Synaptic Targeting of the Postsynaptic Density Protein PSD-95 Mediated by Lipid and Protein Motifs

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Neuron, Vol. 22, 497–509, March, 1999, Copyright 1999 by Cell Press Synaptic Targeting of the Postsynaptic Density Protein PSD-95 Mediated by Lipid and Protein Motifs

Sarah E. Craven, Alaa E. El-Husseini, (GPI proteins; Ledesma et al., 1998), whereas short cyto- and David S. Bredt* solic C-terminal protein sorting motifs can mediate both Department of Physiology and dendritic and basolateral targeting (Dotti and Simons, Program in Neuroscience 1990; Jareb and Bankers, 1998). Whether similar lipid- University of California dependent or C-terminal protein sorting motifs mediate San Francisco, California 94143–0444 protein localization to the postsynaptic membrane is not known. Indeed, there is only limited mechanistic information available on the postsynaptic targeting of any protein in neurons (Kaech et al., 1997). Summary Assembly of some components of the PSD may be mediated by PSD-95/SAP-90 and related membrane-asso- During synaptic development, proteins aggregate at ciated guanylate kinase (MAGUK) proteins (Cho et al., specialized pre- and postsynaptic structures. Mecha- 1992; Kistner et al., 1993; Sheng, 1996; Kornau et al., nisms that mediate protein clustering at these sites 1997; Craven and Bredt, 1998). PSD-95 and related remain unknown. To investigate this process, we ana- MAGUKs contain three PDZ protein–protein interaction lyzed synaptic targeting of a postsynaptic density pro- motifs that bind directly to N-methyl-D-aspartate–(NMDA-) tein, PSD-95, by expressing green fluorescent pro- type glutamate receptors, Shaker-type Kϩ channels tein– (GFP-) tagged PSD-95 in cultured hippocampal (Kim et al., 1995; Kornau et al., 1995), intracellular signal- neurons. We find that postsynaptic clustering relies ing enzymes (Brenman et al., 1996a; Chen et al., 1998; on three elements of PSD-95: N-terminal palmitoyla- Kim et al., 1998), and cell adhesion molecules (Irie et tion, the first two PDZ domains, and a C-terminal tar- al., 1997). These proteins also contain an orphan SH3 geting motif. In contrast, disruptions of PDZ3, SH3, or domain and a C-terminal guanylate kinase–like (GK) do- guanylate kinase (GK) domains do not affect synaptic main that binds to specific proteins of the neuronal cy- targeting. Palmitoylation is sufficient to target the dif- toskeleton (Kim et al., 1997; Naisbitt et al., 1997; Takeu- fusely expressed SAP-97 to synapses, and palmitoyla- chi et al., 1997; Brenman et al., 1998). In addition, the tion cannot be replaced with alternative membrane N terminus of PSD-95 is posttranslationally modified by association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic cluster- the incorporation of palmitate, a 16 carbon saturated ing. The requirements for PDZ domains and a C-termi- fatty acid linked by thioester bonds to specific cysteine nal domain of PSD-95 indicate that protein–protein residues (Milligan et al., 1995; Mumby, 1997). Studies interactions cooperate with lipid interactions in synap- of synaptic development in cultured neurons indicate tic targeting. that PSD-95 localizes and clusters at synapses before other postsynaptic proteins (Rao et al., 1998), suggesting that PSD-95 may play a primary role in organizing and Introduction localizing proteins to the PSD. Consistent with this model, Discs-large (DLG), a MAGUK protein in Drosoph- Proper neuronal development and function require pre- ila, is required for the proper synaptic structure and cise localization of proteins to specialized cellular and function of larval neuromuscular junctions (NMJs) (Bud- plasma membrane domains. The postsynaptic density nik et al., 1996; Thomas et al., 1997), and DLG localizes (PSD), an electron-dense cytoskeletal structure beneath both Kϩ channels and the adhesion molecule fasciclin the plasma membrane of excitatory synapses, is one II to the NMJ (Tejedor et al., 1997; Thomas et al., 1997; such specialization. At the PSD, neurotransmitter recep- Zito et al., 1997). Mechanisms that determine the synap- tors and signaling molecules are clustered and thereby tic clustering of PSD-95, therefore, may be fundamental poised to respond to synaptic stimuli (Walters and Ma- to postsynaptic differentiation. Heterologous expres- tus, 1975; Carlin et al., 1981; Kennedy et al., 1983; Kor- sion studies demonstrate that the N terminus and a nau et al., 1997; Craven and Bredt, 1998). Although single PDZ domain are sufficient for aggregation of PSD- biochemical studies have helped define the protein 95 with Kϩ channels in COS cells (Hsueh et al., 1997), composition of the PSD (Kennedy, 1998), mechanisms but the postsynaptic clustering of PSD-95 in neurons that mediate postsynaptic targeting of these proteins has not been functionally investigated. remain largely unknown. To determine the mechanisms for postsynaptic clus- Some progress has been made, however, in under- tering of PSD-95, we have evaluated trafficking of green standing protein polarization to axonal or dendritic mem- fluorescent protein– (GFP-) tagged PSD-95 in cultured branes in neurons. It has been shown that axonal versus hippocampal neurons. We find that N-terminal palmitoy- dendritic protein sorting shares certain mechanisms lation is essential for synaptic clustering of PSD-95. Fur- with apical versus basolateral targeting in polarized epi- thermore, synaptic targeting is specific to palmitoyla- thelial cells. That is, specialized membrane lipid rafts tion, as addition of alternative membrane anchors to are one route for both axonal and apical sorting of glyco- palmitoylation-deficient PSD-95 does not restore post- sylphosphatidylinositol-anchored membrane proteins synaptic targeting. Using protein chimeras, we find that the N-terminal palmitoylation motif of PSD-95 is sufficient to induce postsynaptic clustering of a closely re- * To whom correspondence should be addressed (e-mail: bredt@ lated but diffusely expressed MAGUK, SAP-97. In addi- itsa.ucsf.edu). tion to palmitoylation, the first two PDZ domains and Neuron 498

12 amino acids within the C terminus of PSD-95 are required for clustering. In contrast, disruptions of the third PDZ, the SH3, and the GK domains do not affect synaptic targeting. This work is the first systematic analysis of the postsynaptic targeting of any protein and suggests that specialized lipid and protein interactions both mediate postsynaptic targeting. As protein palmitoylation and protein interactions with PDZ domains are often dynamically regulated (Milligan et al., 1995; Cohen et al., 1996; Mumby, 1997), this work may have implications for synaptogenesis and synaptic plasticity regulated by the PSD-95 family of proteins.

