calmodulin-dependent serine protein kinase (CASK) and membrane-associated guanylate kinase inverted (MAGI) subfamilies4–6. MAGUK scaffolds are crucial in the development and plasticity of synapses7. Mutations in the genes encoding MAGUKs

| Membrane-associated guanylate kinases (MAGUKs) are a family of scaffold proteins that are highly enriched in synapses and are responsible for organizing the numerous protein complexes required for synaptic development and plasticity. Mutations in genes encoding MAGUKs and their interacting proteins can cause a broad spectrum of human psychiatric disorders. Here, we review MAGUK-mediated synaptic protein complex formation and regulation by focusing on findings from recent biochemical and structural investigations. These mechanistic-based studies show that the formation of MAGUK-organized complexes is often directly regulated by protein phosphorylation, suggesting a close connection between neuronal activity and the assembly of dynamic protein complexes in synapses. NATURE REVIEWS | NEUROSCIENCE VOLUME 17 | APRIL 2016 | 209 REVIEWS © 2016 Macmillan Publishers Limited. All rights reserved. scaffold proteins, cytoskeleton proteins, membrane trafficking proteins and molecular motors, as well as protein synthesis and degradation machineries4,12,13,17 (FIG. 1). EM studies using gold-particle-conjugated antibodies to detect specific PSD proteins14,15 and more recent super-resolution light microscopic imaging studies18–23 have shown that the proteins in PSDs form distinct layers along the axo-dendritic axis of synapses with a sequential order of membrane-spanning glutamate receptors and cell adhesion molecules, MAGUKs, synapseassociated protein 90 (SAP90)/postsynaptic density protein 95 (PSD95)-associated proteins (SAPAPs) and SRC homology 3 (SH3) and multiple ankyrin repeat domains protein (SHANK) scaffolds, and the actin cytoskeleton contacting the interior face of PSDs (FIG. 1). High-resolution EM tomographic investigations have shown that the main PSD scaffold PSD95 (also known as DLG4) is arranged in a pillar-shaped pattern that lies perpendicular to the PSD membrane, with the N terminus palmitoyl group of PSD95 contacting the membrane and its C-terminal guanylate kinase (GK) domain facing the inside of the dendritic spine15 (FIG. 1). Given this arrangement and its extremely high abundance in PSDs (studies have calculated that the concentration of PSD95 at PSDs is ~100 μM, based on the average size of PSDs and the average number of PSD95 mol ecules in each PSD16,24), PSD95 can be viewed as the most crucial organizer of PSD structure and is able to interface with upstream membrane-spanning glutamate receptors and the downstream SAPAP–SHANK complexes, and to shape the architecture of PSDs. The architecture of PSDs is highly dynamic, both during development and in mature neurons25–27. In response to diverse neuronal stimuli, PSD proteins undergo assembly and disassembly, clustering and diffusion, and membrane insertion and removal processes, which are tightly associated with various forms of synaptic plasticity (for example, long-term potentiation (LTP) and long-term depression)25–29. Various super-resolution optical imaging studies of both fixed and living neurons have offered extraordinary rich information on the structure and dynamics of PSD at a resolution of ~25 nm and have revealed several new observations and concepts17,20–23,30,31. First, major scaffold proteins, such as PSD95 and SAPAPs, and glutamate receptors form co-clustered nanodomains that are ~50–80 nm in diameter; each PSD typically contains a few such nano domains. As such, the receptors and scaffold proteins are further enriched in these nanodomains within the already enriched PSD assembly. Second, the juxtaposition of clustered receptors (for example, AMPA-type glutamate receptors (AMPARs)) and presynaptic neuro transmitter-releasing sites positively correlates with the postsynaptic response activity (FIG. 1). Third, even though they are generally less mobile than their counter parts located outside nanodomains, both receptors and scaffolds in nanodomains are still highly dynamic under basal conditions and changes in neuronal activity can further modify their clustering and dynamic properties. Last, increasing or decreasing the levels of scaffold proteins, such as PSD95, in synapses can cause the enlargement or shrinkage of AMPAR nanodomains, respectively. Thus, although the overall morphology of a PSD in a given synapse can be relatively stable within minutes after stimulation, the molecular components and the internal subcompartmental organization within this structure can change rapidly. MAGUKs and their binding partners MAGUK subfamilies. MAGUKs can be classified into several different subfamilies: the DLGs, CASK, the palmitoylated membrane proteins (MPPs), the Zonula occludens (ZO) proteins, the caspase activation and recruitment domain (CARD)-containing MAGUK proteins (CARMAs) and the MAGI subfamily proteins32 (FIG. 2a). The DLG subfamily consists of SAP97 (also known as DLG1), PSD93 (also known as DLG2), SAP102 (also known as DLG3) and PSD95. These proteins are crucial in the dynamic regulation of glutamatergic synaptic signalling and apical–basal cell polarity1,33–35. CASK is found at both the preand postsynaptic sides of excitatory synapses; it modulates synaptic vesicle trafficking and neurotransmitter release as well as post synaptic signalling36,37. The