, γ3, and γ4 proteins, primarily because of glycine–alanine and proline–alanine expansions (shown isolated at bottom). The polyclonal anti-TARP antibody used in this study was directed against the C-terminal 19 aa of mouse Cacng2.

To identify novel proteins interacting with the γ proteins in brain, we performed a series of yeast two-hybrid screens using the cytoplasmic C-terminal tails of γ2, γ3, γ4, γ5, γ7, and γ8 as baits (Fig. 1B). The γ1 and γ6 proteins were excluded because they are expressed at very low levels in brain and have C termini of only 20 aa (Burgess et al., 2001; Chu et al., 2001). We used each bait construct to screen 3.5 × 10⁶ clones of a 9–12 week postnatal mouse brain cDNA prey library using high-stringency selection. This yielded an initial combined total of ∼1200 prey clones, composed of 155 candidate γ2 γ3, γ4, or γ8 (i.e., TARP) interactors and ∼1000 candidate γ5 or γ7 interactors. We sequenced all 155 TARP prey clones and a random sample (n = 64) of γ5 and γ7 prey clones. After eliminating redundancy, false reading frame artifacts, and clones representing E. coli or noncoding genomic DNA contaminants of the cDNA library, 68 prey were selected for additional analysis. Secondary yeast cotransformation tests were performed to identify false-positive interactions between bait and prey attributable to nonspecific interactions. Of the 68 selected prey, 31 interacted with the negative control protein p53 or activated the yeast reporter gene in the absence of bait and were determined to be likely false positives. Among the other 37 prey, four had been identified previously as valid γ protein interactors in brain, including MAP1A, PSD-95, synapse-associated protein 102 (SAP102), and PIST (Dakoji et al., 2003; Cuadra et al., 2004; Ives et al., 2004). These four proteins represented de facto positive controls, suggesting that our screen had been effective at finding true γ interactors and increasing the likelihood that the novel prey we isolated would also be valid.

Two of the novel prey we identified were of particular interest to us for additional evaluation. MAGI-2 and MAGI-3 are multi-PDZ domain-containing proteins that belong to the membrane-associated guanylate kinase with inverted domain organization (MAGI) family of scaffolding proteins. Although the fragments of MAGI-2 and MAGI-3 that we identified as prey in the primary yeast two-hybrid screen were detected by interaction with the γ2 bait probe, secondary yeast cotransformation tests indicated that the MAGI-2 prey could also bind with the γ3 and γ4 baits and the MAGI-3 prey could also bind with the γ4 and γ8 baits (Table 1). The rationale for prioritizing these prey for additional analyses was based on three factors: the ability of both MAGI prey to bind with multiple different γ protein baits, the observation that these interactions were limited specifically to the TARP subset of the γ proteins (γ2 γ3, γ4, and γ8), and the fact that MAGI-2 and MAGI-3 are PDZ domain proteins similar to multiple other previously confirmed γ interacting proteins (PSD-95, PIST, and SAP102). Because MAGI-2 was inferred to bind the γ protein baits with greater affinity than MAGI-3 in the cotransformation tests and MAGI-2 is expressed in brain at much higher levels than MAGI-3, we focused on evaluating MAGI-2 as a candidate TARP interactor in the current study.

Expression of MAGI-2 and TARPs in mouse brain

If the putative MAGI-2/TARP interactions identified by the yeast two-hybrid screens occur in vivo, these genes must be spatiotemporally coexpressed in brain. A detailed study of the developmental expression patterns of all eight Cacng genes in wild-type mouse brain was reported recently (Fukaya et al., 2005) and so was not required here. However, we did confirm by in situ hybridization that the mRNA expression of the TARP genes Cacng3, Cacng4, and Cacng8 was not qualitatively altered by the Cacng2 mutation in stargazer adult mouse brain, because we intended to use that tissue as a control in our experiments (supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

