• Aplysia;
  • Aplysia synapse associated protein;
  • membrane-associated guanylate kinase;
  • post-synaptic density;
  • potassium channel


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The vertebrate post-synaptic density (PSD) is a region of high molecular complexity in which dynamic protein interactions modulate receptor localization and synaptic function. Members of the membrane-associated guanylate kinase (MAGUK) family of proteins represent a major structural and functional component of the vertebrate PSD. In order to investigate the expression and significance of orthologous PSD components associated with the Aplysia sensory neuron-motor neuron synapse, we have cloned an Aplysia Dlg-MAGUK protein, which we identify as Aplysia synapse associated protein (ApSAP). As revealed by western blot, RT-PCR, and immunocytochemical analyses, ApSAP is predominantly expressed in the CNS and is located in both sensory neuron and motor neurons. The overall amino acid sequence of ApSAP is 55–61% identical to Drosophila Dlg and mammalian Dlg-MAGUK proteins, but is more highly conserved within L27, PDZ, SH3, and guanylate kinase domains. Because these conserved domains mediate salient interactions with receptors and other PSD components of the vertebrate synapse, we performed a series of GST pull-down assays using recombinant C-terminal tail proteins from various Aplysia receptors and channels containing C-terminal PDZ binding sequences. We have found that ApSAP selectively binds to an Aplysia Shaker-type channel AKv1.1, but not to (i) NMDA receptor subunit AcNR1-1, (ii) potassium channel AKv5.1, (iii) receptor tyrosine kinase ApTrkl, (iv) glutamate receptor ApGluR1/4, (v) glutamate receptor ApGluR2/3, or (vi) glutamate receptor ApGluR7. These findings provide preliminary information regarding the expression and interactions of Dlg-MAGUK proteins of the Aplysia CNS, and will inform questions aimed at a functional analysis of how interactions in a protein network such as the PSD may regulate synaptic strength.

Abbreviations used



Aplysia NMDA-like receptor


Aplysia synapse associated protein


excitatory post-synaptic current


glutathione S-transferase


guanylate kinase


membrane-associated guanylate kinase


NMDA receptor subunit 2B


phosphate-buffered saline


phosphate buffered saline plus triton


PSD-95, discs-large, zonula occludens-1


post-synaptic density


src homology 3


sensory neuron–motor neuron


Tris buffered saline plus Tween

The vertebrate post-synaptic density (PSD) is an electron dense region of high molecular complexity measuring ∼400 nm long and 40 nm wide (Ziff 1997; Kennedy 2000; Sheng and Kim 2002; Sheng and Hoogenraad 2006). In recent years, significant progress has been made in dissecting this complexity into component molecules and their associated regulation, interactions, and activity. Proteomic studies have identified several hundred PSD proteins (Jordan et al. 2004; Peng et al. 2004; Yoshimura et al. 2004; Chen et al. 2005; Cheng et al. 2006; Collins et al. 2006), which create a physical network between NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), and metabotropic glutamate receptors via a large number of protein interactions. Many of these interactions are mediated by members of the Dlg-membrane-associated guanylate kinase (MAGUK) family of proteins. The vertebrate Dlg-MAGUK protein family contains four identified members: PSD-95/SAP90, Chapsyn-110/PSD-93, SAP97, and SAP102. These proteins, particularly PSD-95, represent major protein components of the vertebrate PSD and are important mediators of the PSD as an interactive protein network (Cho et al. 1992; Chen et al. 2005; Cheng et al. 2006; Schluter et al. 2006; Sheng and Hoogenraad 2006).

The domain organization of Dlg-MAGUK proteins is characterized by three PSD-95, discs-large, zonula occludens-1 (PDZ) domains followed by single src homology 3 (SH3) and guanylate kinase (GK) domains, and a short carboxy terminus. Dlg-MAGUK SAP97 and a low-abundance splice variant of PSD-95 (PSD-95β), as well as Drosophila Dlg (S97N), also contain an amino-terminal L27 domain (Mendoza et al. 2003; Petrosky et al. 2005; Straight et al. 2006). All of these domains are responsible for inter-molecular and in some cases intra-molecular interactions, which collectively form an extensive protein network (for reviews, see Fujita and Kurachi 2000; Kim and Sheng 2004; Armstrong et al. 2006).

Given the high degree of interactions mediated by PSD-95 and other Dlg-MAGUK proteins, a major question has emerged concerning how these molecules and their associated interactions contribute to synaptic function. Genetic studies from Drosophila have provided essential insights into this question. For example, Drosophila Dlg is a tumor-suppressor gene expressed at sites of epithelial and neuronal contacts and is important for development of cellular polarity, as well as synaptic architecture and function (Woods and Bryant 1991; Lahey et al. 1994; Budnik 1996; Budnik et al. 1996; Guan et al. 1996; Mendoza et al. 2003). Synaptic targeting of Dlg is regulated by Ca2+-calmodulin dependent protein kinase II (CaMKII) phosphorylation (Koh et al. 1999) and interactions between Dlg and Drosophila Shaker potassium channel regulate channel trafficking and subcellular distribution (Tejedor et al. 1997; Zito et al. 1997; Ruiz-Canada et al. 2002).

Members of the vertebrate family of Dlg-MAGUK proteins have been extensively implicated in the trafficking and function of ionotropic glutamate receptors. However, the high degree of sequence similarity among family members complicates the delineation of unique functional contributions. Genetic deletion of PSD-95 results in an enhancement of long-term potentiation (Migaud et al. 1998; Beique et al. 2006) but counter-intuitively, produces a deficit in spatial memory (Migaud et al. 1998), while over-expression of PSD-95 leads to an increase in baseline surface AMPA receptors and associated AMPA currents (El-Husseini et al. 2000; Schnell et al. 2002; Stein et al. 2003; Ehrlich and Malinow 2004). Over-expression of PSD-95α or SAP97α (the non-L27 domain-containing isoform) against a PSD-95 null background generated by shRNA similarly leads to an enhancement of AMPA excitatory post-synaptic currents (EPSCs) (Schluter et al. 2006). Moreover, while over-expression of PSD-95β or SAP97β against a knockdown background rescues the impairment of AMPA EPSCs, SAP97β has little or no effect when over-expressed against a non-compromised PSD-95 background (Schnell et al. 2002; Ehrlich and Malinow 2004; Schluter et al. 2006); however, some reports have found a subsequent increase in surface AMPA receptor expression as well as EPSC amplitude (Nakagawa et al. 2004b) and miniature EPSC frequency (Rumbaugh et al. 2003) following SAP97 over-expression. These results underscore how the complexity of the molecular landscape of the PSD, and redundancy among protein family members, can complicate the dissection of individual functional contributions. Thus, a thorough understanding of the structure–function relationships of these proteins and their binding partners remains a focus of considerable experimental attention.

