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1.
Structural requirements for calmodulin binding to membrane-associated guanylate kinase homologs 总被引:1,自引:0,他引:1
Paarmann I Lye MF Lavie A Konrad M 《Protein science : a publication of the Protein Society》2008,17(11):1946-1954
Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies--synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1--and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca2+-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca2+-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK-calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca2+-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton. 相似文献
2.
PSD-95/SAP90 is a member of the MAGUK superfamily. In excitatory synapses, PSD-95 clusters receptors and ion channels at specific sites in the postsynaptic membrane and organizes downstream signaling and cytoskeletal molecules. We have determined the crystal structures of the apo and GMP-bound forms to 2.3 and 2.0 A resolutions, respectively, of a fragment containing the SH3, HOOK, and guanylate kinase (GK) domains of PSD-95. We observe an intramolecular interaction between the SH3 and GK domains involving the formation of a beta sheet including residues N- and C-terminal to the GK domain. Based on amino acid conservation and mutational data available in the literature, we propose that this intramolecular interaction is a common feature among MAGUK proteins. 相似文献
3.
Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding. 相似文献
4.
Wu H Reissner C Kuhlendahl S Coblentz B Reuver S Kindler S Gundelfinger ED Garner CC 《The EMBO journal》2000,19(21):5740-5751
Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners. 相似文献
5.
Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand. 相似文献
6.
Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells. 相似文献
7.
Nix SL Chishti AH Anderson JM Walther Z 《The Journal of biological chemistry》2000,275(52):41192-41200
Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding. 相似文献
8.
Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated. 相似文献
9.
Membrane-associated guanylate kinases (MAGUKs) act as scaffolds to coordinate signaling events through their multiple domains at the plasma membrane. The MAGUK SH3 domain is noncanonical and its function remains unclear. To identify potential binding partners of MAGUK SH3, the synapse-associated protein 102 (SAP102) SH3 domain was used as bait in a yeast two-hybrid screen of a mouse embryonic cDNA library. A mouse homologue of the Drosophila discs large tumor suppressor (Dlg, also known as SAP97) bound preferentially to SAP102 SH3. The 4347bp cDNA sequence encoded an 893 amino acid protein with 94% identity to mouse SAP97. A deleted region (33-aa) strongly suggests this is a novel splice variant, which we call Embryonic-dlg/SAP97 (E-dlg). The interaction of SAP102 and E-dlg was confirmed in mammalian cells. E-dlg can also bind to potassium channel Kv1.4 in a pull-down assay. E-dlg was highly expressed in embryonic and some adult mouse tissues, such as brain, kidney, and ovary. Furthermore, in situ hybridization showed that E-dlg was mostly expressed in olfactory bulb and cerebellum. 相似文献
10.
GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules 总被引:15,自引:2,他引:13 下载免费PDF全文
Eunjoon Kim Scott Naisbitt Yi-Ping Hsueh Anuradha Rao Adam Rothschild Ann Marie Craig Morgan Sheng 《The Journal of cell biology》1997,136(3):669-678
The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH2-terminal PDZ domains, with certain ion channels (NMDA receptors and K+ channels), thereby promoting the clustering of these proteins. Although the function of the NH2-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K+ channels/ NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein– protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK). 相似文献
11.
Multidomain scaffolding proteins are central components of many signaling pathways and are commonly found at membrane specializations. Here we have shown that multiple interdomain interactions in the scaffold Discs Large (Dlg) regulate binding to the synaptic protein GukHolder (GukH). GukH binds the Src homology 3 (SH3) and guanylate kinase-like (GK) protein interaction domains of Dlg, whereas an intramolecular interaction between the two domains inhibits association with GukH. Regulation occurs through a PDZ domain adjacent to the SH3 that allows GukH to interact with the composite SH3-GK binding site, but PDZ ligands inhibit GukH binding such that Dlg forms mutually exclusive PDZ ligand and GukH cellular complexes. The PDZ-SH3-GK module is a common feature of membrane associate guanylate kinase scaffolds such as Dlg, and these results indicate that its supramodular architecture leads to regulation of Dlg complexes. 相似文献
12.
Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102 总被引:3,自引:0,他引:3
Seabold GK Burette A Lim IA Weinberg RJ Hell JW 《The Journal of biological chemistry》2003,278(17):15040-15048
The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102. 相似文献
13.
Masuko N Makino K Kuwahara H Fukunaga K Sudo T Araki N Yamamoto H Yamada Y Miyamoto E Saya H 《The Journal of biological chemistry》1999,274(9):5782-5790
NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses. 相似文献
14.
A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia 下载免费PDF全文
Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. We recently identified a new protein-protein interaction domain, called the L27 domain, which is present twice in mLin-2/CASK. In this report, we further define the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identify the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Our colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. We also report the identification of a novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, which contains a region highly related to the SAP97 N terminus and which binds Camguk, a Drosophila orthologue of mLin-2/CASK. Our data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice. 相似文献
15.