We transfected full-length mouse γ2, γ3, γ4, and γ8 expression constructs into cultured HEK-293T cells and performed immunoblot analysis on crude cell lysates and membrane fractions to test the ability of a polyclonal anti-TARP antibody to recognize each of these proteins. This commercial antibody was raised against a C-terminal γ2 antigen that is partially conserved (Fig. 1B) among all four TARPs and recognizes each of these proteins (Fig. 2). As expected from the amino acid alignments, the anti-TARP antibody recognized γ2 with the highest affinity and the other TARPS with lower affinities. Whereas immunoreactive bands corresponding to γ2, γ3, and γ4 were close to the expected sizes of these proteins based on primary amino acid sequences (323, 315, and 327 aa, respectively), the γ8 specific band was smaller than predicted given its larger amino acid length of 423 aa. Identical results were obtained by Inamura et al. (2006), who expressed γ2 and γ8 in HEK-293T cells and examined their sizes by immunoblotting with antibodies different from the anti-TARP antibody we used here. In that study, the apparent size discrepancy was interpreted as a likely difference in glycosylation between γ2 and γ8 in this cell line. The amount of immunoreactive protein contained in large-molecular-weight aggregates demonstrates that the γ proteins are relatively insoluble under the mild extraction conditions often required to examine protein–protein interactions in vivo.

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Figure 2.

Recognition of a conserved γ2, γ3, γ4, and γ8 epitope by anti-TARP antibody. Crude lysates (A–C) or membrane fraction proteins (D) were prepared from HEK-293T cells transiently expressing γ2, γ3, γ4, γ8, or a GFP–γ8 fusion protein and used for immunoblotting with polyclonal antibodies directed against C-terminal amino acids 305–323 of the γ2 protein (KDSKDSLHANTANRRTTPV). This antigen is partially conserved among all four TARPs (see Fig. 1B), resulting in an antibody that reacts strongly with γ2 protein and moderately with γ3, γ4, and γ8. A, A short exposure shows that anti-TARP antibodies effectively recognized a γ2 doublet of the expected size, although much of the protein appeared to be trapped in insoluble aggregates near the top of the gel. B, A longer exposure of the same immunoblot shows the antibody also detected γ4, γ8, and GFP–γ8 proteins of the expected sizes. The γ3 protein from cell lysate appeared to be highly insoluble. The arrows in A and B are identical to facilitate comparison. C, The anti-β-actin immunoblot demonstrates equal sample loading in A and B. D, Membrane proteins were prepared from HEK-293T cells expressing either γ2, γ3, or γ4 using the CelLytic MEM Protein kit (CE0050; Sigma) and immunoblotted with anti-TARP. Increasing amounts of the γ3 and γ4 membrane fraction were loaded on the gel. Although an improved yield of soluble γ3 or γ4 protein was obtained from membrane fractions relative to crude lysates, substantial amounts remained insoluble. Double bands of the TARPs represent glycosylated and unglycosylated isoforms. Molecular mass markers are indicated at the left. The anti-TARP antibody did not react with endogenous HEK-293T proteins (Mock).

MAGI-2 is expressed exclusively in brain in three prominent isoforms (α, β, and γ) (Hirao et al., 1998, 2000), but the distribution of these isoforms among major brain subdivisions is unknown. We used immunoblot analysis to examine the expression of MAGI-2 protein in cerebral cortex, cerebellum, and hippocampus in both wild-type and stargazer mouse brain using an antibody that recognizes all three isoforms (Fig. 3). In these tissues, MAGI-2 α and γ expression were most prominent in cerebellum, which did not exhibit any detectable MAGI-2 β. Conversely, MAGI-2 β was the most prominent isoform in cerebral cortex and hippocampus, which each expressed lower levels of MAGI-2 α and γ. An examination of mouse genomic sequences from NCBI databases corresponding to MAGI-2 revealed that the α, β, and γ isoforms each initiate translation from methionine start codons on distinct exons (exons 1, 2, and 4, respectively), suggesting that the β and γ isoforms result from the use of alternate transcriptional promoters in the preceding introns, which are 387 and 140 kbp in size (NCBI accession number NC_000071.3). No differences in the expression of any MAGI-2 isoforms were detected between wild type and stargazer in these brain regions.

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Figure 3.