To relate specific PSD proteins to synaptic function requires a preparation that is amenable to a cellular and molecular analysis. Aplysia californica provides a powerful simplified system in which to investigate cellular mechanisms responsible for synaptic function and plasticity, because of its capacity to demonstrate simple forms of memory, and a well-characterized CNS amenable to cellular and molecular manipulation and analysis (Antzoulatos and Byrne 2004; Hawkins et al. 2006; Reissner et al. 2006). While the post-synaptic specialization at Aplysia CNS synapses is more narrow and less pronounced than the vertebrate PSD, electron microscopy studies have reported the presence of an electron-dense region of the post-synaptic membrane directly associated with the pre-synaptic active zone (Bailey et al. 1979, 1981). Thus, Aplysia presents a unique opportunity to more closely examine the relationship between molecular networks and synaptic function. In this system, it is possible to directly explore questions of how the regulation of protein interactions can mediate cellular responses to a well specified stimulus. But to exploit this feature of Aplysia, information regarding orthologous constituents of the post-synaptic specialization is required before associated functional questions can be addressed. Toward that end, we sought to identify and characterize Dlg-MAGUK proteins associated with the Aplysia sensory neuron–motor neuron (SN–MN) synapse. We here report the identification of a single Dlg-MAGUK protein, Aplysia synapse associated protein (ApSAP). ApSAP has a domain organization which allows it to be included among the Dlg-MAGUK family of proteins. ApSAP is expressed in both sensory and MNs, and is enriched in synaptosomes. Moreover, ApSAP forms a physical association with the C-terminal tail of Aplysia Shaker-type potassium channel AKv1.1, but not with other PDZ binding sequences including potassium channel AKv5.1, multiple glutamate receptors, and receptor tyrosine kinase ApTrkl. However, ApSAP can bind to the C-terminal region of rat NMDA receptor subunit 2B (NR2B), indicating that the structure of ApSAP is conserved for this interaction. These results provide insight into the molecular evolution of the PSD, and will allow further investigation of the functional roles of this identified MAGUK member at a well characterized synapse.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

General methods

Wild-caught Aplysia californica (120–250 g) were obtained commercially (Marinus, Long Beach, CA, USA). Animals were anesthetized by injection of isotonic MgCl2, and isolated ganglia were pinned and desheathed in dishes containing 1 : 1 MgCl2/artificial seawater (containing in mM: 460 NaCl, 55 MgCl2, 11 CaCl2 10 KCl, and 10 Tris, pH 7.6).

Protein quantification was performed using the BCA Protein Assay (Pierce, Rockford, IL, USA). Western blots were performed by electrophoresis using the Invitrogen (Carlsbad, CA, USA) NuPAGE gel system. Blotting was performed in 3% Blotto in Tris buffered saline plus Tween (TBST) for 1 h at 25°C. Blots were probed overnight in primary antibody (1 : 1000) in blocking solution, followed by 1 h at 25°C in horseradish peroxidase-conjugated secondary antibody at 1 : 2500. Blots were developed using the ECL western blotting analysis system by Amersham (Piscataway, NJ, USA).

ApSAP cloning

Multiple sets of degenerate primers were initially designed within PDZ, SH3, and GK domains based on alignments of PSD-95, SAP97, Chapsyn-110, and Drosophila Dlg. Primers were used to amplify cDNA obtained from Aplysia total RNA extracted from pleural and pedal ganglia. An initial product of the anticipated size was obtained by PCR from forward and reverse primer sequences GGITTYAAYATHGTNGGNGG and GCYTGCCACCAYTCNTCRTC, respectively. From here, Aplysia gene-specific primers were coupled with T3 or T7 vector primers to amplify cDNA from Aplysia SN and CNS libraries (kind gifts of Kelsey Martin and Wayne Sossin, respectively). Typically, PCR products were cloned into the Invitrogen pCR2.1 sequencing vector and submitted for DNA sequencing (Laguna Scientific, Laguna Beach, CA, USA). This approach was continued until the entire reading frame was completed. The full-length sequence of ApSAP was confirmed by multiple rounds of amplification and cDNA sequence analysis with overlapping primers. Determination of domains was predicted by sequence analysis at

Semi-quantitative RT-PCR

Pleural and pedal ganglia were dissected into Trizol for preparation of RNA. In all cases, reverse transcription was performed using 3.0 μg total RNA, and 1.0 μL cDNA was used in the PCR reaction. Cycle number was optimized for each primer set, to ensure amplification was kept within the linear range for comparison between control and experimental groups.

Antibody characterization

A panel of commercially available PSD-95 and PDZ family antibodies were tested for reactivity in Aplysia, including Novus#NB300-294 (clone 6G6-1C9; Littleton, CO, USA), Cell Signaling#2507 (Danvers, MA, USA), Upstate#05-494 and 05-427 (Billerica, MA, USA), and Chemicon#1598 (clone 7E3-1B8; Billerica, MA, USA) and #9634. Initial characterization of antibodies was performed by western blotting as described. For further characterization of 6G6-1C9 antibody reactivity, 10 ng of purified bacterial recombinant glutathione S-transferase (GST) or GST-ApSAP (full-length) was separated for western blot, as was 10 μg Aplysia CNS or rat extract. The 6G6-1C9 antibody was pre-adsorbed with either phosphate-buffered saline (PBS) or equimolar excess of purified recombinant GST or GST-ApSAP (full-length) in PBS. Pre-adsorption was performed in 3% Blotto with 1 : 1000 6G6-1C9 overnight and probed against 20 μg Aplysia CNS or rat brain extract.

Synaptosome preparation, tissue distribution, and immunocytochemistry

Synaptosomes were prepared by the two-step protocol as described by Chin et al. (1989). For western blots, 20 μg of P2 fractions were analyzed by western blot as described (Thompson et al. 2004). For ApSAP tissue distribution, homogenates were made from various tissues by sonication in phosphate buffered saline plus 1.0% triton X-100 (PBST) followed by centrifugation at 10 000 g for 10 min, and 20 μg extract was separated and probed with primary antibody.

For whole ganglia immunocytochemistry, intact Aplysia pleural-pedal ganglia were fixed with 4% formaldehyde at 4°C overnight. The ganglia were then desheathed and permeablized with 4% Triton X-100 in PBS/30% sucrose for 1 h. Free aldehydes were quenched with 1 M glycine for 15 min and non-specific binding sites were blocked with 10% normal goat serum for 1 h. The ganglia were incubated with 6G6-1C9 antibody for 1.5 days at 4°C in blocking solution at 1 : 100. AlexaFluor-647 conjugated anti-mouse secondary antibody was applied at 1 : 200 for 1 h and fluorescent signal was visualized using a Zeiss LSM510M Confocal Microscope (Thornwood, NY, USA) with Plan-Neofluor 20×/0.5NA lens. The images are presented as the mid-sagittal plane of the neurons.

Cloning and recombinant protein expression

The last 107 amino acids Aplysia NMDA-like receptor (AcNR1-1) were cloned into the EcoRI and NotI sites of the pGEX GST-fusion expression vector (Amersham). The C-terminal tails of AKv5.1 (Swiss-Prot K9H3M0, terminal 82 amino acids), AKv1.1 (Swiss-Prot P16388, terminal 78 amino acids), ApGluR1/4 (Swiss-Prot Q38JW0 and Q7Z1H6, terminal 67 amino acids), ApGluR2/3 (Swiss-Prot Q38JV9 and Q7Z1H7, terminal 73 amino acids), ApGluR7 (Swiss-Prot Q7Z1H3, terminal 64 amino acids), ApTrkl (Swiss-Prot Q51J68, terminal 93 amino acids), and rat NR2B (Swiss-Prot Q00960, terminal 94 amino acids) were cloned into the pENTR and pDEST15 N-terminal GST fusion vectors by the Gateway cloning system (Invitrogen). Full-length ApSAP was also cloned into the Gateway pENTR and pDEST vectors. The accuracy of all constructs was confirmed by DNA sequencing.

