Functional analysis of the guanylate kinase‐like domain in the synapse‐associated protein SAP97

Kuhlendahl, Sven; Spangenberg, Oliver; Konrad, Manfred; Kim, Eunjoon; Garner, Craig C.

doi:10.1046/j.1432-1327.1998.2520305.x

Cited by 47 publications

(41 citation statements)

References 11 publications

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“…The GK domain is catalytically inactive, although structurally closely related to authentic guanylate kinases as we have shown in previous work (18,19).…”

supporting

confidence: 58%

Formation of Complexes between Ca2+·Calmodulin and the Synapse-associated Protein SAP97 Requires the SH3 Domain-Guanylate Kinase Domain-connecting HOOK Region

Paarmann

Spangenberg

Lavie

et al. 2002

Journal of Biological Chemistry

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Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre-and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calciumdependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K d of 122 nM for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.Membrane-associated guanylate kinase homologs (MAGUKs) 1 have been implicated in the assembly of synapses and tight junctions. These are multidomain proteins consisting of one or more PDZ domains, an Src homology 3 (SH3) domain, and a guanylate kinase (GK)-like domain (for reviews, see Refs. 1-4). Rat SAP97 belongs to the MAGUK subfamily comprising Drosophila Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102/NE-Dlg, and PSD93/Chapsyn110. SAP97 has earlier been reported to be presynaptic (5), but it has recently been shown also to be present at postsynaptic sites in cerebral cortex (6); it is found both at the post-synaptic density (PSD) region and in the cytoplasm of hippocampal synapses, suggesting a role for SAP97 in ionotropic glutamate receptor trafficking (7). Recently it was discovered that SAP97, via its guanylate kinase (GK)-like domain, directly regulates the function of an inwardly rectifier potassium channel (8). In addition to its crucial role as a scaffolding protein in neuronal cells, SAP97 is an essential component of the basolateral membrane cytoskeleton in a variety of epithelial cells (5).Each domain in the SAP subfamily of MAGUKs is highly conserved and has been shown to be a site of protein-protein interaction: PDZ domains bind voltage-and ligand-gated ion channels (3, 9); SH3 domains interact with proline-rich, PXXPR-like sequences (3, 4, 10) as w...

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“…The GK domain is catalytically inactive, although structurally closely related to authentic guanylate kinases as we have shown in previous work (18,19).…”

supporting

confidence: 58%

Formation of Complexes between Ca2+·Calmodulin and the Synapse-associated Protein SAP97 Requires the SH3 Domain-Guanylate Kinase Domain-connecting HOOK Region

Paarmann

Spangenberg

Lavie

et al. 2002

Journal of Biological Chemistry

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“…The analogy with Src family kinases prompts us to speculate that an intramolecular interaction between the SH3 domain and the GK region may be involved in regulating the activity of the SH3 or GK domain in MAGUK proteins. Currently, no enzymatic activity is attributed to the GK domain of PSD-95 family proteins (Kuhlendahl et al, 1998). However, the "closed" (cis-interacting) conformation of the SH3-GK region might prevent binding of ligands to the SH3 domain or to the GK domain.…”

Section: Potential Functions Of Intramolecular Sh3-gk Region Interactionmentioning

confidence: 99%

An Intramolecular Interaction between Src Homology 3 Domain and Guanylate Kinase-Like Domain Required for Channel Clustering by Postsynaptic Density-95/SAP90

et al. 2000

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Members of the postsynaptic density-95 (PSD-95)/SAP90 family of membrane-associated guanylate kinase (MAGUK) proteins function as multimodular scaffolds that organize proteinsignaling complexes at neuronal synapses. MAGUK proteins contain PDZ, Src homology 3 (SH3), and guanylate kinase (GK)-like domains, all of which can function as sites for specific protein-protein interactions. We report here a direct proteinprotein interaction between the SH3 domain and the GK region in the PSD-95 family of MAGUKs. The SH3 domain of the PSD-95 family appears to have an atypical binding specificity, because the classical SH3 binding (-P-X-X-P-) motif is absent from the GK domain. Although SH3-GK binding can occur in either an intramolecular or intermolecular manner, the intramolecular mode is preferred, possibly because of additional tertiary interactions available when the SH3 and GK domains are adjacent in the same polypeptide. Mutations disrupting the intramolecular SH3-GK interaction do not interfere with PSD-95 association with the K ϩ channel Kv1.4 or with the GK domain-binding protein GKAP. The same mutations, however, inhibit the clustering of Kv1.4 by PSD-95, suggesting that the intramolecular SH3-GK interaction may modulate the clustering activity of PSD-95. Key words: PDZ domain; ion channel clustering; postsynaptic density; Src tyrosine kinase; polyproline helix; disks largeSynaptic transmission in neuronal synapses requires the proper organization of ion channels, neurotransmitter receptors, and signaling molecules in the synaptic junction. An emerging theme for the micro-organization of synapses is that functionally coupled proteins are placed in close proximity to each other via their interactions with scaffold proteins. The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) plays such a role at postsynaptic sites of glutamatergic synapses by interacting with a variety of ion channels, receptors, cytoskeletal components, and intracellular signaling proteins (Sheng and Wyszynski, 1997;Ziff, 1997;Craven and Bredt, 1998;Kennedy, 1998;O'Brien et al., 1998).The PSD-95 family of proteins (PSD-95/SAP90, SAP97/hdlg, chapsyn-110/PSD-93, and SAP102) share a common domain organization, consisting of three PDZ domains in their N-terminal half, a central Src homology 3 (SH3) domain, and a guanylate kinase (GK) domain at their C terminus. The PDZ domains of PSD-95 family proteins interact with the C terminal sequence motif (consensus -X-T/S-X-V) found in a variety of membrane and intracellular proteins, including Shaker K ϩ channels, NMDA receptors, and SynGAP (Kim et al., 1995Kornau et al., 1995;Doyle et al., 1996;Chen et al., 1998). In addition, PSD-95 binds to neuronal nitric oxide synthase (nNOS) through a PDZ-PDZ/␤-finger interaction (Brenman et al., 1996a;Hillier et al., 1999). In vitro and in vivo evidence suggests that these PDZmediated interactions are involved in the clustering of specific membrane proteins at synaptic sites (Kim et al., 1995Thomas et al., 1997).The GK domain of the PSD-95 family of MA...

