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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.
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.
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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.