GST pull-down and overlay assays

Expression plasmids were transformed into BL21(Star)DE3 cell (Invitrogen) and expression was induced by the addition of 0.5 mM Isopropyl β-D-1-thiogalacto pyranosive (IPTG) at 32°C. Soluble protein was recovered by sonication of bacterial cell pellets in bacterial lysis buffer followed by centrifugation at 16 000 g for 5 min. Recombinant proteins were bound to glutathione–agarose (Pierce) by affinity chromatography, eluted with free glutathione, and dialyzed into PBS. Dialyzed recombinant proteins were concentrated by microcon concentrators (Millipore, Billerica, USA). Five microgram of recombinant protein was coupled to glutathione–agarose beads overnight, and unbound material was washed three times in PBST prior to the addition of Aplysia CNS extract.

Aplysia CNS extracts were prepared from pleural, pedal, and abdominal ganglia into PBS + 1.0% Triton X-100, 1 : 100 Sigma Mammalian Protease Inhibitor Cocktail (St. Louis, MO, USA), 3 mM Na o-vanadate, and 30 mM NaF. Ganglia from four to six animals were homogenized with a glass-teflon motor driven homogenizer into 1.0–1.2 mL PBST plus inhibitors, and centrifuged for 10 min at 10 000 g; 1.0 mg CNS extract was applied to each glutathione-coupled protein on a rotator for 4 h at 4°C, following which unbound material was removed by three washes of PBST plus inhibitors. Bound material was eluted by addition of sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer and boiling for 5 min, followed by analysis by western blot. Separate lanes containing 2 μL of each sample were run for western blot with an anti-GST antibody, to confirm equivalent recombinant proteins bound to glutathione–agarose.

Overlay assays were performed similarly, except that purified recombinant proteins were blotted directly onto nitrocellulose, blocked for 1 h at 25°C with 3% Blotto in TBST, then washed with ∼5 mg pleural, pedal, abdominal extract in PBST for 1 h at 25°C. Following three washes (10 min each) in TBST, the membrane was probed with 6G6-1C9 primary antibody as described for other western blots.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

ApSAP is a Dlg-MAGUK protein expressed in the CNS of Aplysia

In order to obtain an initial cDNA fragment from an Aplysia Dlg-MAGUK family member, multiple degenerate primers were designed based on an alignment of vertebrate and Drosophila Dlg-MAGUK proteins. RT-PCR was performed on RNA prepared from Aplysia pleural and pedal ganglia, and one primer set resulted in amplification of a product close to the expected size. In this case, the forward primer was located within one of the three conserved PDZ domains, whereas the reverse primer was designed from a sequence within the SH3 domain, thereby increasing the likelihood of a true Dlg-MAGUK target, rather than a PDZ domain-containing protein from another family member. The translated sequence of the amplified product provided 132 amino acids which was 69% identical to Drosophila Dlg and murine PSD-95, 77% identical to murine SAP97 and SAP102, and 79% identical to murine Chapsyn-110. Aplysia gene-specific primers were then designed within this sequence and used to amplify progressively larger portions of the gene, until the entire open reading frame was obtained (Fig. 1). Sequence analysis indicated that the protein contains an N-terminal L27 domain followed by three PDZ domains, an SH3 domain, and a GK domain, similarly to the Dlg-MAGUK family of proteins. The full-length protein is 863 amino acids and shares an overall sequence identity of 55%, 61%, 56%, 60%, and 55% with Drosophila Dlg and murine PSD-95/SAP90, SAP97, SAP102, and PSD-93/Chapsyn-110, respectively (Fig. 2). Moreover, identity between ApSAP and other family members within L27, PDZ, SH3, and GK domains is higher, between 61% and 82% (Fig. 2). Based on the domain similarity with vertebrate family members, we have designated this molecule as ApSAP.


Figure 1.  Alignment of ApSAP with other Dlg-MAGUK family members. ClustalW alignment of Aplysia ApSAP with Drosophila Dlg, and murine Chapsyn-110, PSD-95, SAP97, and SAP102. Identical amino acids are shaded in gray, and L27, PDZ, SH3, and GK domains are highlighted in yellow, purple, green, and blue, respectively. The L27-domain containing form of Drosophila Dlg (S97N) is shown. The nucleotide sequence for ApSAP is available in the Genbank database under accession number EU375312.

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Figure 2.  Amino acid identity between ApSAP and other Dlg-MAGUK family members. Percentage identity is indicated based on ClustalW alignments of full-length Dlg-MAGUK family members or individual domains. Overall sequence identity with ApSAP is indicated to the left of each protein, while identity within domains is indicated vertically for each domain. The exact spacing between domains varies in some vertebrate Dlg-MAGUK members, but in this illustration the domains are arranged in columns to assist comparison between family members.

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Characterization of ApSAP antibody reactivity

Because of the overall sequence similarity between ApSAP and mammalian MAGUK family members, commercially available antibodies raised against recombinant mammalian MAGUK proteins were tested for reactivity in Aplysia. Three antibodies, Novus Biologicals PSD-95 antibody (catalog #NB300-556, clone 6G6-1C9), Cell Signaling #2507, and Upstate PDZ family antibody (#05-427) recognized bands in the anticipated size range in Aplysia CNS extract (Fig. 3a), while Upstate #05-494 and Chemicon AB9634 and MAB 1598 did not (data not shown). Cell Signaling antibody #2507 detected two bands near and below rat PSD-95 (Fig. 3a, middle panel). However, the sequence of ApSAP contains 863 amino acids, compared with 724 amino acids in rat PSD-95. Thus, the protein detected by this antibody is likely not the same identified in our cloning screen. However, antibody 6G6-1C9 and Upstate antibody #05-427 recognized a protein of ∼110 kDa. The Upstate PDZ family antibody recognizes PSD-95, SAP97, and Chapsyn-110/PSD-93 in rat extracts. In comparison, this antibody recognizes the same major band at ∼110 kDa from Aplysia as seen with the 6G6-1C9 antibody, as well as two bands of lesser intensity at ∼95 and ∼70 kDa, raising the possibility that these might represent additional family members, splice variants, or proteolytic products. Interestingly, the addition of protease inhibitor N-ethylmaleimide (NEM) in homogenization buffer substantially reduces the presence of the ∼95 kDa band detected by the PDZ family antibody (Fig. 3a, right panel), suggesting this is likely a proteolytic product. Collectively these results indicate that the 110 kDa band recognized by 6G6-1C9 is a good candidate for ApSAP.


Figure 3.  Characterization of ApSAP-reactive antibodies. (a) To compare reactivity of commercially available antibodies, rat brain and Aplysia CNS extract were probed by western blot with antibodies 6G6-1C9 (left), Cell Signaling #2507 (middle), and Upstate PDZ family antibody (right). The position of PSD-95 in rat extracts is indicated by an asterisk. The ∼95 kDa band detected in Aplysia extracts by the PDZ family antibody is sensitive to the protease inhibitor NEM. (b) Aplysia CNS extract and purified recombinant GST or GST-ApSAP were probed with monoclonal 6G6-1C9 antibody. (c) 6G6-1C9 antibody was pre-adsorbed overnight with PBS or equimolar amounts of purified recombinant GST or GST-ApSAP, and used to detect ApSAP in rat or Aplysia extracts. Pre-adsorption sample is indicated above each gel panel.