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“…The guanylate kinase-like domains of MAGUKs exhibit little or no guanylate kinase activity (23), and their principal function seems to serve as a protein-protein interaction motif (18,24). Besides GAKIN, several proteins are reported to interact with the GUK domains of MAGUKs.…”

mentioning

confidence: 99%

Direct Interaction with a Kinesin-related Motor Mediates Transport of Mammalian Discs Large Tumor Suppressor Homologue in Epithelial Cells

Asaba¹,

Hanada²,

Takeuchi

et al. 2003

Journal of Biological Chemistry

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Membrane-associated guanylate kinase homologues (MAGUKs) are generally found under the plasma membrane of cell-cell contact sites and function as scaffolding proteins by linking cytoskeletal and signaling molecules to transmembrane receptors. The correct targeting of MAGUKs is essential for their receptorclustering function; however, the molecular mechanism of their intracellular transport is unknown. Here, we show that the guanylate kinase-like domain of human discs large protein binds directly within the amino acids 607-831 of the stalk domain of GAKIN, a kinesin-like protein of broad distribution. The primary structure of the binding segment, termed MAGUK binding stalk domain, is conserved in Drosophila kinesin-73 and some other motor and non-motor proteins. This stalk segment is not found in GKAP, a synaptic protein that interacts with the guanylate kinase-like domain, and unlike GKAP, the binding of GAKIN is not regulated by the intramolecular interactions within the discs large protein. The recombinant motor domain of GAKIN is an active microtubule-stimulated ATPase with k cat ‫؍‬ 45 s ؊1 , K 0.5 (MT) ‫؍‬ 0.1 M. Overexpression of green fluorescent protein-fused GAKIN in Madin-Darby canine kidney epithelial cells induced long projections with both GAKIN and endogenous discs large accumulating at the tip of these projections. Importantly, the accumulation of endogenous discs large was eliminated when a mutant GAKIN lacking its motor domain was overexpressed under similar conditions. Taken together, our results indicate that discs large is a cargo molecule of GAKIN and suggest a mechanism for intracellular trafficking of MAGUK-laden vesicles to specialized membrane sites in mammalian cells.Membrane-associated guanylate kinase homologues (MAGUKs) 1 are a family of proteins composed of one or more PDZ domains, an SH3 domain, and a guanylate kinase-like (GUK) domain (1). They are thought to play scaffolding functions at specialized membrane sites, such as synaptic membrane, tight junction, and adherens junction (2-4). Drosophila Dlg is a MAGUK encoded by lethal (1) discs large-1 tumorsuppressor gene (dlg), and mutations of dlg cause neoplastic overgrowth of the imaginal discs (5). The Dlg protein localizes to the septate junctions in epithelial cells where it regulates cell proliferation, apical-basal cell polarity, and the organization of junctional structure (6 -8). The human homologue of Drosophila Dlg, termed hDlg, and its rat counterpart SAP97, localize at the pre-and postsynaptic membrane sites as well as the basolateral membrane of epithelial cells (9, 10) and are proposed to perform scaffolding functions by linking cytoskeletal components to the transmembrane proteins (11). In addition to the scaffolding function, mounting evidence now indicates that hDlg regulates cell proliferation and could be involved in tumorigenesis. For example, hDlg interacts with viral oncoproteins such as high-risk human papillomavirus E6 and human T-cell leukemia virus type 1 (HTLV-1) Tax (12-14). Similarly, hDlg forms a com...

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Functional analysis of the guanylate kinase‐like domain in the synapse‐associated protein SAP97

Cited by 47 publications

References 11 publications

Formation of Complexes between Ca2+·Calmodulin and the Synapse-associated Protein SAP97 Requires the SH3 Domain-Guanylate Kinase Domain-connecting HOOK Region

Formation of Complexes between Ca2+·Calmodulin and the Synapse-associated Protein SAP97 Requires the SH3 Domain-Guanylate Kinase Domain-connecting HOOK Region

An Intramolecular Interaction between Src Homology 3 Domain and Guanylate Kinase-Like Domain Required for Channel Clustering by Postsynaptic Density-95/SAP90

Direct Interaction with a Kinesin-related Motor Mediates Transport of Mammalian Discs Large Tumor Suppressor Homologue in Epithelial Cells

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