Differential expression of MAGI-2 isoforms in brain. A, Three distinct isoforms of MAGI-2 (α, β, and γ) have been described previously (Hirao et al., 2000). The fragment of MAGI-2 that we identified by yeast two-hybrid screening contains 277 C-terminal amino acids that are present in all major MAGI-2 isoforms and encodes the last 10 amino acids of the PDZ4 domain and all of the PDZ5 domain. The numbers flanking each model indicate the first and last amino acids of each native isoform and the prey clone relative to the largest known isoform (MAGI-2 α). GK, Guanylate kinase. B, Homogenates of cerebral cortex (CTX), cerebellum (CRB), and hippocampus (HPC) from adult wild-type (WT) and γ2-null homozygous stargazer (STG) mice were used for immunoblot analysis with either anti-MAGI-2 (at 1:500; M2441; Sigma) or anti-β-actin (at 1:5000; A5316; Sigma). Anti-MAGI-2 recognized the expected MAGI-2 isoforms of 180 (α), 160 (β), and 140 (γ) kDa (arrows). The smaller band of 105 kDa (asterisk) recognized by this antibody may correspond to a yet undescribed MAGI-2 isoform. The anti-β-actin immunostaining shows the relative protein loading from each brain region on the blot.

Coimmunoprecipitation of MAGI-2 and TARPs from mouse brain

We performed coimmunoprecipitation experiments to test whether TARPs interacted with MAGI-2 in vivo in mouse brain (Fig. 4). The anti-TARP antibody coimmunoprecipitated with two prominent MAGI-2 isoforms from wild-type and stargazer cerebral cortex but not from cerebellum or hippocampus from either mouse. These isoforms correspond to MAGI-2 α and β based on their sizes. In wild-type cerebral cortex, γ2, γ3, and γ8 were also immunoprecipitated so any of these, or some combination, might be responsible for interacting with MAGI-2. In γ2-null stargazer cerebral cortex, only γ3 and γ8 were immunoprecipitated, indicating that one or both of these proteins interacted with MAGI-2 in vivo. Although γ2 was immunoprecipitated from wild-type cerebellum and hippocampus, MAGI-2 was not coimmunoprecipitated from these brain regions at detectable levels.

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Figure 4.

MAGI-2 interacts with TARPs in mouse cerebral cortex. Homogenates of cerebral cortex (CTX), cerebellum (CRB), and hippocampus (HPC) from wild-type (WT) and γ2-null stargazer (STG) mouse brain were used for immunoprecipitation with the anti-TARP antibody (IP:anti-TARP; 5 μg/reaction). Immunoprecipitated proteins were separated on a 4–20% SDS-PAGE gel for immunoblot analysis with either anti-MAGI-2 (1:500) or anti-TARP (1 μg/μl) antibodies. The anti-MAGI-2 blot shows that anti-TARP coimmunoprecipitated MAGI-2 from both wild-type and stargazer cerebral cortex but not from cerebellum or hippocampus. The anti-TARP blot shows that anti-TARP immunoprecipitated γ2, γ3, and γ8 from wild-type cortex but only γ3 and γ8 from stargazer cerebral cortex. The anti-TARP antibody immunoprecipitated detectable amounts of γ2 only from wild-type cerebellum and hippocampus. The IgG panel shows that equal amounts of preimmune sera and anti-TARP sera were used for the immunoprecipitation reactions. The results shown were replicated in duplicate experiments.

PDZ domain interactions between stargazin and MAGI-2

To further characterize protein–protein interactions between the TARPs and MAGI-2, we performed in vitro binding assays using γ2 protein and fragments of MAGI-2 fused to GST (Fig. 5). The GST fusion protein containing only the guanylate kinase domain and WW domains of MAGI-2 was unable to bind γ2. However, GST fused to full-length MAGI-2, MAGI-2 PDZ domains 1–5, or MAGI-2 PDZ domains 1–3 did bind γ2. These results suggest that one or more of the PDZ domains mediate basic γ2 binding and that the GK and WW domains are not necessary for this function. The three GST–MAGI-2 fusion proteins each bound three distinct γ2 immunoreactive bands, presumably representing different posttranslational modifications of γ2 as it was expressed in HEK-293T cells (Fig. 5B). The smaller γ2 bands were identical in size. However, the largest γ2 band retained from interactions with full-length MAGI-2 was slightly larger than the largest γ2 band retained by interactions with the PDZ domain 1–5 or 1–3 GST fusions, suggesting that some posttranslational control over γ2–Magi-2 binding selectivity is possible.

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Figure 5.