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In order to more thoroughly confirm this possibility, we expressed and purified a recombinant GST fusion protein with full-length ApSAP. Either 10 ng GST or GST-ApSAP was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis for western blot, together with 10 μg of Aplysia CNS extract. Antibody 6G6-1C9 recognized the ∼130 kDa GST-ApSAP band, but not GST alone (Fig. 3b). Western blot of purified recombinant GST-ApSAP resulted in two separate bands of ∼130 and 100 kDa. These bands were similarly recognized by antibody 6G6-1C9, as well as an anti-GST antibody (data not shown). Thus, while the higher band matches the predicted size for the recombinant fusion protein, the smaller band likely represents a C-terminal truncation or other proteolytic product. Finally, antibody 6G6-1C9 was pre-adsorbed with either PBS, or equimolar amounts of GST or GST-ApSAP, and used in western blot against Aplysia CNS or rat brain extracts (Fig. 3c). While pre-incubation of the 6G6-1C9 antibody with either PBS or GST had no effect on immunoreactivity, pre-incubation with GST-ApSAP completely blocked signal from either rat brain or Aplysia CNS extract. Collectively, these results indicate that the band recognized by the 6G6-1C9 antibody is ApSAP.

These findings, together with the lack of evidence for additional Dlg-MAGUK by PCR screening, suggest that ApSAP is likely the predominant Dlg-MAGUK protein of the Aplysia CNS. Moreover, a BLAST search of the recently available Aplysia expressed sequence tag database (Moroz et al. 2006) using mouse PSD-95 revealed a sequence from a single Dlg-MAGUK gene matching approximately half of the ApSAP coding sequence. While other PDZ domain-containing and MAGUK proteins (e.g. p55 MAGUK proteins) are present in the database, no other members of the Dlg-MAGUK family are present. Nonetheless, given the splice variants identified in other Dlg-MAGUK proteins, including Drosophila, the possibility exists for additional ApSAP splice products. This possibility will provide an important avenue for future study.

Cellular and subcellular distribution of ApSAP

We next used the 6G6-1C9 antibody to investigate the tissue distribution of ApSAP by western blotting (Fig. 4a). As expected, a strong signal was observed from a CNS extract. There was also a very faint signal from heart, but no signal from buccal mass, muscle, kidney, gill, and ovotestis. RT-PCR analysis also indicated that ApSAP is primarily expressed within the CNS. Some message was detected in tissues outside the CNS (Fig. 4b), but results from western blot indicated that very little if any protein is found in these tissues. These results may be because of differences in the level of detection by RT-PCR and western blot, or perhaps because of untranslated message in tissue outside the CNS. Nonetheless, these results agree with the general conclusion that expression of ApSAP is heavily concentrated within the CNS relative to other tissues. Mammalian studies of Dlg-MAGUK expression indicate that PSD-95 is exclusively localized to the CNS, while SAP97 is predominately, but not exclusively localized to the nervous system (Cho et al. 1992; Muller et al. 1995). For analysis of subcellular localization, we prepared synaptosomes by the two-step method as previously described (Chin et al. 1989). ApSAP is enriched in the synaptosomal fraction as would be expected (Fig. 4c).


Figure 4.  Cellular and Subcellular distribution of ApSAP. (a) Tissue distribution as determined by western blotting. A total of 20 μg protein extract from different tissues was prepared and blotted with 6G6-1C9 antibody. The lower portion of the western blot was probed with anti-β-actin (Abcam, Cambridge, MA, USA) as loading control. (b) Relative message level was assessed by RT-PCR. cDNA was simultaneously amplified for ApSAP and Aplysia histone H4. (c) ApSAP is enriched in synaptosomes. VAMP positive control (Synaptic Systems, Goettingen, Germany) and ApSAP were blotted from crude homogenate and P2 synaptosome fraction of an individual preparation. (d) Immunocytochemical detection of ApSAP in Aplysia pleural and pedal ganglia. Controls missing primary antibody, or using mouse serum substituted for primary antibody, are shown on right. Pleural ganglion containing sensory neurons (SN) is shown at top, and pedal ganglion containing motor neurons (MN) is shown at bottom.

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To determine expression in SNs and MNs, immunocytochemistry was performed on pleural and pedal ganglia. We observed strong and diffuse signals in both cell types. Staining was present in both the cell body (absent nucleus) with a peri-membranous localization. Staining was also particularly abundant in neuropil, without apparent punctuate distribution (Fig. 4d). This finding is consistent with the diffuse, rather than punctuate, staining previously observed for some vertebrate MAGUKS (Cho et al. 1992; Kim et al. 1996; Sans et al. 2001; Schluter et al. 2006). Interestingly, results from immunohistochemistry of vertebrate MAGUKS suggest that the L27 domain-containing β-isoforms of PSD-95 and SAP97 (as found in ApSAP) demonstrate diffuse somato-dendritic staining, compared with the double cysteine α-isoforms, suggesting that the N-terminal sequence may influence subcellular distribution (Schluter et al. 2006).

Selective interaction between ApSAP and recombinant C-terminal tail of AKv1.1

The conserved domain organization of ApSAP with other Dlg-MAGUK proteins allows for comparison of functional interactions between receptors and intracellular binding partners. Such information can significantly inform evolutionary and functional questions regarding interactions between orthologous components of the vertebrate PSD which modulate receptor trafficking and plasticity. Despite the conserved domain structure and overall sequence similarity among the four vertebrate Dlg-MAGUK family members, many of these interactions are protein-specific and not common to all family members. For example, while all four vertebrate MAGUK proteins bind directly to various NMDA receptor subunits, SAP97 specifically binds to the GluR1 AMPA receptor subunit (Kornau et al. 1995; Kim et al. 1996; Lau et al. 1996; Muller et al. 1996; Niethammer et al. 1996; Leonard et al. 1998; Bassand et al. 1999; Sans et al. 2001; Wang et al. 2005).

A single NMDA receptor gene has been identified in Aplysia by Boulter et al. (NCBI accession code AY163562) and Ha et al. (2006), with one alternative splice variant (AcNR1-1 and AcNR1-2, respectively). One notable difference between Aplysia AcNR1-1 and mammalian NMDA receptors is found at the extreme C-terminus. The C-terminal tails of mammalian NR2 and some NR1 NMDA receptor splice variants (Hollmann et al. 1993) contain a Class I PDZ binding sequence (X-S/T-X-ϕ), while AcNR1-1 and AcNR1-2 do not. However, AcNR1-1 terminates with the sequence −NPDNVV, leaving open the possibility for a PDZ domain interaction.

Besides AcNR1-1, we sought also to identify possible interactions between ApSAP and other identified receptors and channels in Aplysia containing candidate PDZ binding sequences. Seven different ionotropic non-NMDA glutamate receptors have been provided to the NCBI database by Boulter et al. and Li et al. Three of these contain C-terminal Class I or II PDZ binding sequences (Table 1) and were thus used to test for an interaction with ApSAP. In addition, two identified Aplysia potassium channels, AKv1.1 and AKv5.1, also contain a C-terminal Class I PDZ-binding domain (Pfaffinger et al. 1991; Zhao et al. 1994). Lastly, the Aplysia receptor tyrosine kinase ApTrkl contains a PDZ binding domain at the C-terminus.