The beta-subunit of voltage-gated Ca(2+) channels plays a dual role in chaperoning the channels to the plasma membrane and modulating their gating. It contains five distinct modular domains/regions, including the variable N- and C-terminus, a conserved Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a connecting variable and flexible HOOK region. Recent crystallographic studies revealed a highly conserved interaction between the GK domain and alpha interaction domain (AID), the high-affinity binding site in the pore-forming alpha(1) subunit. Here we show that the AID-GK domain interaction is necessary for beta-subunit-stimulated Ca(2+) channel surface expression and that the GK domain alone can carry out this function. We also examined the role of each region of all four beta-subunit subfamilies in modulating P/Q-type Ca(2+) channel gating and demonstrate that the beta-subunit functions modularly. Our results support a model that the conserved AID-GK domain interaction anchors the beta-subunit to the alpha(1) subunit, enabling alpha(1)-beta pair-specific low-affinity interactions involving the N-terminus and the HOOK region, which confer on each of the four beta-subunit subfamilies its distinctive modulatory properties. 相似文献
16.
Zhe Wei Blake Behrman Wei-Hua Wu Bo-Shiun Chen 《The Journal of biological chemistry》2015,290(8):5105-5116
Synapse-associated protein 102 (SAP102) is a scaffolding protein abundantly expressed early in development that mediates glutamate receptor trafficking during synaptogenesis. Mutations in human SAP102 have been reported to cause intellectual disability, which is consistent with its important role during early postnatal development. SAP102 contains PDZ, SH3, and guanylate kinase (GK)-like domains, which mediate specific protein-protein interactions. SAP102 binds directly to N-methyl-d-aspartate receptors (NMDARs), anchors receptors at synapses, and facilitates transduction of NMDAR signals. Proper localization of SAP102 at the postsynaptic density is essential to these functions. However, how SAP102 is targeted to synapses is unclear. In the current study we find that synaptic localization of SAP102 is regulated by alternative splicing. The SAP102 splice variant that possesses a C-terminal insert (I2) between the SH3 and GK domains is highly enriched at dendritic spines. We also show that there is an intramolecular interaction between the SH3 and GK domains in SAP102 but that the I2 splicing does not influence SH3-GK interaction. Previously, we have shown that SAP102 expression promotes spine lengthening. We now find that the spine lengthening effect is independent of the C-terminal alternative splicing of SAP102. In addition, expression of I2-containing SAP102 isoforms is regulated developmentally. Knockdown of endogenous I2-containing SAP102 isoforms differentially affect NMDAR surface expression in a subunit-specific manner. These data shed new light on the role of SAP102 in the regulation of NMDAR trafficking. 相似文献
17.
Membrane-associated guanylate kinases (MAGUKs) regulate the formation and
function of molecular assemblies at specialized regions of the membrane.
Allosteric regulation of an intramolecular interaction between the Src
homology 3 (SH3) and guanylate kinase (GK) domains of MAGUKs is thought to
play a central role in regulating MAGUK function. Here we show that a mutant
of the Drosophila MAGUK Discs large (Dlg), dlgsw,
encodes a form of Dlg that disrupts the intramolecular association while
leaving the SH3 and GK domains intact, providing an excellent model system to
assess the role of the SH3-GK intramolecular interaction in MAGUK function.
Analysis of asymmetric cell division of maternal-zygotic
dlgsw embryonic neuroblasts demonstrates that the
intramolecular interaction is not required for Dlg localization but is
necessary for cell fate determinant segregation to the basal cortex and
mitotic spindle alignment with the cortical polarity axis. These defects
ultimately result in improper patterning of the embryonic central nervous
system. Furthermore, we demonstrate that the sw mutation of Dlg results in
unregulated complex assembly as assessed by GukHolder association with the
SH3-GK versus PDZ-SH3-GK modules of Dlgsw. From these
studies, we conclude that allosteric regulation of the SH3-GK intramolecular
interaction is required for regulation of MAGUK function in asymmetric cell
division, possibly through regulation of complex assembly.The membrane-associated guanylate kinase
(MAGUK)2 superfamily
consists of ubiquitous scaffolding proteins that are composed of a common core
of contiguously linked modular domains (protein-protein interaction domains,
PDZ and SH3 domains, and a domain with homology to the yeast guanylate kinase,
GK domain). MAGUKs are concentrated at sites of cell-cell contact
(1) and organize a variety of
cell adhesion molecules, cytoskeletal proteins, receptors, ion channels, and
associated signaling molecules at specialized regions of the membrane
(2). Protein complex
organization by MAGUKs has been thought to occur, at least in part, through
allosteric regulation that arises from an intramolecular interaction between
the SH3 and GK domains. This interaction has been shown to regulate binding of
numerous MAGUK ligands in vitro
(3–5).