Results

Exogenous PSD-95–GFP Is Correctly Localized in Hippocampal Neurons To determine mechanisms for postsynaptic clustering of PSD-95, a variety of wild-type and mutant protein constructs of PSD-95 were expressed in cultured hippocampal neurons. Initial experiments were conducted to determine whether exogenously expressed wild-type PSD-95 would be targeted in neurons appropriately. A plasmid encoding PSD-95 as a C-terminal fusion protein with GFP was transfected into hippocampal neurons, and the neurons were allowed to develop in culture for 5–16 days. Exogenously expressed PSD-95–GFP forms clusters in the dendrites of transfected neurons after 5–6 days in culture and remains clustered for as long as expression can be detected, up to 2.5 weeks, similar to endogenous PSD-95 (Figure 1A). In contrast, GFP alone is expressed diffusely throughout the neurons (data not shown). After 1 week in culture, clusters of PSD-95–GFP become synaptic and opposed to presynaptic terminals stained for synaptophysin (Figure 1B), Figure 1. Exogenous PSD-95–GFP Is Targeted to Synapses in a as has been reported for endogenous PSD-95 (Rao et Manner Indistinguishable from Endogenous PSD-95 al., 1998). These data indicate that exogenous PSD- (A) Comparison of the clustering of exogenous PSD-95–GFP (left) 95–GFP is targeted to the postsynaptic membrane in a in unfixed cells with untransfected cells stained for PSD-95 (right) manner indistinguishable from endogenous PSD-95 and at various times in vitro: day in vitro (div) 6, div 11, and div 16, from that this system can be exploited to elucidate the re- top to bottom. gions of PSD-95 that are involved in postsynaptic tar- (B) Exogenous PSD-95–GFP clusters are synaptic. An example of geting. a cell transfected with PSD-95–GFP double stained for GFP (left; scale bar, 10 ␮m) and the presynaptic marker synaptophysin (right) at div 16. Palmitoylation Is Necessary for Postsynaptic Localization of PSD-95 N-terminal palmitoylation is important for targeting PSD- 95 to cell membranes, where PSD-95 clusters and asso- either of the two N-terminal cysteines also block palmi- ciates with interacting transmembrane proteins in heter- toylation of PSD-95 (Topinka and Bredt, 1998), and con- ologous cells (Topinka and Bredt, 1998). To determine structs of PSD-95 with mutations of the third (C3S) or if palmitoylation is also necessary for the postsynaptic the fifth (C5S) cysteine also are expressed diffusely in targeting of PSD-95, hippocampal neurons were trans- the processes of cultured neurons (Figure 2A). fected with a PSD-95–GFP protein construct in which One trivial explanation for the differences in target- the N-terminal palmitoylated cysteines were mutated to ing between wild-type and cysteine mutant constructs serines (C35S), thereby abolishing palmitoylation of the could be differences in expression levels. Quantitative protein (Topinka and Bredt, 1998; Figure 7A). In contrast immunofluorescence indicates that exogenous wild- to 10-fold higher -5ف to wild-type PSD-95, C35S PSD-95 does not form clus- type PSD-95 is expressed at levels ters or become synaptic when expressed in hippocam- than endogenous PSD-95 protein (see Experimental pal neurons. Instead, the protein is expressed diffusely Procedures). If the cysteine mutant constructs express throughout the processes at all times in culture (Figure at even higher levels, this could conceivably saturate the 2A), though the distribution of NMDA receptors and syn- targeting machinery and result in a diffuse distribution. aptophysin remain unaffected, suggesting that syn- However, we observed a range of expression levels in apses are normal (data not shown). Single mutations of the population of transfected cells for all the PSD-95 Postsynaptic Targeting of PSD-95 499

differences in expression levels as an explanation for differences in targeting. These data show that the N-terminal cysteines are necessary for the targeting and clustering of PSD-95 at the PSD. However, it remains formally possible that the cysteine residues are critical, not because they are palmitoylated, but rather because of another modification or interaction that requires both cysteines. To assess better the importance of palmitoylation, a single amino acid mutation was identified that disrupts palmitoylation but leaves the cysteine residues intact. This mutation changes the fourth amino acid in PSD-95 from leucine to alanine (L4A) and reduces palmitoylation significantly to Ͻ10% of wild-type levels in heterologous cells (Figure 7A). When this construct is transfected into hippocampal neurons, it resembles the palmitoylation-deficient mutants, with the protein diffusely expressed (Figure 2B; Table 1). Conversely, a construct containing an N-terminal mutation in which the second amino acid of PSD-95, an aspartic acid, was changed to an alanine (D2A) did not disrupt palmitoylation (Figure 7A), and this mutant protein is synaptically targeted, like wild-type PSD-95 (Figure 2B; Table 1). These results are consistent with the idea that palmitoylation rather than other N-terminal interactions is necessary for synaptic clustering of PSD-95.

Palmitoylation of PSD-95 Cannot Be Functionally Replaced with Other Lipid Associations Because palmitoylation targets PSD-95 to cell membranes, it is possible that disrupting membrane association could account for the loss of postsynaptic clustering. We therefore determined whether restoring membrane association through modifications other than palmitoylation could rescue the targeting of palmitoylation-deficient PSD-95. To determine whether integral membrane association of PSD-95 could functionally Figure 2. Palmitoylated Cysteines 3 and/or 5 Are Necessary for substitute for palmitoylation, chimeric molecules were Postsynaptic Clustering of PSD-95 created with the human T lymphocyte type I transmem- (A) The seven N-terminal amino acids of PSD-95 include the palmi- brane protein CD8 (Littman et al., 1985) attached to the toylated cysteines at positions 3 and 5. Palmitoylation-deficient PSD-95 proteins are expressed diffusely in the dendrites of unfixed N terminus of both wild type (CD8–WT) and C35S PSD- transfected cells: C35S PSD-95–GFP (upper left; div 8; scale bar, 95 (CD8–C35S). Though addition of the CD8 transmem- 10 ␮m), C3S PSD-95–GFP (upper right; div 11), and C5S PSD-95 brane domain is sufficient to localize C35S PSD-95 to the (bottom left; div 15). For comparison, the synaptic clustering of wild- membrane and restore its association with interacting type PSD-95–GFP is shown (lower right; div 11) in a transfected cell transmembrane proteins when expressed in heterolo- stained for GFP. (B) Two N-terminal PSD-95 mutants that preserve gous cells (Topinka and Bredt, 1998), it does not rescue cysteines 3 and 5 were generated, one that does not affect palmitoylation (D2A) and one that significantly disrupts palmitoylation (L4A; postsynaptic clustering when CD8–C35S is expressed see Figure 7A). D2A–GFP protein in unfixed neurons is clustered in in hippocampal neurons (Figure 3A). CD8–WT is still of wild-type levels and still yields %50ف a manner indistinguishable from wild-type PSD-95–GFP (top left; palmitoylated to div 10). These clusters are synaptic as they colocalize with the some clusters (Figures 3A and 7A). However, neurons presynaptic marker, synaptophysin (GFP, top right; synaptophysin, transfected with either CD8 fusion appear less devel- middle right; merged, bottom right; div 11; scale bar, 3 ␮m). Synapto- oped than neurons transfected with any other construct, physin staining associated with neurites of untransfected cells in the same field can also be seen. In contrast, L4A–GFP protein is suggesting that expression of these proteins may be expressed diffusely in the dendrites (bottom left; div 10). deleterious to the cells. Although the CD8 fusion did not restore synaptic targeting of palmitoylation-deficient PSD-95, a transmem- constructs used and found cellular targeting to be simi- brane domain is quite different from a lipid anchor and lar at all expression levels. To quantitate PSD-95 expres- may not allow for proper orientation of the protein within sion of wild-type versus C35S PSD-95, the normalized the cell membrane or may sterically interfere with other total fluorescence intensity per cell was measured (see protein interactions. To restore membrane association Experimental Procedures). This analysis found no sig- in a manner more analogous to palmitoylation, we used nificant difference in the expression levels between two other lipid modifications, myristoylation and iso- wild-type and C35S PSD-95 (Table 1), arguing against prenylation. Myristate is a 14 carbon saturated fatty acid Neuron 500