The PDZ domains of MAGI-2 are required for γ2 interaction in vitro. A, The molecular organization of MAGI-2 β and derivative GST–MAGI-2 fusion proteins used in the experiment (1–5) is illustrated. B, Proteins corresponding to GST or GST fused to various MAGI-2 β fragments were immobilized on glutathione-Sepharose beads and incubated with membrane proteins prepared from HEK-293T cells expressing γ2. Bound protein complexes were analyzed by SDS-PAGE and immunoblotted with anti-TARP antibody. The numbers of the GST or GST–MAGI-2 fusion proteins in A correspond to numbered lanes in B. Coomassie blue staining of the original gel shows the relative amount, purity, and expected size (arrows) of GST or GST–MAGI-2 fusion proteins. The smaller Coomassie blue-stained fragments in each lane represent partial proteolytic products. The anti-TARP immunoblot shows that GST fusion proteins containing full-length MAGI-2, PDZ1–5, or PDZ1–3 were able to bind γ2 protein effectively, whereas GST alone or the GST–GKW fusion protein did not. The existence of multiple anti-TARP immunoreactive bands likely reflects differential posttranslational modifications (glycosylation and phosphorylation) of the γ2 protein. Whereas the GST–MAGI-2 fusion proteins in lanes 2, 4, and 5 each interacted with three distinct γ2 bands, the largest γ2 band bound by full-length MAGI-2 (lane 2) was different in size than the largest γ2 bands bound by the MAGI-2 PDZ domains (lanes 4, 5). The Input lane contains membrane protein prepared from HEK-293T cells expressing γ2 before incubation with GST fusion proteins. The results shown are representative of duplicate experiments.

We generated fusion proteins of GST with each of the five MAGI-2 PDZ domains to determine which was responsible for binding γ2 in vitro. For this experiment, we used only the C-terminal 121 aa of γ2 linked to N-terminal His₆ and c-myc epitopes to facilitate purification. The results showed that PDZ domains 1, 3, and 5, but not PDZ domains 2 and 4, bound to the cytoplasmic γ2 C-terminal domain (Fig. 6A,B). PDZ1 appeared to exhibit the strongest affinity and PDZ5 the weakest. A subsequent multiple sequence alignment and phylogenetic reconstruction showed that PDZ domains 1, 3, and 5 of MAGI-2 were most similar to each other and were distinct from PDZ domains 2 and 4, consistent with the preferential binding pattern observed (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Because many subclasses of PDZ domains typically interact with only the C-terminal few amino acids of their ligand proteins, we tested whether this was also true for the γ2–MAGI-2 interaction. We repeated the experiments using a recombinant γ2 protein missing the final four amino acids (-TTPV) and showed that this alteration obliterated binding to the MAGI-2 PDZ domains (Fig. 6C,D). The observation that these four C-terminal amino acids are perfectly conserved among all TARP proteins (γ2, γ3, γ4 and γ8) (Fig. 1B) raises the possibility that MAGI-2 could interact with each TARP in vivo via the same PDZ domains and a similar binding mechanism.

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Figure 6.

The C-terminal -TTPV motif of γ2 is a ligand for PDZ domains 1, 3, and 5 of MAGI-2 in vitro. GST or GST fusion proteins of PDZ1, PDZ2, PDZ3, PDZ4, or PDZ5 were immobilized on glutathione-Sepharose beads and incubated with either His₆–myc–γ2 (amino acids 204–323) (A, B) or His₆–myc–γ2ΔTTPV (amino acids 204–319) (C, D). Bound protein complexes were analyzed by SDS-PAGE and immunoblotted with anti-myc antibodies (B, D). Coomassie blue staining shows the relative amount and purity of the recombinant GST fusion proteins (A, C). The anti-myc blots show that GST fusion proteins with PDZ1, PDZ3, or PDZ5 pull down His₆–myc–γ2 (amino acids 204–323) effectively (B) but do not pull down His₆–myc–γ2ΔTTPV (amino acids 204–319) (D). GST does not pull down either γ2 or γ2ΔTTPV. Equal amounts of GST fusion proteins (∼2 μg/μl) were used for the pull-down assays (A, C). Input was purified His₆–myc–γ2 (amino acids 204–323) (A, B) or His₆–myc–γ2ΔTTPV (amino acids 204–319) (C, D) before incubation with GST or GST fusion proteins. The results shown are representative of duplicate experiments.