MAGI subfamily proteins are broadly expressed in various tissues and cells, such as the brain (for example, MAGI2 is expressed at inhibitory synapses) and various epithelia, and they are known to have crucial roles in the proper development and function of tissues6,38. The MPP subfamily has an important role in establishing and maintaining cell polarity in many cell populations, including neurons39,40. The functions of the ZO and CARMA subfamilies of MAGUKs are better known in tissues other than brain and will not be covered in this Review. Not surprisingly, mutations in genes encoding the MAGUK family proteins are linked to human diseases, including many forms of cancer and neurological disorders3,8,41. Domain organization of MAGUKs. By definition, every member of the MAGUK family contains a GK-like domain (FIG. 2a). Except for the MAGI subfamily members, the rest of MAGUKs invariably contain a PDZ–SH3–GK tandem (PSG) in their C-terminal end (FIG. 2a). In addition to this common domain architecture, some MAGUKs contain LIN2–LIN7 (L27), CARD, CaM kinase-like, WW and further PDZ domains (FIG. 2a). These domains are all known to function as protein–protein interaction modules42–45 and thus MAGUKs are ideally suited as scaffold proteins for assembling large signalling complexes. Except for the CaM kinase-like domain in CASK, which can function both as a specific protein–protein interaction module45 and as a protein kinase specifically phosphorylating neurexins46, none of the other domains found in MAGUKs has catalytic activities. Targets on PSD membranes. Many synaptic transmembrane proteins — including glutamate receptors (both NMDA-type glutamate receptors (NMDARs) and AMPARs), ion channels and synaptic adhesion mol ecules — contain conserved PDZ-binding motifs (PBMs) at their C-terminal cytoplasmic tails (FIG. 1). Interactions between these PBMs and the PDZ domains of MAGUKs are known to be crucial in the trafficking, clustering, targeting or R E V I E W S 210 | APRIL 2016 | VOLUME 17 www.nature.com/nrn © 2016 Macmillan Publishers Limited. All rights reserved. © 2016 Macmillan Publishers Limited. All rights reserved. Figure 1 | Scaffold protein-mediated protein complex organizations in excitatory synapses. Scaffold proteins, in particular membrane-associated guanylate kinases (MAGUKs), have central roles in interfacing synaptic membrane receptors or channels with signalling enzymes and cytoskeletal proteins deep in the postsynaptic density (PSD). The domain structures of Discs large homologue (DLG) MAGUKs and members of two of their interacting postsynaptic scaffold proteins, synapse-associated protein 90 (SAP90)/PSD95-associated protein (SAPAP) and SRC homology 3 (SH3) and multiple ankyrin repeat domains protein (SHANK), are shown in the figure because the DLG–SAPAP–SHANK complex is thought to be one of the most critical proteins that determine the overall architecture of PSDs (the rest of the proteins are drawn using simple schematics). The DLG–SAPAP–SHANK complex and glutamate receptors often form co-clustered dynamic nanodomains (the AMPA-type glutamate receptor (AMPAR) cluster shown at the centre of the postsynaptic side represents one of these nanodomains), which are aligned with presynaptic neurotransmitter release sites, allowing strong postsynaptic responses following neurotransmitter release. The clustering of AMPARs may be facilitated by the multimerization of DLG MAGUKs, as indicated by the two-way arrow. A growing list of synaptic adhesion proteins serves to connect preand postsynaptic membranes and to regulate synaptic development and plasticity. Many such synaptic adhesion proteins (for example, interleukin-1 receptor accessory protein-like 1 (IL1RAPL1)) can bind to MAGUKs by their cytoplasmic tails. The synaptic protein complexes that are organized by scaffold proteins are often dynamic and can be regulated by synaptic activity-induced protein modifications such as phosphorylation (indicated by red circles with a ‘P’ at the centre). In addition, several small GTPase regulatory proteins such as SYNGAP, RAP guanine nucleotide exchange factor (RAPGEF), spine-associated RAP GTPase activating protein (SPAR) and kalirin 7 are also known to be intimately involved in the dynamic assemblies of synaptic protein complexes. Selected presynaptic protein complexes involved in neurotransmitter release and synaptic adhesion are also shown here. The domain structure for the presynaptic MAGUK calcium/calmodulin-dependent serine protein kinase (CASK) is also shown. AKAP, A-kinase anchor protein; CaMKII, calcium/calmodulin-dependent protein kinase II; CaN, calcineurin; Cav2.2, voltage-gated calcium channel subunit-α Cav2.2; EphR, ephrin receptor; GBR, GK-binding region; GK, guanylate kinase-like; GRIP, glutamate receptor-interacting protein; L27, LIN2–LIN7; LAR, leukocyte common antigen related; LRRTM, leucine-rich repeat transmembrane protein; MAGI, membrane-associated guanylate kinase inverted; MAP1A, microtubule-associated protein 1A; mGluR, metabotropic glutamate receptor; NGL-3, netrin-G3 ligand; NMDAR, NMDA-type glutamate receptor; PBM, PDZ-binding motif; PDZ, PSD95–DLG1–Zonula occludens 1; PKA, protein kinase A; PKC, protein kinase C; PICK1, protein interacting with C kinase 1; SAM, sterile α-motif; TARP, transmembrane AMPAR regulatory protein; VELI1, vertebrate lin-7 homologue 1. Neurexin Nature Reviews | Neuroscience Synaptic vesicle Glutamate