Table 1. Synaptic Clustering Ratio (SCR) of PSD-95–GFP Fusion Proteins Expression Fluorescence Construct n SCR Ϯ SEM Category Intensity Ϯ SEM Wild-type PSD-95 9 0.030 Ϯ 0.010†† S 1800 Ϯ 300 C35S 9 1.0 Ϯ 0.0** D 1800 Ϯ 350 D2A 8 0.070 Ϯ 0.027†† S L4A 8 0.92 Ϯ 0.062** D Myr-95 7 0.86 Ϯ 0.069** D Ger-95 7 0.97 Ϯ 0.018** D 1-PDZ2 (L) 7 0.83 Ϯ 0.076** D 2100 Ϯ 150 1-PDZ2 (H) 9 0.87 Ϯ 0.059** D 5600 Ϯ 450** 1-PDZ2ϩ25 8 0.30 Ϯ 0.047*(ϩ)†† SC 1-PDZ3 7 0.45 Ϯ 0.040**†† SC 1-SH3 9 0.41 Ϯ 0.041**†† SC 2200 Ϯ 220 1-SH3ϩ25 9 0.057 Ϯ 0.020(ϩ)†† S 2200 Ϯ 250 ⌬25 7 0.37 Ϯ 0.048*†† SC 2000 Ϯ 240 ⌬12 6 0.033 Ϯ 0.021†† S ⌬PDZs 7 0.99 Ϯ 0.010** D 1000 Ϯ 140 ⌬PDZ2&3 10 0.41 Ϯ 0.051**†† SC ⌬PDZ1&3 9 0.38 Ϯ 0.048*†† SC ⌬PDZ3 8 0.073 Ϯ 0.024†† S 2300 Ϯ 350 SH3 m30 9 0.040 Ϯ 0.011†† S SAP-97 8 0.93 Ϯ 0.053** D 95–97 5 0.066 Ϯ 0.017†† S The synaptic clustering ratios (SCRs) for neurons transfected with the various expression constructs are presented. The SCR quantitates the intensity of fluorescent protein in the dendrites compared with that at synaptic clusters, with 0 indicative of complete synaptic clustering and 1 indicative of no clusters (see Experimental Procedures). Values are mean Ϯ SE for the number of cells indicated. (H) and (L) indicate high- and low-expressing cells, respectively. A one-way ANOVA was performed with Bonferroni correction for multiple comparisons; double asterisk indicates statistical significance from wild-type (p Ͻ 0.001), asterisk indicates p Ͻ 0.01, plus sign indicates statistical significance from constructs lacking the last 25 amino acids (p Ͻ 0.001), and double dagger indicates statistical significance from C35S (p Ͻ 0.001). A one letter code indicates the three expression patterns observed: S, synaptic targeting that is not statistically significant from wild-type; D, diffuse targeting that is not statistically different from C35S; and SC, semiclustered targeting that is statistically different from both wild-type and C35S. Fluorescence intensity is the total cellular GFP fluorescence in arbitrary units (see Experimental Procedures). that is cotranslationally attached to certain proteins at GFP is expressed in cultured hippocampal neurons, it an N-terminal glycine. To replace the palmitoylation of forms synaptic clusters in a manner similar to PSD- PSD-95 with myristoylation, the N-terminal 13 amino 95 (data not shown). In contrast, SAP-97 is expressed acids of PSD-95 were exchanged for those of 43K/ diffusely throughout the processes (Figure 3B), consis- rapsyn, which contains a consensus for myristoylation tent with the notion that palmitoylation is necessary for (Frail et al., 1988). This chimeric protein is myristoylated the synaptic targeting of the PSD-95 family of proteins when expressed in COS cells, whereas wild-type PSD- in hippocampal neurons. To determine whether forced 95 is not (Figure 7B). Despite being myristoylated, the palmitoylation of SAP-97 could induce postsynaptic tar- chimeric protein is expressed diffusely in neurons, iden- geting, we designed a series of chimeric constructs in tical to other palmitoylation-deficient protein constructs which the first 13 amino acids of PSD-95 were fused to (Figure 3A; Table 1). Similarly, addition of a consensus the N terminus of SAP-97. Replacing the first 13 or 36 sequence for geranylgeranylation, a 20 carbon isopren- amino acids of SAP-97 with the first 13 amino acids of oid that is added posttranslationally at the C terminus, PSD-95 produced chimeric proteins that are not palmi- resulted in isoprenylation on the protein when expressed toylated when expressed in heterologous cells and are in COS cells (Figure 7B) but did not rescue postsynaptic not synaptically targeted when expressed in neurons targeting of palmitoylation-deficient PSD-95 (Figure 3A; (data not shown), again suggesting that the presence Table 1). The inability of myristoylation or isoprenylation of the first 13 amino acids of PSD-95 is not sufficient to rescue palmitoylation mutants of PSD-95 suggests for synaptic targeting in the absence of palmitoylation. that properties of palmitoylation beyond simple mem- However, replacing the entire unique N terminus of SAP- brane association are important for synaptic targeting. 97 with the first 13 amino acids of PSD-95 yielded a chimeric protein (95–97) that is palmitoylated (Figure Addition of a Palmitoylation Motif Induces 7A) and, when expressed in hippocampal neurons, is Postsynaptic Targeting of SAP-97 synaptically clustered, just like wild-type PSD-95 (Figure Closely related to PSD-95 are two additional members 3B; Table 1). of the MAGUK family of proteins, PSD-93/Chapsyn-110 and SAP-97 (Lue et al., 1994; Kim et al., 1995; Muller et PDZ Domains and a C-terminal Targeting Motif al., 1995; Brenman et al., 1996b). Like PSD-95, PSD-93 of PSD-95 Are Required for Synaptic Targeting contains N-terminal cysteines that are palmitoylated, To determine systematically the domains of PSD-95 while SAP-97 has an extended N terminus of 108 amino that, together with palmitoylation, mediate synaptic lo- acids that is not palmitoylated (Topinka and Bredt, 1998; calization, a series of progressively longer N-terminal Figure 7A; data not shown). When PSD-93 tagged with constructs were introduced into primary hippocampal Postsynaptic Targeting of PSD-95 501