Stargazin recruits MAGI-2 to cell membranes

Protein interactions characterized in vitro may not be representative of interactions between the same molecules in the more complex cellular environment. To test this for γ2–MAGI-2, we expressed three fluorescently tagged fusion proteins in cultured HEK-293T cells: MAGI-2 (GFP–MAGI-2), γ2 (HcRed–Cacng2), and γ2 minus the conserved C-terminal PDZ interaction motif (HcRed–Cacng2ΔTTPV) (Fig. 7). When expressed alone, HcRed–Cacng2 and HcRed–Cacng2ΔTTPV were each localized to plasma membranes with the highest levels observed at points of cell–cell contact. GFP–MAGI-2, expressed alone, exhibited a diffuse localization throughout the cytoplasm. The coexpression of GFP–MAGI-2 with HcRed-Cacng2ΔTTPV did not appreciably alter the cellular distribution of either molecule, consistent with the requirement of the γ2 -TTPV motif for binding between these molecules. Coexpression of GFP–MAGI-2 with HcRed–Cacng2, however, dramatically altered the distribution of both molecules. In these cells, GFP–MAGI-2 was recruited to the plasma membrane in which it overlapped closely with HcRed–Cacng2, whereas HcRed–Cacng2 exhibited a more punctate and membrane-specific pattern than when it was expressed in the absence of GFP–MAGI-2. These cellular data are consistent with the results of the yeast two-hybrid in vitro binding and in vivo coimmunoprecipitation results and support a physical interaction between MAGI-2 and Cacng2.

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Figure 7.

Recruitment of MAGI-2 to plasma membranes requires the C-terminal -TTPV motif of γ2. Deconvolution micrographs are shown of HEK-293T cells transiently transfected for expression of HcRed wild-type Cacng2 (γ2), HcRed–Cacng2ΔTTPV, or GFP–MAGI-2 fusion proteins, individually (A–D) or in combinations (E–J). Nuclei are stained with DAPI (blue). Cacng2 is membrane-associated and concentrated at cell–cell contact sites in the presence (A) or absence (B, C) of the C-terminal -TTPV amino acid motif. MAGI-2 expressed alone (D) is generally diffuse throughout the cytoplasm and only occasionally concentrated near membranes (arrow). E–G show cells cotransfected with HcRed–Cacng2 and GFP–MAGI-2 and visualized for HcRed–Cacng2 (E), GFP–MAGI-2 (F), or both images merged (G). H–J show cells cotransfected with HcRed–Cacng2ΔTTPV and GFP–MAGI-2 and visualized for HcRed–Cacng2ΔTTPV (H), GFP–MAGI-2 (I), or both images merged (J). Efficient recruitment of MAGI-2 to plasma membranes requires the C-terminal -TTPV motif of γ2, which we showed is a binding target for PDZ domains 1, and, 5 of MAGI-2 (F, I). Wild-type Cacng2 expressed alone (A) exhibits a membrane localization similar to mutant Cacng2ΔTTPV coexpressed with MAGI-2 (H). However, when wild-type Cacng2 is coexpressed with MAGI-2 (E), Cacng2 localization in the membrane appeared more concentrated and punctate than when Cacng2 was expressed alone, suggesting clustering of Cacng2 by the multi-PDZ domain MAGI-2 protein. The circled letters K–N in A, D, G, and J indicate the positions of 3-pixel-wide digital transects (dashed lines) corresponding to fluorescence intensity measured across representative cells shown in K–N (0–255 in grayscale arbitrary units, A.U.). K, HcRed–Cacng2 expressed alone is concentrated at cell membranes. L, GFP–MAGI-2 expressed alone appears diffuse in the cytoplasm. M, When HcRed–Cacng2 is coexpressed with GFP–MAGI-2, the fluorescence intensity transects overlap and both are concentrated at cell membranes. N, When HcRed–Cacng2ΔTTPV is coexpressed with GFP–MAGI-2, the fluorescence intensity transects do not overlap; HcRed–Cacng2ΔTTPV is concentrated at cell membranes, but GFP–MAGI-2 is not recruited to plasma membranes. Scale bars, 10 μm.