Table 1.   Comparison of PDZ interaction sequences in Aplysia and mouse proteins
NMDA receptorsC-terminal SequencePDZ interaction motif
  1. The extreme C-terminus of Aplysia NMDA-like receptor AcNR1-1 terminates in a hydrophobic residue, but does not contain a typical PDZ interaction sequence, in comparison with vertebrate NR2 and some NR1 splice variants. However, Aplysia Shaker-type potassium channel AKv1.1 contains a Class I PDZ binding sequence, as does AKv5.1 and some vertebrate potassium channels. Three identified Aplysia glutamate receptors also contain PDZ binding sequence, although none are perfectly conserved with murine AMPA/kainite glutamate receptors. ApTrk-l also contains a Class I PDZ-binding sequence. AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionate.

 Mouse NR2ESDVClass I
 Mouse NR 1–3 and 1–4STVVClass I
Potassium channels
 AKv1.1ETDVClass I
 AKv5.1ETVVClass I
 Mouse Kv1.1LTDVClass I
 Mouse Kv1.6LTEVClass I
 Mouse Kv1.4ETDVClass I
non-NMDA GluRs
 Mouse GluR1ATGLClass I
 Mouse GluR2SVKIClass II
 Mouse GluR3SVKIClass II
 Mouse GluR4SDLP 
 Mouse GluR6ETVAClass I
 Mouse GluR6ETMAClass I
 ApGluR1/4QTLVClass I
 ApGluR2/3HTEVClass I
 ApGluR7EVYAClass II
Receptor tyrosine kinase
 ApTrk1HSLLClass I

In order to test for the interactions between these receptors/channels and ApSAP, bacterial expression constructs were made in which C-terminal tails of AcNR1-1, AKv1.1, AKv5.1, ApGluR1/4, ApGluR2/3, ApGluR7, and ApTrkl were expressed as fusion proteins with GST and used in GST pull-down assays. Soluble bacterial protein was purified over glutathione–agarose, dialyzed into PBS, and equal microgram amounts were coupled to glutathione–agarose. Aplysia CNS extract was prepared and added to recombinant proteins. Equal amounts of bound material were loaded from each pull-down sample and probed for ApSAP with the 6G6-1C9 antibody. The results indicate that ApSAP was specifically co-precipitated with AKv1.1, but not with AcNR1-1, AKv5.1, ApTrkl, ApGluR1/4, ApGluR2/3, or ApGluR7 (Fig. 5). In some experiments, a faint ApSAP signal was detected following GST pull-down using GST-ApGluR2/3 as bait (Fig. 5a), indicating a possible low-affinity in vitro interaction of unlikely physiological relevance. Ha et al. (2006) have pointed out that AcNR1-1 amino acids 893–896 contain a Class I PDZ binding domain which may serve as a putative interaction site. However, this region is included in the GST-AcNR1-1 construct we expressed, suggesting a lack of interaction between ApSAP and AcNR1-1, and therefore a lack of homologous NMDA receptor complex with AcNR1-1 in Aplysia. Moreover, we generated a GST fusion protein with the C-terminal tail of rat NR2B, and found a robust interaction with ApSAP (Fig. 5b), indicating the ability of ApSAP to form MAGUK-NMDA receptor interactions as observed in mammalian systems.


Figure 5.  ApSAP selectively binds to AKv1.1 and NR2B in GST pull-down assays. (a) Purified recombinant GST fusion proteins (indicated at bottom) were used as bait with Aplysia CNS extract, and ApSAP binding was assessed by western blot using the 6G6-1C9 antibody. Eluted proteins from the same samples were also probed with anti-GST to confirm the presence of recombinant proteins. The size of fusion proteins ranged between ∼26 kDa (GST alone) and ∼40 kDa (GST-AcNR1-1). Proteolysis of recombinant proteins was noticeable; however, the largest band in each individual lane corresponds to the predicted intact size for that particular recombinant protein. (b) While GST pull-down with Aplysia NMDA-like receptor AcNR1-1 did not yield an interaction with ApSAP from CNS extract, an interaction was observed between ApSAP and the C-terminal region of rat NR2B. (c) Mutation of AKv1.1 C-terminal amino acid disrupts interaction with ApSAP. Substitution of alanine for the extreme C-terminal valine in AKv1.1 completely abolished interaction with ApSAP. Detection of recombinant proteins in eluted samples is indicated with GST antibody, shown at bottom.

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The selective interaction between ApSAP and the Shaker-type potassium channel AKv1.1 was particularly interesting, given the demonstrated interaction between vertebrate Shaker channels and Dlg-MAGUK proteins, as well as the findings that localization of Drosophila Shaker channels at the neuromuscular junction and within the CNS is regulated by an interaction with Dlg (Kim et al. 1995, 1996; Tejedor et al. 1997; Zito et al. 1997; Ruiz-Canada et al. 2002). Because the extreme C-terminal amino acid has been repeatedly demonstrated to be critical for PDZ interactions (Kim et al. 1995; Kornau et al. 1995; Niethammer et al. 1996; Songyang et al. 1997), we generated an AKv1.1 GST fusion protein in which alanine was substituted for the C-terminal valine. Results shown in Fig. 5c indicate, as predicted, that the interaction between AKv1.1 and ApSAP is completely abolished following this individual amino acid substitution, confirming the necessity of the C-terminal valine in this interaction.

Because of the susceptibility of C-terminal constructs to proteolytic degradation, we performed a complimentary series of overlay experiments. In this case, purified recombinant proteins were blotted onto a nitrocellulose membrane and probed with an Aplysia CNS extract. Following washes, binding was assessed by western blot for ApSAP. As observed for the pull-downs, a robust interaction was observed by overlay between ApSAP and AKv1.1, as well as rat NR2B, but not with other proteins tested (Fig. 6). Moreover, as in the pull-down experiments, a faint signal was detected between ApSAP and ApGluR2, although far below that observed for either AKv1.1 or NR2B.


Figure 6.  ApSAP selectively recognizes AKv1.1 and NR2B in overlay assay. (a) ApSAP signal from 6G6-1C9 antibody following overlay indicates a strong interaction with AKv1.1 and rat NR2B. (b) Membrane was stripped and reprobed for GST to ensure equal loading. Because of strong GST signal, a separate gel with 2 μL per sample was also separately run and probed for GST, shown here. Recombinant proteins separated in each lane are indicated at bottom.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dlg-MAGUK proteins represent major components of the PSD and are important mediators of protein complex formation in the mammalian CNS. However, the high degree of structural similarity between MAGUK proteins has complicated the ability to make specific functional attributions to unique Dlg-MAGUK proteins and affiliated binding partners. Thus, interpretation of results from genetic deletion of any one or two proteins within a family must include possible compensatory mechanisms by other related molecules (Migaud et al. 1998; McGee et al. 2001; Tao et al. 2003; Yao et al. 2004; Beique et al. 2006; Elias et al. 2006; Vickers et al. 2006; Cuthbert et al. 2007). Because of this, a clear understanding of how individual Dlg-MAGUK proteins, as integral components of a protein network, contribute to synaptic function and plasticity remains elusive. In contrast, the relative simplicity of Aplysia, coupled to the ability to directly address structure–function questions at the single cell level, provides a unique opportunity to more deeply understand the activity of MAGUK proteins within the PSD.