not shown), indicating that palmitoylation, even with the addition of a PDZ domain, is not sufficient to target proteins to the PSD. When the fusion protein is extended further to include the first two PDZ domains (1-PDZ2), all three PDZ domains (1-PDZ3), and the SH3 domain (1-SH3), a tendency toward clustering occurs (Figure 4; Table 1; see Experimental Procedures). Although clusters are observed with these three proteins, and these clusters colocalize with synaptophysin (Figure 4; data not shown), protein distribution is different from that of wild-type PSD-95. First, clustering of the proteins requires longer times in vitro to occur (10 rather than 5 days). Second, most of the protein remains diffuse in the cell bodies and dendrites, whereas wild-type PSD- 95 is almost entirely restricted to synaptic sites. This delayed, partial clustering may reflect a passive association of these fusion proteins with other synaptic proteins as opposed to active targeting of the full-length PSD- 95, and we define this distribution as “semiclustered” (see below). In fact, deleting as few as 25 amino acids from the C terminus of PSD-95 (⌬25) disrupts wild-type synaptic clustering and yields this semiclustered phenotype (Figure 4; Table 1), indicating that a signal within these last amino acids is involved in synaptic targeting. To determine if the very C terminus of PSD-95 contains this targeting signal, the final 12 amino acids were deleted (⌬12). This truncation, however, does not affect synaptic targeting (Figure 4; Table 1). Rather, an internal deletion in which the last 13–25 C-terminal amino acids were deleted and the last 12 were present disrupts synaptic targeting and results in a semiclustered phenotype indistinguishable from that of ⌬25 (data not shown). These data indicate that there is a signal within the last 13–25 amino acids that is necessary for the proper synaptic targeting of PSD-95. To quantitate the extent of synaptic targeting, we cal- Figure 3. Synaptic Clustering of PSD-95 Requires Membrane Asso- ciation via Palmitoylation culated a synaptic clustering ratio (SCR) for each con- (A) Addition of the CD8 transmembrane domain does not rescue struct (Table 1). The SCR is a measure of the average synaptic targeting of the palmitoylation-deficient protein C35S PSD- protein expression in the dendrites compared with that 95–GFP (upper right; div 11). Addition of this transmembrane domain at synaptic clusters (see Experimental Procedures). The to wild-type PSD-95–GFP decreases the number of clusters (upper SCR calculated for wild-type PSD-95 is close to zero, left; div 11; scale bar, 10 ␮m). In both cases, the cells look smaller 0.030 Ϯ 0.010, indicating that the average fluorescence and less healthy then other transfected cells. Addition of alternative in the dendrites is close to background fluorescence lipid modifications to PSD-95, myristoylation (bottom left; div 15) or geranylgeranylation (bottom right; div 12), also could not functionally and that almost all of the protein is at synapses. In replace palmitoylation, and these proteins are expressed diffusely contrast, the SCR for C35S PSD-95 is 1.0 Ϯ 0.0, indi- throughout the cells. cating that the protein is expressed evenly throughout (B) The N-terminal palmitoylated domain of PSD-95 can mediate the processes without clusters. The smallest trun- postsynaptic clustering of SAP-97. When transfected into neurons, cation construct that subjectively yields any clustering the nonpalmitoylated PSD-95 family member, SAP-97, is expressed is 1-PDZ2, which has an SCR of 0.83 Ϯ 0.076. This diffusely throughout the cell (upper left; div 11). Replacing the unique N terminus of SAP-97 with the N-terminal palmitoylation domain value is significantly different from that of the wild-type of PSD-95 changes the localization of the protein from diffuse to PSD-95 (p Ͻ 0.001) but not from that of C35S (p Ͼ 0.05), clustered, like wild-type PSD-95 (upper right; div 11). These clusters so this construct is classified as diffuse. On the other also colocalize with synaptophysin (bottom left, GFP; bottom mid- hand, 1-PDZ3, 1-SH3, and ⌬25 have SCRs of 0.45 Ϯ dle, synaptophysin; bottom right, merged; div 11). Again, synapto- 0.40, 0.41 Ϯ 0.041, and 0.31 Ϯ 0.048, respectively, which physin staining associated with the neurites of untransfected cells within the same field can be seen. are not statistically significant from each other but are significantly different from both wild-type PSD-95 and other synaptically targeted constructs (D2A, 95–97, and cultures. A GFP fusion containing only the first 64 amino ⌬12) and from the diffusely expressed C35S. These con- acids of PSD-95 (1–64) or the first 157 amino acids structs (1-PDZ3, 1-SH3, and ⌬25), therefore, represent through the first PDZ domain (1-PDZ1) is not synaptically an intermediate expression category, which we call localized but expressed diffusely in neurons (Figure 4; semiclustered (Table 1). data not shown). These constructs are palmitoylated In addition, the 1-PDZ2 construct shows a very large when expressed in heterologous cells (Figure 7A; data range of expression levels and was therefore used to Neuron 502

Figure 4. Postsynaptic Clustering of PSD-95 Requires the C Terminus of PSD-95 A domain map of PSD-95 is shown at the top. Expression of the palmitoylated N terminus and a single PDZ domain is not sufficient for synaptic clustering (1-PDZ1, top left; div 11; scale bar, 10 ␮m). Addition of the second PDZ domain (1-PDZ2, top middle; div 11), third PDZ domain (1-PDZ3, top right; div 11), the SH3 (1-SH3, bottom left; div 11), and all but the final 25 amino acids (⌬25, bottom middle; div 11) of PSD-95 results in an increase in clustering, but not to the level of wild-type PSD-95. Rather, these truncated proteins show a semiclustered phenotype, and most of the protein remains diffusely distributed in the cell soma and dendrites (see Table 1 for quantitation). The clusters that do occur with these constructs are synaptic and colocalize with synaptophysin, as is shown for 1-SH3 (under 1-SH3: GFP, top; synaptophysin, middle; merged, bottom; div 11). Deletion of the last 12 amino acids (⌬12, bottom right; div 11) results in wild-type synaptic clustering (under ⌬12: GFP, top; synaptophysin, middle; merged, bottom; div 11; scale bar, 4 ␮m).