Regional specificity of TARP–MAGI-2 interactions in brain

Several factors likely influence which TARPs interact with MAGI-2 in particular brain regions, including coexpression, competition, and posttranslational modification. The fundamental importance of coexpression is shown by the result that anti-TARP antibodies immunoprecipitated detectable amounts of MAGI-2 from adult mouse cerebral cortex (high MAGI-2 levels) but not from cerebellum (very low MAGI-2 levels), although both tissues expressed a similar amount of TARPs. Ultimately, an analysis of the coexpression of MAGI-2 and TARPs in individual cells, using high-resolution in situ or laser microdissection approaches, will be required to determine which specific TARP–MAGI-2 interactions are possible in a particular cell type.

Competition for protein binding sites may influence TARP–MAGI-2 interactions in two general ways: competition between different PDZ domain proteins for binding to the same TARP and competition between different TARPs (and other proteins) for binding to the same PDZ domain protein. In the first case, it was reported previously that TARPs associate with the MAGUK proteins PSD-95 and PSD-93 in brain (Dakoji et al., 2003). Here we show that the same TARPs associate with MAGI-2 in brain. Because PSD-95, PSD-93, and MAGI-2 each bind the same C-terminal -TTPV motif, competition between coexpressed PDZ proteins for binding to TARPs is likely. The significance of this competition remains to be explored. Regarding the second type of competition, we determined that γ2 could bind PDZ domains 1, 3, and 5 of MAGI-2 with an apparent preference for PDZ domain 1. If other TARPs exhibit similar binding affinities, which we suspect, there could be competition among the TARPs for access to individual MAGI-2 PDZ domains. Additional competition could come from the many other known MAGI-2 interactors. For example, four neuroligin proteins (Nlgn1–Nlgn4) each terminate with the PDZ binding amino acid motif -TTRV, which is quite similar to the terminal motif of the TARPs (-TTPV) because position −1 is considered of minor importance for PDZ binding specificity (van Ham and Hendriks, 2003). Neuroligins are clustered by MAGI-2 (Iida et al., 2004), and the C terminus of neuroligin-1 interacts specifically with PDZ domain 1 (Hirao et al., 1998). Other proteins interacting with PDZ domain 1 include the β1-adrenergic receptor (Xu et al., 2001) and neural GDP/GTP exchange protein Rapgef2 (Ohtsuka et al., 1999). Measuring the relative access of TARPs and other proteins to different PDZ domains of MAGI-2 in vivo will be an important topic of future investigation.

Posttranslational modification of TARPs is another logical mechanism for regulating MAGI-2 interactions in vivo. For example, PKA phosphorylation of a threonine residue (T321) in the -TTPV motif of γ2 disrupts binding by PSD-95 (Chetkovich et al., 2002; Choi et al., 2002). Thus, phosphorylation by PKA or other kinases is a reasonable candidate for regulating the synaptic targeting of γ2 and associated AMPARs by other PDZ proteins in vivo, including MAGI-2. Interestingly, a recent study suggests that phosphorylation of γ2 and γ8 by different sets of kinases and phosphatases in cortical neurons may regulate the association of these TARPs with different PDZ proteins and their localization to biochemically distinct membrane compartments (Inamura et al., 2006). A role for PKC phosphorylation in controlling the binding of MAGI-2 PDZ domains to other ligands has been documented (Yap et al., 2003), but the regulation of TARP–MAGI-2 interactions by similar posttranslational modifications remains to be tested.

A role for MAGI-2 ligands in the AMPA receptor pathway?

TARPs bind AMPAR subunits in vivo and are increasingly recognized as critical regulators of AMPAR trafficking and biophysical properties in neurons (Nicoll et al., 2006). The significance of this relationship is highlighted by the fact that mutations in the Cacng2 gene result in AMPAR dysfunction associated with severe epilepsy and ataxia in stargazer mice (Letts et al., 1998), and targeted inactivation of the mouse γ8 gene (Cacng8) alters hippocampal AMPAR distribution and synaptic plasticity (Rouach et al., 2005). Previous studies of MAGI-2 revealed interactions with a large and diverse array of interesting brain proteins. An important outcome of our current study, which indicates that TARPs might act as a physical link between AMPARs and MAGI-2, will be the promotion of some of these MAGI-2 ligands as plausible candidates for involvement in the AMPAR signaling pathway.