While the vertebrate excitatory PSD is pronounced and readily identified by electron microscopy, initial efforts to identify such a region in Aplysia yielded negative results; some early studies reported the absence of an observable post-synaptic specialization (Graubard 1978; Tremblay et al. 1979). However, Bailey et al. (1981) used a modified tissue preparation and demonstrated a post-synaptic membrane specialization, smaller and less pronounced than vertebrate type 1 PSDs, but nonetheless apparent and apposed to the pre-synaptic active zone (Bailey et al. 1981). An electron dense region of complex membrane folding, the subsynaptic reticulum, is also observed on the post-synaptic side of type Ib glutamatergic neuromuscular synapses in Drosophila (Atwood et al. 1993; Lahey et al. 1994). Thus, excitatory synapses in both Drosophila and Aplysia possess electron dense regions of post-synaptic specialization comparable to, although less pronounced than, the vertebrate PSD, and perhaps represent an evolutionary precursor to the mammalian PSD.

It is reasonable to speculate that the reduced complexity of post-synaptic specialization in Drosophila and Aplysia may also reflect reduced complexity at the molecular level. Consequently, invertebrate models such as these can provide simplified systems in which to study the evolution and function of orthologous components of the vertebrate PSD, and consequently represent a unique opportunity to investigate contributions of specific proteins and their affiliated interactions. Aplysia neurons are quite large, making intracellular recording and injection of peptides, proteins, oligonucleotides, and drugs quite straightforward. Thus, perturbation of protein interactions can be used to identify functional consequences at the single cell level. However, information regarding the structure, expression, and interactions of PSD components in Aplysia is required before any functional analysis can be initiated. We have therefore begun a characterization of Dlg-MAGUK proteins in Aplysia, in order to use this system as a model for understanding protein networks in synaptic function.

Amplification of Aplysia cDNA using paired degenerate primers from vertebrate PDZ and SH3 domains led to the identification of a single member of the Dlg-MAGUK family in Aplysia, ApSAP, which is recognized by commercially available antibodies. ApSAP shares some specific common features with several other Dlg-MAGUK proteins. For example, in addition to the canonical Dlg-MAGUK PDZ, SH3, and GK domains, ApSAP also contains an amino-terminal L27 domain. L27 domains are so named based on their identification in Caenorhabditis  elegans LIN-2 and LIN-7 proteins, and have been identified as regions of heteromultimerization between a number of different L27 domain-containing proteins (Nakagawa et al. 2004a; Petrosky et al. 2005; Straight et al. 2006). L27 domains are particularly found within proteins important for organization of cell polarity and assembly of scaffolds at multicellular junctions (Budnik 1996). Within the Dlg-MAGUK family of proteins, this domain is also observed in mammalian SAP97 and a low abundance splice variant of PSD-95 (PSD-95β), as well as at the N-terminus of Drosophila Dlg splice variant, S97N, a splice variant critical for neuronal development (Mendoza et al. 2003). ApSAP also contains a Hook domain, a protein interaction domain found in mammalian SAP97, required for interaction with calmodulin (Paarmann et al. 2002). Particularly in the case of ApSAP, the possibility exists for splice variants not yet identified, such as in the case of N-terminal α- and β-isoforms of PSD-95 and SAP97 (Chetkovich et al. 2002; Sierralta and Mendoza 2004; Schluter et al. 2006).

Similarly to the exclusive CNS distribution previously identified for mammalian PSD-95 and CNS-enriched expression of SAP97 (Cho et al. 1992; Kistner et al. 1993; Muller et al. 1995; Aoki et al. 2001), ApSAP is primarily expressed within the CNS. At the cellular level, ApSAP is expressed in both SN and MNs, consistent with pre-synaptic and post-synaptic localization, similar to that described for Drosophila Dlg and some vertebrate Dlg-MAGUKs (Lahey et al. 1994; Koulen et al. 1998; Aoki et al. 2001). Moreover, while some vertebrate MAGUK proteins demonstrate punctuate and synapse-enriched localization, a diffuse staining pattern has been reported for vertebrate SAP97.

Results from GST pull-down and overlay experiments demonstrate that ApSAP binds to Aplysia Shaker-type channel AKv1.1, but not to NMDA-like receptor AcNR1-1, or other candidate receptors and channels. The lack of ApSAP binding to the Aplysia NMDA-like receptor appears to be because of the binding properties of the receptor, as ApSAP readily binds to the mammalian NR2B receptor. A comparison of C-terminal 4 amino acids between Aplysia and murine receptors and channels in shown in Table 1. While no sequences are absolutely conserved, the presence of Class I and II PDZ binding sequences in a number of Aplysia proteins (minus AcNR1-1) is apparent. Thus, despite the presence of C-terminal PDZ binding domains in these proteins, ApSAP appears to selectively interact with the Shaker potassium channel, and further studies are warranted to identify other PDZ domain-containing proteins which may assemble at these sites. Notably, fragments of numerous other PDZ domain containing proteins can be found within the Aplysia EST database, leaving open the possibility for glutamate receptor interactions with these proteins.

Our results now allow for exploration of the functional significance of the specific interaction of ApSAP with AKv1.1. Several questions can now be addressed: does modulation of this interaction have consequence for channel trafficking, synaptic clustering, channel kinetics, or cellular excitability? Does the phosphorylation state of ApSAP or its binding partners affect any of these properties? Can antibodies directed against different MAGUK protein interaction domains be used to identify other binding partners? Finally, how does a change in phosphorylation state in response to a stimulus influence these interactions and their cellular function? Investigation of these and related questions can help to further elucidate the significance of ApSAP-dependent interactions.