further address whether expression levels affect the pro- SH3 domain may play a role in synaptic targeting. To tein distribution in the transfected neurons. This was address this, three separate mutations were introduced done by calculating the SCR for cells that express into the SH3 domain. Two of the mutations (L460P and 1-PDZ2 at a level similar to that of wild-type PSD-95 W471A) correspond to changes that are predicted to and for cells that express 1-PDZ2 at more than twice destroy the structure of the SH3 domain, and one muta- this level. Both low expressors and high expressors of tion (W470F) was designed to specifically block binding 1-PDZ2 had nearly identical SCR ratios (0.83 Ϯ 0.076 of the SH3 domain to proline-containing peptides (Lim versus 0.87 Ϯ 0.059), again arguing against differences and Richards, 1994). However, none of these mutations in cellular expression level as an explanation for differ- introduced into the full-length protein disrupts the syn- ences in synaptic targeting. aptic clustering of PSD-95 (Table 1; data not shown). To define further the role of the C terminus of PSD- Similarly, the elimination of the GK domain in the con- 95 in synaptic targeting, the last 25 amino acids of PSD- struct 1-SH3ϩ25 does not disrupt synaptic localization 95 were added to constructs encoding the N terminus of PSD-95 (Figure 5), indicating that neither the SH3 or of PSD-95 through the second or third PDZ domain GK domain is involved in postsynaptic targeting. (1-PDZ2ϩ25 and 1-PDZ3ϩ25, respectively). Strikingly, To examine the role of the PDZ domains in the synap- addition of these amino acids mediates synaptic tar- tic targeting of PSD-95, constructs were made lacking geting of both of these proteins. Adding the 25 C-termi- various combinations of the PDZ domains. When all nal amino acids to 1-PDZ2 changes the categorical three PDZ domains are deleted (⌬PDZs), the resulting distribution from diffuse (SCR ϭ 0.83 Ϯ 0.076) to semi- fusion protein is diffusely expressed (SCR ϭ 0.99 Ϯ clustered (SCR ϭ 0.30 Ϯ 0.047; Figure 5; Table 1; data 0.10), indicating that at least one PDZ domain is required not shown). Furthermore, addition of the C terminus to for synaptic targeting of PSD-95 (Figure 6). Deleting PDZ the construct encoding the N terminus through the SH3 domains 2 and 3 (⌬PDZ2&3) or PDZ domains 1 and 3 domain (yielding 1-SH3ϩ25) changes the localization (⌬PDZ1&3) also disrupts wild-type clustering but yields from semiclustered (SCR ϭ 0.41 Ϯ 0.041) to a wild-type a semiclustered distribution (Figure 6). The clustering of synaptic distribution (SCR ϭ 0.057 Ϯ 0.020; Figure 5; ⌬PDZ2&3 and ⌬PDZ1&3 appears similar to that of the Table 1). Again, though there is a difference in synaptic C-terminal truncation mutants of PSD-95; the clusters targeting, there is no statistical difference in the expres- form later during maturation of the cultures (div 10 ver- sion levels between these two constructs (Table 1). In sus 5), they have SCRs between diffusely expressed addition, the presence of the SH3 domain in 1-SH3ϩ25 and synaptically targeted constructs (0.41 Ϯ 0.051 and versus 1-PDZ2ϩ25 or 1-PDZ3ϩ25 makes a small differ- 0.38 Ϯ 0.048, respectively) (Table 1), and they are still ence in the synaptic distribution, suggesting that the synaptic and colocalize with synaptophysin (data not Postsynaptic Targeting of PSD-95 503

Figure 5. The C Terminus of PSD-95 Contains a Synaptic Targeting Motif Figure 6. Postsynaptic Clustering of PSD-95 Requires the First and Addition of the final 25 amino acids of PSD-95 to the truncated Second, But Not the Third, PDZ Domains protein 1-PDZ2 (top left; div 11; scale bar, 10 ␮m) to yield 1-PDZ2ϩ25 (bottom left; div 11) increases targeting from diffuse to semiclus- Deletion of all three PDZ domains of PSD-95 (⌬PDZs, top; div 11; tered. Under 1-PDZ2ϩ25 is shown the colocalization of 1-PDZ2ϩ25 scale bar, 10 ␮m) completely disrupts synaptic clustering. Deletion clusters with synaptophysin (GFP, top; synaptophysin, middle; of the second and third PDZ domains (⌬PDZ2&3, middle left; div merged, bottom; div 11; scale bar, 4 ␮m). Addition to 1-SH3 (top 11) or of the first and third PDZ domains (⌬PDZ1&3, middle right; right; div 11) to yield 1-SH3ϩ25 (bottom right; div 11) changes tar- div 10) results in a semiclustered phenotype, whereas deletion of geting from semiclustered to wild-type clustering that is again syn- only the third PDZ domain (⌬PDZ3, bottom; div 11) results in wild- aptic (under 1-SH3ϩ25: GFP, top; synaptophysin, middle; merged, type synaptic clustering (bottom right ⌬PDZ3: GFP, top; synapto- bottom; div 11; scale bar, 3 ␮m). physin, middle; merged, bottom; div 11; scale bar, 3 ␮m). shown). On the other hand, deleting PDZ3 (⌬PDZ3) does Lipid-Dependent Interactions in Synaptic Sorting not disrupt wild-type synaptic localization (Figure 6; Ta- A striking finding of this work is the essential and specific ble 1). These data suggest that synaptic targeting of role of palmitoylation in the postsynaptic clustering of PSD-95 requires both the first and second, but not the PSD-95. The requirement for palmitoylation of PSD-95 third, PDZ domain. cannot be replaced by alternative membrane association motifs, suggesting that specialized lipid interactions Discussion at the PSD are involved in clustering. Palmitate is a 16 carbon saturated fatty acid that is linked by thioester This study describes the first systematic analysis of pro- bonds to specific cysteine residues of certain proteins tein domains that underlie postsynaptic sorting in neu- (Milligan et al., 1995; Mumby, 1997). Protein palmitoyla- rons. The primary finding is that postsynaptic clustering tion can influence the function of a protein by altering its of PSD-95 relies critically on three elements: N-terminal subcellular targeting and protein interactions. Previous palmitoylation, the first two PDZ domains, and a C-ter- work has demonstrated that PSD-95 is heavily palmitoy- minal targeting motif. PSD-95 proteins lacking these lated on cysteines 3 and/or 5 and that this modification elements are targeted diffusely or are only semiclustered of PSD-95 is essential for membrane association and in dendrites, whereas disruptions in PDZ3, SH3, or GK interaction with an ion channel–binding partner, Kv 1.4 domains have no affect on synaptic targeting. Because (Topinka and Bredt, 1998). In COS cells, the interaction postsynaptic clustering of PSD-95 occurs early during of PSD-95 with Kv 1.4 can be rescued by substituting an neuronal development (Rao et al., 1998), the specialized artificial transmembrane domain for the palmitoylation lipid, PDZ, and C-terminal protein interactions identified (Topinka and Bredt, 1998). In neurons, however, we were here may represent fundamental steps in postsynaptic unable to restore postsynaptic clustering of PSD-95 by differentiation. replacing the palmitoylation site with a transmembrane Neuron 504

Figure 7. Palmitoylation, Myristoylation, and Isoprenylation of PSD-95 Proteins Transfected COS cells were metabolically labeled with [3H] palmitic acid, myristic acid, or mevalonolactone. Cells lysates were separated by SDS–PAGE and analyzed by fluorography (top or left) or immunoblotting with anti- GFP antibodies (bottom or right). (A) Proteins analyzed for palmitoylation from left to right: wild-type PSD-95 (WT), C35S PSD-95, 1-PDZ1, D2A, L4A, SAP-97, PSD- 95/SAP-97 (95–97), CD8–WT, and CD8–C35S. Though all proteins were expressed (bottom), only wild-type PSD-95, 1-PDZ1, D2A, and 95–97 are robustly palmitoylated. CD8–WT is palmitoylated at approximately one-half the level of wild type, and L4A is only very weakly palmitoylated (top). (B) Myristoylation of myristoyl–PSD-95 (Myr- 95) versus wild-type PSD-95 (WT) is shown on the left and the geranylgeranylation of Ger- 95 versus C35S on the right. The weakness of the prenylation signal is likely attributed to the poor uptake of [3H]mevalonolactone by cells compared with [3H]palmitic and [3H]myristic acid.