Perhaps most exciting is the recent discovery that synaptic clustering of NMDA receptors in cultured ventral spinal neurons and hippocampal interneurons is dependent on the presence of synaptic AMPA receptors and that the synaptic AMPA and NMDA receptors are physically linked by stargazin and some unidentified PDZ protein (Mi et al., 2004). Hirao et al. (1998, 2000) demonstrated that PDZ domain 5 of MAGI-2 interacted with the NMDA receptor subunits 2A and 2C. Consequently, our demonstration that stargazin binds PDZ domain 1 of MAGI-2 suggests that MAGI-2 could be the missing link between synaptic AMPA and NMDA receptors. Other known MAGI-2 ligands that might be associated with the AMPA receptor pathway via TARP–MAGI-2 interactions include the following: atrophin-1, activin receptor-2A, axin, β1-adrenergic receptors, β- and δ-catenin, Cnksr2 (connector enhancer of kinase suppressor of Ras 2)/Maguin-1, dasm1 (dendrite arborization and synapse maturation 1), δ-1/Dll1 (delta-like 1), dendrin, hyperpolarization-activated cation channel 2, neuroligins, PTEN (phosphatase and tensin homolog), RapGEF2 (Rap guanine nucleotide exchange factor 1), and SAP-associated protein (Hirao et al., 1998; Wood et al., 1998; Ide et al., 1999; Ohtsuka et al., 1999; Yao et al., 1999; Shoji et al., 2000; Wu et al., 2000; Xu et al., 2001; Nishimura et al., 2002; Pfister et al., 2003; Hirabayashi et al., 2004; Iida et al., 2004; Kimura et al., 2004; Shi et al., 2004; Kremerskothen et al., 2006).

MAGI-2 and neuronal cell adhesion

Although the reliance of the glutamate signaling pathway on PDZ protein interactions at synapses is well documented, an important association with cell adhesion molecules is now emerging. NMDA receptors, for example, are linked to the synaptic adhesion protein neuroligin through the PDZ protein PDS-95 and were recently discovered to also interact with a novel family of PDZ domain-containing proteins called synaptic adhesion-like molecules, or SALMs (Wang et al., 2006). Our identification of interactions between the AMPA receptor regulating TARPs and the PDZ protein MAGI-2 expands on this concept.

Neuronal synapses have much in common with the intercellular adhesive junctions formed by other cell types (Yamagata et al., 2003). We recently reported that TARPs are evolutionarily related to a large family of proteins known as claudins, which regulate cell adhesion and paracellular permeability as fundamental components of non-neuronal tight junctions (Price et al., 2005). In that study, we showed that expression of stargazin in cultured fibroblasts results in cell aggregation similar to that produced by claudins. We speculated that stargazin and other TARPs might act as cell–cell adhesion molecules at neuronal adhesive junctions in vivo, reminiscent of the role of claudins at classical tight junctions. Our identification of the TARP–MAGI-2 interaction supports and extends that hypothesis. First, MAGI-1 and MAGI-3, which are paralogs of MAGI-2, serve as scaffolding proteins at epithelial cell tight junctions in which they colocalize with claudins, which are paralogs of TARPs (Ide et al., 1999; Mino et al., 2000; Wu et al., 2000; Yamada et al., 2004). Second, MAGI-2 was identified as a component of perisynaptic adherens junctions between mossy fiber terminals and the dendrites of CA3 pyramidal cells in mouse hippocampus, in which it interacts with the adhesion proteins β-catenin, nectin-1, and neuroligin (Yamada et al., 2003; Subauste et al., 2005). We predict that the TARPs will also be localized here through their association with MAGI-2. In fact, immunoelectron microscopic studies recently showed γ2 and γ8 situated at the edge of postsynaptic densities in asymmetrical synapses (Inamura et al., 2006), consistent with the location of some adherens junctions (Nishioka et al., 2000).

Together, these observations raise the intriguing possibility of a common ancestral origin for synaptic adherens junctions and epithelial tight junctions. Both structures serve as docking sites for numerous proteins that exploit the implicit proximity of adjoining cells to maximize their own signaling or structural functions. This similarity might be the product of convergent evolution or it could reflect divergence from a common origin. The presence of MAGI and TARP proteins at neuronal adherens junctions, and MAGI and claudin proteins at epithelial tight junctions, would support a common origin. It is relevant to note that both neurons and epithelial cells are ultimately derived from the same simple ectoderm during early embryonic development. It will be exciting to discover whether this emerging paralogous relationship between neuronal and non-neuronal adhesive cell junctions will be found to encompass additional molecules.