The precedent for involvement of PSD proteins in synaptic function, plasticity, and memory is well established in both invertebrates and vertebrates. Drosophila Dlg is required not only for structural integrity of the subsynaptic reticulum, but also for clustering of K+ channels, synaptic function, and structural plasticity of the neuromuscular junction in association with post-synaptic muscle growth (Lahey et al. 1994; Budnik et al. 1996; Guan et al. 1996; Ruiz-Canada et al. 2002). Moreover, numerous studies of the mammalian PSD indicate the requirement of affiliated components in synaptic function and plasticity (for review, see Funke et al. 2005). Thus, identification of an orthologous family member in Aplysia and its associated interactions now allows for the use of Aplysia as a well-characterized model system to further elucidate how these ubiquitous proteins contribute to cellular mechanisms of synaptic plasticity and memory.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by NIH Grant R01 MH14-10183 to TJC. We thank members of the Carew lab and Lisa Lyons for helpful scientific discussions, and Jim Boulter for consultation regarding Aplysia glutamate receptors. We also thank Kelsey Martin and Wayne Sossin for use of SN and CNS Aplysia libraries.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Antzoulatos E. G. and Byrne J. H. (2004) Learning insights transmitted by glutamate. Trends Neurosci. 27, 555560.
  • Aoki C., Miko I., Oviedo H., Mikeladze-Dvali T., Alexandre L., Sweeney N. and Bredt D. S. (2001) Electron microscopic immunocytochemical detection of PSD-95, PSD-93, SAP-102, and SAP-97 at postsynaptic, presynaptic, and nonsynaptic sites of adult and neonatal rat visual cortex. Synapse 40, 239257.
  • Armstrong J. D., Pocklington A. J., Cumiskey M. A. and Grant S. G. (2006) Reconstructing protein complexes: from proteomics to systems biology. Proteomics 6, 47244731.
  • Atwood H. L., Govind C. K. and Wu C. F. (1993) Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J. Neurobiol. 24, 10081024.
  • Bailey C. H., Thompson E. B., Castellucci V. F. and Kandel E. R. (1979) Ultrastructure of the synapses of sensory neurons that mediate the gill-withdrawal reflex in Aplysia. J. Neurocytol. 8, 415444.
  • Bailey C. H., Kandel P. and Chen M. (1981) Active zone at Aplysia synapses: organization of presynaptic dense projections. J. Neurophysiol. 46, 356368.
  • Bassand P., Bernard A., Rafiki A., Gayet D. and Khrestchatisky M. (1999) Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97. Eur. J. Neurosci. 11, 20312043.
  • Beique J. C., Lin D. T., Kang M. G., Aizawa H., Takamiya K. and Huganir R. L. (2006) Synapse-specific regulation of AMPA receptor function by PSD-95. Proc. Natl Acad. Sci. USA 103, 1953519540.
  • Budnik V. (1996) Synapse maturation and structural plasticity at Drosophila neuromuscular junctions. Curr. Opin. Neurobiol. 6, 858867.
  • Budnik V., Koh Y. H., Guan B., Hartmann B., Hough C., Woods D. and Gorczyca M. (1996) Regulation of synapse structure and function by the Drosophila tumor suppressor gene Dlg. Neuron 17, 627640.
  • Chen X., Vinade L., Leapman R. D., Petersen J. D., Nakagawa T., Phillips T. M., Sheng M. and Reese T. S. (2005) Mass of the postsynaptic density and enumeration of three key molecules. Proc. Natl Acad. Sci. USA 102, 1155111556.
  • Cheng D., Hoogenraad C. C., Rush J. et al. (2006) Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum. Mol. Cell Proteomics 5, 11581170.
  • Chetkovich D. M., Bunn R. C., Kuo S. H., Kawasaki Y., Kohwi M. and Bredt D. S. (2002) Postsynaptic targeting of alternative postsynaptic density-95 isoforms by distinct mechanisms. J. Neurosci. 22, 64156425.
  • Chin G. J., Shapiro E., Vogel S. S. and Schwartz J. H. (1989) Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions. J. Neurosci. 9, 3848.
  • Cho K. O., Hunt C. A. and Kennedy M. B. (1992) The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9, 929942.
  • Collins M. O., Husi H., Yu L., Brandon J. M., Anderson C. N., Blackstock W. P., Choudhary J. S. and Grant S. G. (2006) Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome. J. Neurochem. 97 (Suppl. 1), 1623.
  • Cuthbert P. C., Stanford L. E., Coba M. P., Ainge J. A., Fink A. E., Opazo P., Delgado J. Y., Komiyama N. H., O’Dell T. J. and Grant S. G. (2007) Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies. J. Neurosci. 27, 26732682.
  • Ehrlich I. and Malinow R. (2004) Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. J. Neurosci. 24, 916927.
  • El-Husseini A. E., Schnell E., Chetkovich D. M., Nicoll R. A. and Bredt D. S. (2000) PSD-95 involvement in maturation of excitatory synapses. Science 290, 13641368.
  • Elias G. M., Funke L., Stein V., Grant S. G., Bredt D. S. and Nicoll R. A. (2006) Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins. Neuron 52, 307320.
  • Fujita A. and Kurachi Y. (2000) SAP family proteins. Biochem. Biophys. Res. Commun. 269, 16.
  • Funke L., Dakoji S. and Bredt D. S. (2005) Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu. Rev. Biochem. 74, 219245.
  • Graubard K. (1978) Serial synapses in Aplysia. J. Neurobiol. 9, 325328.
  • Guan B., Hartmann B., Kho Y. H., Gorczyca M. and Budnik V. (1996) The Drosophila tumor suppressor gene, Dlg, is involved in structural plasticity at a glutamatergic synapse. Curr. Biol. 6, 695706.
  • Ha T. J., Kohn A. B., Bobkova Y. V. and Moroz L. L. (2006) Molecular characterization of NMDA-like receptors in Aplysia and Lymnaea: relevance to memory mechanisms. Biol. Bull. 210, 255270.
  • Hawkins R. D., Kandel E. R. and Bailey C. H. (2006) Molecular mechanisms of memory storage in Aplysia. Biol. Bull. 210, 174191.
  • Hollmann M., Boulter J., Maron C., Beasley L., Sullivan J., Pecht G. and Heinemann S. (1993) Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron 10, 943954.
  • Jordan B. A., Fernholz B. D., Boussac M., Xu C., Grigorean G., Ziff E. B. and Neubert T. A. (2004) Identification and verification of novel rodent postsynaptic density proteins. Mol. Cell Proteomics 3, 857871.
  • Kennedy M. B. (2000) Signal-processing machines at the postsynaptic density. Science 290, 750754.
  • Kim E. and Sheng M. (2004) PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771781.
  • Kim E., Niethammer M., Rothschild A., Jan Y. N. and Sheng M. (1995) Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378, 8588.
  • Kim E., Cho K. O., Rothschild A. and Sheng M. (1996) Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins. Neuron 17, 103113.
  • Kistner U., Wenzel B. M., Veh R. W., Cases-Langhoff C., Garner A. M., Appeltauer U., Voss B., Gundelfinger E. D. and Garner C. C. (1993) SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene Dlg-A. J. Biol. Chem. 268, 45804583.
  • Koh Y. H., Popova E., Thomas U., Griffith L. C. and Budnik V. (1999) Regulation of DLG localization at synapses by CaMKII-dependent phosphorylation. Cell 98, 353363.
  • Kornau H. C., Schenker L. T., Kennedy M. B. and Seeburg P. H. (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 17371740.
  • Koulen P., Fletcher E. L., Craven S. E., Bredt D. S. and Wassle H. (1998) Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina. J. Neurosci. 18, 1013610149.
  • Lahey T., Gorczyca M., Jia X. X. and Budnik V. (1994) The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure. Neuron 13, 823835.