domain or with alternative lipid anchoring modifications, synapse and helps specify protein aggregation. Addi- including N-terminal myristoylation or C-terminal geranyl- tionally, palmitoylation, unlike myristoylation and gera- geranylation. Palmitoylation, therefore, does not merely nylgeranylation, is a reversible modification, and the dy- function as a lipid anchor but plays a specific role in the namic nature of palmitoylation may explain why stable postsynaptic clustering of PSD-95. cell membrane association cannot substitute for palmi- In epithelial cells, palmitoylated proteins are often tar- toylation in synaptic targeting. geted to caveolae, cave-like invaginations of the plasma membrane enriched in cholesterol (Anderson, 1993; Li- santi et al., 1995). Caveolae accumulate numerous re- Synaptic Clustering Compared with Sorting ceptors and enzymes involved in signal transduction in Nonneuronal Cells pathways. Caveolin, the major protein constituent of Previous expression studies in nonneuronal cells have caveolae, is itself heavily palmitoylated and, like PSD- suggested the importance of the N terminus and PDZ 95, functions as a molecular scaffold by interacting with domains in mediating the targeting and clustering of diverse receptors and cytosolic enzymes (Simons and MAGUK proteins. In epithelial cells, members of the Ikonen, 1997). While caveolae have not previously been MAGUK family are localized to sites of cell–cell contact. identified in neurons, there are data that suggest that In Drosophila, the localization of the MAGUK protein specialized lipid domains are involved in protein traffick- DLG to epithelial septate junctions requires the HOOK ing in neurons. Proper axonal sorting of viral glycoprotein region between the SH3 domain and GK for cell mem- hemagglutinin and endogenous Thy-1 may involve spe- brane interaction and the second PDZ domain for specialized sphingolipid/cholesterol-rich rafts (Ledesma et cific localization to the septate junctions (Hough et al., al., 1998), which are diffusionally restricted to the axon 1997). In contrast, the N terminus of SAP-97, the mam- by the initial segment (Kobayashi et al., 1992; but see malian DLG homolog, is required for subcellular tar- Winckler and Poo, 1996). That postsynaptic targeting of geting to sites of epithelial cell contact (Wu et al., 1998). PSD-95 specifically requires palmitoylation may indicate Regarding PSD-95, transfection of deletion mutants into that a specialized lipid environment also occurs at the COS cells demonstrates that only the N terminus and a Postsynaptic Targeting of PSD-95 505

single PDZ domain from PSD-95 are required for cluster- third PDZ domain is dispensable. As PDZ domains are ing with a coexpressed Kϩ channel subunit (Hsueh et protein interaction interfaces, the requirement for these al., 1997). Similarly, genetic manipulations of Drosophila domains suggests that the association of PSD-95 with DLG, which mediates postsynaptic clustering of Shaker another synaptic protein mediates postsynaptic cluster- Kϩ channel subunits at the larval NMJ, show that expres- ing. The first and second PDZ motifs of PSD-95 have sion of the N terminus and PDZ domains of DLG is similar binding specificities (Kim et al., 1995; Kornau et sufficient for clustering of Shaker Kϩ channels on the al., 1995), whereas the third PDZ domain interacts with muscle membrane (Tejedor et al., 1997). However, the a different set of proteins (Irie et al., 1997; Niethammer Kϩ channel clusters formed by this truncated DLG pro- et al., 1998). This differential binding specificity may tein are not synaptically localized, indicating that other explain the requirement for the first and second PDZ domains mediate targeting to the NMJ (Tejedor et al., domains but not the third. Because PSD-95 localizes to 1997). synapses earlier than known binding partners for the In contrast, the presence of the N terminus and the first two PDZ domains, such as the NMDA receptors PDZ domains is not sufficient for wild-type clustering (Rao et al., 1998), the PDZ-binding protein(s) involved of PSD-95 in neurons (Figure 4). Instead, synaptic clus- in synaptic targeting likely remains unidentified. We also tering also requires a signal within the last 13–25 find that NMDA receptors are synaptically clustered in C-terminal amino acids of PSD-95. The additional re- transfected neurons that diffusely express C35S or quirement we find for a C-terminal sequence indicates 1-PDZ2. This suggests that the presence at synapses that another targeting step or protein interaction is in- of a binding partner for PDZ1 and PDZ2 of PSD-95 is volved in clustering in neurons as compared with non- not sufficient for synaptic targeting of PSD-95. neuronal cells. The role of the C terminus of PSD-95 Alternatively, the involvement of PDZ domains in syn- is reminiscent of basolateral and dendritic targeting of aptic targeting may be independent of classical PDZ transmembrane proteins, which can be mediated by binding and involve the surrounding linker regions. short C-terminal tyrosine- or leucine-based cytosolic Though only a short linker region exists between PDZ1 sorting motifs (Matter et al., 1992; Ohno et al., 1995; and PDZ2, this region, rather than the PDZ binding inter- Jareb and Bankers, 1998). Some of these sequences face, could be important for synaptic targeting. More associate with the clathrin adapter protein AP-2, which complex possibilities can also be imagined. For exam- directs membrane proteins into clathrin-coated pits, ple, a combination of regions within PDZ1 and PDZ2 suggesting a role for these sequences in endocytosis (which includes the 67 amino acid linker region between (Ohno et al., 1995; Dietrich et al., 1997). The 13–25 PDZ2 and PDZ3) could be involved in synaptic targeting, C-terminal amino acids of PSD-95 contain a consensus so that the presence of one or the other yields semiclus- sequence for such a tyrosine-based signal that con- ters, as with ⌬PDZ1&3 and ⌬PDZ2&3, whereas the pres- forms to the canonical motif YXXH, where Y is a tyrosine, ence of both, as with ⌬PDZ3, is sufficient for wild-type X is any amino acid, and H is a hydrophobic amino acid; clustering (Table 1). The identification of binding part- however, the relevance of these signals for cytosolic ners involved in the synaptic targeting of PSD-95 will proteins has not been well established. The cytosolic help to clarify these remaining uncertainities. Finally, the Nef protein of the simian immunodeficiency virus (SIV) 40 amino acid linker region between PDZ3 and the SH3 has been found to interact with AP-2 adaptors, leading domain is absent from all constructs with deletions of to the internalization of the transmembrane protein CD4 the PDZ domains and therefore is dispensable for syn- (Piguet et al., 1998). It is thought that viral Nef may mimic aptic targeting. endogenous molecules that are involved in linking cell Numerous synaptic protein interactions with the third surface proteins to components of the endocytic ma- PDZ and with the GK domain have been identified. The chinery. Whether clathrin-coated pits are involved in the third PDZ interacts with the cell adhesion protein, neu- cellular trafficking that mediates synaptic targeting of roligin (Irie et al., 1997) and the cytoskeletal protein, PSD-95 warrants future study. CRIPT (Niethammer et al., 1998), and the GK domain Another possible role for the C terminus of PSD-95 in interacts with additional proteins of the neuronal cy- synaptic targeting is suggested by the work of Rongo toskeleton (Kim et al., 1997; Naisbitt et al., 1997; Takeu- et al. (1998) in which the PDZ-containing protein LIN-10 chi et al., 1997; Brenman et al., 1998). These interactions was found to be involved in the postsynaptic targeting of with PSD-95 have been proposed to help link the synap- the GLR-1 glutamate receptors in Caenorhabditis ele- tic extracellular matrix to the underlying cytoskeleton. gans. While LIN-10 does not interact with GluR1, it does However, we find that association of PSD-95 with the bind directly to the MAGUK protein, LIN-2, forming a extracellular space or with the cytoskeleton through ternary complex with LIN-2 and a third PDZ-containing these protein interactions is not necessary for synaptic protein, LIN-7, a complex that is conserved in mammals targeting. Although no binding partners have been found (Butz et al., 1998; Kaech et al., 1998). PSD-95 may be yet for the SH3 domain of PSD-95, it is clear from our in a similar complex containing a targeting protein like studies that classical SH3 domain binding does not play LIN-10 that interacts with the C terminus of PSD-95. a role in synaptic clustering. Yet, the presence of the SH3 domain in the construct containing the last 25 amino acids of PSD-95 (1-SH3ϩ25) did change the synaptic Roles for the PDZ, SH3, and GK Domains targeting from semiclustered with 1-PDZ3ϩ25 to wild- in Synaptic Sorting type, suggesting that there may be a signal within the Finally, proper synaptic targeting of PSD-95 also re- primary sequence of the SH3 domain involved in synap- quires the first and second PDZ domains, whereas the tic targeting. However, addition of the SH3 domain in Neuron 506