  • Lau L. F., Mammen A., Ehlers M. D., Kindler S., Chung W. J., Garner C. C. and Huganir R. L. (1996) Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102. J. Biol. Chem. 271, 2162221628.
  • Leonard A. S., Davare M. A., Horne M. C., Garner C. C. and Hell J. W. (1998) SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit. J. Biol. Chem. 273, 1951819524.
  • McGee A. W., Topinka J. R., Hashimoto K., Petralia R. S., Kakizawa S., Kauer F. W., Aguilera-Moreno A., Wenthold R. J., Kano M. and Bredt D. S. (2001) PSD-93 knock-out mice reveal that neuronal MAGUKs are not required for development or function of parallel fiber synapses in cerebellum. J. Neurosci. 21, 30853091.
  • Mendoza C., Olguin P., Lafferte G., Thomas U., Ebitsch S., Gundelfinger E. D., Kukuljan M. and Sierralta J. (2003) Novel isoforms of Dlg are fundamental for neuronal development in Drosophila. J. Neurosci. 23, 20932101.
  • Migaud M., Charlesworth P., Dempster M. et al. (1998) Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433439.
  • Moroz L. L., Edwards J. R., Puthanveettil S. V. et al. (2006) Neuronal transcriptome of Aplysia: neuronal compartments and circuitry. Cell 127, 14531467.
  • Muller B. M., Kistner U., Veh R. W., Cases-Langhoff C., Becker B., Gundelfinger E. D. and Garner C. C. (1995) Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein. J. Neurosci. 15, 23542366.
  • Muller B. M., Kistner U., Kindler S. et al. (1996) SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo. Neuron 17, 255265.
  • Nakagawa T., Futai K., Lashuel H. A., Lo I., Okamoto K., Walz T., Hayashi Y. and Sheng M. (2004a) Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions. Neuron 44, 453467.
  • Niethammer M., Kim E. and Sheng M. (1996) Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J. Neurosci. 16, 21572163.
  • Ormond J., Hislop J., Zhao Y., Webb N., Vaillaincourt F., Dyer J. R., Ferraro G., Barker P., Martin K. C. and Sossin W. S. (2004) ApTrkl, a Trk-like receptor, mediates serotonin-dependent ERK activation and long-term facilitation in Aplysia sensory neurons. Neuron 44, 715728.
  • Paarmann I., Spangenberg O., Lavie A. and Konrad M. (2002) Formation of complexes between Ca2+ calmodulin and the synapse-associated protein SAP97 requires the SH3 domain-guanylate kinase domain-connecting HOOK region. J. Biol. Chem. 277, 4083240838.
  • Peng J., Kim M. J., Cheng D., Duong D. M., Gygi S. P. and Sheng M. (2004) Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry. J. Biol. Chem. 279, 2100321011.
  • Petrosky K. Y., Ou H. D., Lohr F., Dotsch V. and Lim W. A. (2005) A general model for preferential hetero-oligomerization of LIN-2/7 domains: mechanism underlying directed assembly of supramolecular signaling complexes. J. Biol. Chem. 280, 3852838536.
  • Pfaffinger P. J., Furukawa Y., Zhao B., Dugan D. and Kandel E. R. (1991) Cloning and expression of an Aplysia K+ channel and comparison with native Aplysia K+ currents. J. Neurosci. 11, 918927.
  • Reissner K. J., Shobe J. L. and Carew T. J. (2006) Molecular nodes in memory processing: insights from Aplysia. Cell Mol. Life Sci. 63, 963974.
  • Ruiz-Canada C., Koh Y. H., Budnik V. and Tejedor F. J. (2002) DLG differentially localizes Shaker K+-channels in the central nervous system and retina of Drosophila. J. Neurochem. 82, 14901501.
  • Rumbaugh G., Sia G. M., Garner C. C. and Huganir R. L. (2003) Synapse-associated protein-97 isoform-specific regulation of surface AMPA receptors and synaptic function in cultured neurons. J. Neurosci. 23, 45674576.
  • Sans N., Racca C., Petralia R. S., Wang Y. X., McCallum J. and Wenthold R. J. (2001) Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway. J. Neurosci. 21, 75067516.
  • Schluter O. M., Xu W. and Malenka R. C. (2006) Alternative N-terminal domains of PSD-95 and SAP97 govern activity-dependent regulation of synaptic AMPA receptor function. Neuron 51, 99111.
  • Schnell E., Sizemore M., Karimzadegan S., Chen L., Bredt D. S. and Nicoll R. A. (2002) Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc. Natl Acad. Sci. USA 99, 1390213907.
  • Sheng M. and Hoogenraad C. C. (2006) The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu. Rev. Biochem. 76, 823847.
  • Sheng M. and Kim M. J. (2002) Postsynaptic signaling and plasticity mechanisms. Science 298, 776780.
  • Sierralta J. and Mendoza C. (2004) PDZ-containing proteins: alternative splicing as a source of functional diversity. Brain Res. Brain Res. Rev. 47, 105115.
  • Songyang Z., Fanning A. S., Fu C., Xu J., Marfatia S. M., Chishti A. H., Crompton A., Chan A. C., Anderson J. M. and Cantley L. C. (1997) Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275, 7377.
  • Stein V., House D. R., Bredt D. S. and Nicoll R. A. (2003) Postsynaptic density-95 mimics and occludes hippocampal long-term potentiation and enhances long-term depression. J. Neurosci. 23, 55035506.
  • Straight S. W., Pieczynski J. N., Whiteman E. L., Liu C. J. and Margolis B. (2006) Mammalian lin-7 stabilizes polarity protein complexes. J. Biol. Chem. 281, 3773837747.
  • Tao Y. X., Rumbaugh G., Wang G. D. et al. (2003) Impaired NMDA receptor-mediated postsynaptic function and blunted NMDA receptor-dependent persistent pain in mice lacking postsynaptic density-93 protein. J. Neurosci. 23, 67036712.
  • Tejedor F. J., Bokhari A., Rogero O., Gorczyca M., Zhang J., Kim E., Sheng M. and Budnik V. (1997) Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo. J. Neurosci. 17, 152159.
  • Thompson K. R., Otis K. O., Chen D. Y., Zhao Y., O’Dell T. J. and Martin K. C. (2004) Synapse to nucleus signaling during long-term synaptic plasticity; a role for the classical active nuclear import pathway. Neuron 44, 9971009.
  • Tremblay J. P., Colonnier M. and McLennan H. (1979) An electron microscope study of synaptic contacts in the abdominal ganglion of Aplysia californica. J. Comp. Neurol. 188, 367389.
  • Vickers C. A., Stephens B., Bowen J., Arbuthnott G. W., Grant S. G. and Ingham C. A. (2006) Neurone specific regulation of dendritic spines in vivo by post synaptic density 95 protein (PSD-95). Brain Res. 1090, 8998.
  • Wang L., Piserchio A. and Mierke D. F. (2005) Structural characterization of the intermolecular interactions of synapse-associated protein-97 with the NR2B subunit of N-methyl-D-aspartate receptors. J. Biol. Chem. 280, 2699226996.
  • Woods D. F. and Bryant P. J. (1991) The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451464.
  • Yao W. D., Gainetdinov R. R., Arbuckle M. I., Sotnikova T. D., Cyr M., Beaulieu J. M., Torres G. E., Grant S. G. and Caron M. G. (2004) Identification of PSD-95 as a regulator of dopamine-mediated synaptic and behavioral plasticity. Neuron 41, 625638.
  • Yoshimura Y., Yamauchi Y., Shinkawa T., Taoka M., Donai H., Takahashi N., Isobe T. and Yamauchi T. (2004) Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry. J. Neurochem. 88, 759768.
  • Zhao B., Rassendren F., Kaang B. K., Furukawa Y., Kubo T. and Kandel E. R. (1994) A new class of noninactivating K+ channels from Aplysia capable of contributing to the resting potential and firing patterns of neurons. Neuron 13, 12051213.
  • Ziff E. B. (1997) Enlightening the postsynaptic density. Neuron 19, 11631174.
  • Zito K., Fetter R. D., Goodman C. S. and Isacoff E. Y. (1997) Synaptic clustering of Fascilin II and Shaker: essential targeting sequences and role of Dlg. Neuron 19, 10071016.