1-SH3 did not enhance the synaptic targeting of 1-PDZ3, subcloned into the HindIII and EcoRI of GW1 (British Biotechnology; as both are semiclustered (Table 1). Therefore, the re- Topinka and Bredt, 1998). GFP was subcloned in-frame at the C quirement of the SH3 may be a matter of spacing be- terminus of PSD-95 at the EcoRI site. Deletion mutants of PSD-95 (1–64; 1-PDZ1 to amino acid 157; 1-PDZ2 to amino acid 309, which tween the domains involved in synaptic targeting, so includes the 67 amino acid linker region between PDZ2 and PDZ3; that the C-terminal motif interaction(s) is not too close 1-PDZ3 to amino acid 433, which includes the 40 amino acid linker to those of the PDZ domains. region between PDZ3 and the SH3 domain; 1-SH3 to amino acid 502, ⌬25 to amino acid 699, and ⌬12 to amino acid 712) were gener- Implications for Synaptic Development ated by PCR and subcloned into GW1 at the HindIII and KpnI sites and Plasticity in-frame with GFP that was subcloned into GW1 at the KpnI and Although our work defines essential elements for clus- EcoRI sites. Deletion mutants containing the last 25 C-terminal amino acids of PSD-95 (1-PDZ2ϩ25, 1-PDZ3ϩ25, and 1-SH3ϩ25) tering of PSD-95, it is not yet clear how the number and were generated by PCR and subcloned into PSD-95–GFP GW1 with location of PSD-95 clusters are determined. To help the HindIII of GW1 and the internal BglII site of PSD-95. The PDZ address these critical questions, it may be valuable to deletion constructs ⌬PDZs, ⌬PDZ2&3, and ⌬PDZ3 were generated consider differentiation of the NMJ. In developing by using PSD-95 1–64, 1-PDZ1, and 1-PDZ2, respectively, as vectors muscle, nicotinic acetylcholine receptors (nAChRs) be- in GW1 GFP and by subcloning in-frame at the KpnI site a PCR come clustered at postsynaptic muscle membranes in product encoding amino acids 436–724 of PSD-95. The PDZ dele- response to axonal release of agrin, which activates tion constructs ⌬PDZ1&3 was generated by using PSD-95 1–64 in GW1 as a vector and by subcloning in a PCR product encoding a muscle-specific tyrosine kinase (MuSK) (McMahan, PDZ2 (amino acids 158–309, which includes the 67 amino acid linker 1990; DeChiara et al., 1996; Glass et al., 1996). This region between PDZ2 and PDZ3), along with amino acids 436–724 tyrosine kinase cascade induces clustering of nAChRs of PSD-95. at the synapse in association with a cytoskeletal protein, CD8–PSD-95 constructs are described in Topinka and Bredt rapsyn. Decisive genetic evidence for this model derives (1998). GFP was added in-frame to these constructs at a C-terminal from studies of rapsyn, MuSK, and agrin knockout mice, EcoRI site. Myristoylated PSD-95 was generated by PCR with an N-terminal primer encoding the first 12 amino acids of rapsyn and all of which lack properly clustered nAChRs (Gautam et subcloned into GW1 at the HindIII and EcoRI sites. Adding the 4 al., 1995, 1996; DeChiara et al., 1996). It is not yet known amino acid motif CAAL to the C terminus of PSD-95–GFP generated whether axon-derived signals or tyrosine kinase cas- geranylgeranyl–PSD-95 (Ger-95). cades mediate postsynaptic differentiation of synapses Wild-type SAP-97 construct was generated by PCR and sub- in brain. However, clustering of PSD-95 may be one of cloned into GW1 at the SalI and KpnI sites in-frame to GFP that was the early events downstream of such a signal that helps subcloned into GW1 at the KpnI and EcoRI sites. SAP-97 constructs set up the postsynaptic membrane for synaptic forma- containing the first 13 amino acids of PSD-95 (95–97) were generated by PCR with an N-terminal primer encoding these amino acids and tion. Therefore, identifying signals that affect the forma- subcloned as done for wild-type SAP-97 into GW1. tion of PSD-95 clusters in neurons may help to elucidate the cascades that mediate synaptogenesis in the central Primary Neuronal Culture and Transfection nervous system. Neuronal cultures were prepared from hippocampi of E18/E19 rats. Taken together, our findings indicate that multiple The hippocampi were dissociated by enzyme digestion with papain specific elements, including protein–lipid and protein– followed by brief mechanical trituration. Cells were plated on poly- protein interactions, are necessary for clustering of PSD- D-lysine– (Sigma) treated glass coverslips (12 mm in diameter). Cul- 95 at the synapse. Considering the central role that PSD- tures were plated and maintained in Neurobasal media (Gibco) sup- 95 and related MAGUKs play at the synapse (Sheng, plemented with B27, penicillin, streptomycin, and L-glutamine as 1996; Tejedor et al., 1997; Thomas et al., 1997; Zito described in Brewer et al. (1993). Hippocampal cultures were transfected by lipid-mediated gene transfer based on the protocol de- et al., 1997), the specificity that determines their own scribed in Kaech et al. (1996). Briefly, cells were transfected just synaptic targeting is understandable. In addition to pro- before plating in a balanced salt solution at 1 million cell/0.25 mL. viding a stable scaffold, the postsynaptic cytoskeleton Two micrograms of DNA and 10 ␮l of DOTAP (Boehringer-Mann- must retain plasticity. Reorganization of the postsynap- heim) were mixed in 25 ␮l of HBS (150 mM NaCl, 20 mM HEPES tic elements occurs dramatically in regenerating central [pH 7.4]) and added to the cells with immediate and gentle mixing. synapses (de la Cruz et al., 1996), and subtle alterations The cells were incubated for 1 hr at 37ЊC and then plated at a density 2 in postsynaptic structure are thought to underlie aspects of 600/mm on glass coverslips (Fisher) in 24-well plates (Falcon). To visualize transfected cells, coverslips were removed from the of synaptic plasticity (Geinisman et al., 1991). Consistent wells and mounted live onto slides (Frost Plus slides; Fisher) with with this, the elements that determine synaptic cluster- Fluoromount-G (Southern Biotechnology Associates). Transfection ing of PSD-95 can all be reversibly regulated. Protein efficiency was never Ͼ0.01%, and on average, 15 transfected cells phosphorylation has been previously shown to regulate were obtained for each independent transfection. PDZ domain interactions with PSD-95 (Cohen et al., 1996). Similarly, protein palmitoylation occurs through Immunofluorescent Labeling a labile thioester linkage and is often dynamically con- Coverslips were removed from culture wells and fixed in Ϫ20ЊC trolled by extracellular stimuli (Milligan et al., 1995; methanol for 15–20 min. After washing with Tris-buffered saline Mumby, 1997). It will now be important to dynamically containing 0.1% Triton-X-100 (TBST) three times for 5 min, the cells were incubated in TBST containing 3% normal goat serum for 1 hr monitor protein and lipid interactions with PSD-95 and at room temperature to block nonspecific antibody interactions. determine how these influence synaptogenesis and syn- Primary antibodies against PSD-95 (monoclonal 046; Affinity Biore- aptic plasticity. agents, Golden, CO), GFP (monoclonal; Quantum), or synaptophysin (polyclonal rabbit; Sigma) were added in block solution for 1 hr at Experimental Procedures room temperature followed by donkey anti-mouse or goat anti-rabbit secondary antibodies conjugated to Cy2 or Cy3 fluorophores cDNA Cloning and Mutagenesis (diluted 1:200 in block solution) for 1 hr at room temperature. Cov- Wild-type and N-terminal mutant full-length PSD-95 constructs erslips were then mounted on slides (Frost Plus slides; Fisher) with (PSD-95–GFP, C35S, C3S, and C5S) were generated by PCR and Fluoromount-G (Southern Biotechnology Associates), and images Postsynaptic Targeting of PSD-95 507

were taken under fluorescence microscopy with a 100ϫ oil immer- the National Association for Research on Schizophrenia and De- sion objective (numerical aperture, 1.4) affixed to a Leica upright pression, the EJLB, and the Beckman and Culpeper Foundations. microscope. Received November 2, 1998; revised January 20, 1999. Quantitative Measurement of GFP Expression Transfected neurons for quantitation were chosen randomly by se- References lecting neurons from 2 to 5 coverslips from 2 to 3 independent transfections of each construct to obtain 5–10 cells per construct. Anderson, R.G. (1993). Caveolae: where incoming and outgoing The total number of transfected neurons qualitatively examined for messengers meet. Proc. Natl. Acad. Sci. USA 90, 10909–10913. each construct ranged from 10 to 50, depending on transfection Brenman, J.E., Chao, D.S., Gee, S.H., McGee, A.W., Craven, S.E., efficiency. Images were acquired with an exposure time adjusted Santillano, D.R., Huang, F., Xia, H., Peters, M.F., Froehner, S.C., and to limit photobleaching and such that maximum pixel value was Bredt, D.S. (1996a). Interaction of nitric oxide synthase with the at least half saturation. For quantitation of expression levels, the postsynaptic density protein PSD-95 and ␣-1 syntrophin mediated exposure time was identical for each cell. The average background by PDZ motifs. Cell 84, 757–767. fluorescence of untransfected cells was subtracted. The SCR was determined by dividing the average pixel intensity Brenman, J.E., Christopherson, K.S., Craven, S.E., McGee, A.W., and in the dendrites by that of the synaptic clusters in individual cells. Bredt, D.S. (1996b). Cloning and characterization of postsynaptic The perimeters of the dendrites were traced (excluding clusters) density 93, a nitric oxide synthase interacting protein. J. Neurosci. and the average pixel intensity calculated in NIH Image. Similarly, 16, 7407–7415. the perimeters of clusters were traced and the average pixel intensity Brenman, J.E., Topinka, J.R., Cooper, E.C., McGee, A.W., Rosen, obtained. The ratio of the average pixel intensity in dendrites versus J., Milroy, T., Ralston, H.J., and Bredt, D.S. Localization of PSD-93 to clusters was defined as the SCR, with a ratio of zero indicating dendritic microtubules and interaction with microtubule-associated complete clustering, such that dendritic pixel intensity was not dif- protein 1A. J. Neurosci., 1998. ferent from background fluorescence, and a ratio of 1 indicating Brewer, G.J., Torricelli, J.R., Evege, E.K., and Prince, P.J. (1993). diffuse dendritic fluorescence with no clusters. When no clusters Optimized survival of hippocampal neurons in B-27-supplemented were distinguishable, an SCR of 1 was assigned. The data were Neurobasal, a new serum-free medium combination. J. Neurosci. analyzed by one-way ANOVA with Bonferroni correction for multiple Res. 35, 567–576. comparison with Prism software (Graph Pad, San Diego, CA). To Budnik, V., Koh, Y.H., Guan, B., Hartman, B., Hough, C., Woods, compare the expression levels in neurons transfected with different D., and Gorczyca, M. (1996). Regulation of synapse structure and constructs, the total pixel intensity of individual transfected cells function by the Drosophila tumor suppressor gene dlg. Neuron 17, was measured by tracing the perimeter of the entire cell and calcu- 627–640. lating the total pixel intensity in NIH Image. To compare the expression levels of exogenous and endogenous PSD-95, transfected cells Butz, S., Okamoto, M., and Sudhof, T.C. (1998). A tripartite protein were immunofluorescently labeled with antibodies to PSD-95, and complex with the potential to couple synaptic vesicle exocytosis to the total pixel intensity of transfected versus untransfected cells cell adhesion in brain. 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The rat brain post- mCi/mL [3H]palmitic acid (50 Ci/mmol; New England Nuclear), myris- synaptic density fraction contains a homolog of the Drosophila tic acid (49 Ci/mmol; New England Nuclear), or mevalonolactone discs-large tumor suppressor protein. Neuron 9, 929–942. (15 Ci/mmol; American Radiolabeled Chemicals), the precursor of Cohen, N.A., Brenman, J.E., Snyder, S.H., and Bredt, D.S. (1996). all isoprenoid biosynthesis, with 40 ␮M Mevastatin (ICN), an inhibitor Binding of the inward rectifier Kϩ channel Kir 2.3 to PSD-95 is regu- of endogenous mevalonate biosynthesis, for 4–5 hr. Cells were lated by protein kinase A phosphorylation. Neuron 17, 759–767. washed with ice-cold PBS and scraped off culture wells into 0.4 mL Craven, S.E., and Bredt, D.S. (1998). PDZ proteins organize synaptic of TEE (50 mM Tris-HCl [pH 7.4], 1 mM EDTA, and 1 mM EGTA) signaling pathways. Cell 93, 495–498. with 150 mM NaCl and 0.2% SDS. 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