However, whereas the SH3-GK interaction has been extensively characterized
biochemically
(6–8),
very little data exists regarding its physiological relevance. Here we
investigate the role of the SH3-GK intramolecular interaction of Discs large
(Dlg), a prototype of the MAGUK superfamily.Loss of dlg activity results in overgrowth of imaginal discs and
tumor formation (9). Dlg
localizes to septate and neuromuscular junctions and is essential for
establishing and maintaining apicobasal polarity of Drosophila
epithelia (10). Dlg also plays
an important role in regulating the process of asymmetric cell division (ACD)
(11–13).
ACD is a mechanism for generating cellular diversity via unequal mitotic
divisions of progenitor cells. For instance, in wild-type Drosophila
neuroblasts, cell signaling networks interact to allow for asymmetric
segregation of basal cell fate determinants, followed by alignment of the
mitotic spindle along the apical-basal cortical polarity axis (see Refs.
14 and
15) for review). ACD results
in the formation of a self-renewing, stem-cell like neuroblast and a smaller
ganglion mother cell, which has neuronal or glial fate. In dlg
germline clone (dlgGLC) embryonic metaphase neuroblasts,
ganglion mother cell fate determinants are not restricted to the basal cortex
(11,
12) and the mitotic spindle
does not reliably align with the apical-basal cortical polarity axis
(13). Defects in neurogenesis
have also been observed for dlgGLC embryos
(16), as well as embryos
treated with RNA interference against an alternatively spliced isoform of Dlg
(17), providing evidence for
the function of Dlg in neuronal differentiation and axon guidance. Such
defects in neurogenesis are thought to be attributed to defective localization
of basal cell fate determinants during ACD
(17). Although Dlg function is
important in a broad range of dynamic cellular processes, the role of the Dlg
SH3-GK intramolecular interaction in Dlg activity is poorly understood.One potential role for the SH3-GK intramolecular association is to regulate
MAGUK complex assembly. In vitro binding assays demonstrated that
mutations disrupting this intramolecular interaction allowed mutant SH3-GK
modules to associate with SH3 or GK domains of various MAGUK proteins in
trans, providing a mechanism of regulating oligomerization of MAGUKs
in vivo (6,
8). A role in clustering of ion
channels was also observed as mutations that disrupted the intramolecular
association, whereas having no effect on association with the potassium
channel KV1.4 or homo-oligomerization of PSD-95, resulted in loss
of channel clustering in vivo
(18). Furthermore, multiple
in vitro studies support its regulation of binding of protein ligands
with the GK domain of MAGUKs: examples include interaction of GK domains of
Dlg with GukHolder (GukH) (3),
SAP97 with guanylate kinase-associated protein
(4), and PSD-93 with the
microtubule-associated protein 1A
(5). These studies suggest that
allosteric modulation of the SH3-GK intramolecular interaction is important
for regulation of complex assembly, yet little evidence exists that such
regulation of MAGUKs is required for their function in vivo.In crystal structures of the SH3-GK module of PSD-95, two β-strands
that emerge from the SH3 and GK domains appear to mediate the interaction
between the domains (Fig.
1A) (19,
20). Functional studies of the
interaction of these β-strands demonstrate that a COOH-terminal
truncation of the strand following the GK domain abrogates the intramolecular
association of the SH3-GK module. This 13-amino acid truncation of PSD-95 is
comparable with a dlg allele that had previously been identified in a
genetic screen, dlgsw
(21). Drosophila
containing only the sw form of Dlg (refer to COOH-terminal sequences in
Fig. 1B) die in the
latter stages of embryonic development due to failure of dorsal closure and
terminal defects (21). We
hypothesized that this mutation disrupts the intramolecular interaction while
leaving the SH3 and GK domains largely intact, making the
dlgsw allele an excellent model system for assessing the
role of this interaction in MAGUK function.Open in a separate windowFIGURE 1.The dlgsw allele disrupts the SH3-GK intramolecular
interaction formed by interacting E and Fβ-strands. A, a
ribbon diagram of the SH3-GK module of PSD-95 (Protein Data Bank 1KJW
(19)) created using MOLSCRIPT
(58) and RASTER3D
(59), highlighting the SH3
(red), HOOK (gray), and GK (blue) domains. The
intramolecular interaction between the SH3 and GK domains is mediated by the
interaction of two β-strands (shown in black), one strand is
contributed by the linker region following the SH3 domain (strand E), whereas
the second strand is contributed by a COOH-terminal strand that follows the GK
domain (strand F). B, sequence comparison of the COOH-terminal
regions of WT Dlg versus Dlgsw. C, GST pull-downs
of the Dlg SH3 domain using GST fusions of WT and mutant Dlg GK domains.
Association of protein components was detected by an anti-His antibody that
recognized an NH2-terminal on the SH3 domain of Dlg. D,
circular dichroism wavelength scans, monitoring the change in ellipticity as a
function of wavelength. Changes in α-helical content are indicated by
the change in ellipticity at 222 nm (see black dashed line).
Comparison of CD spectra of WT PDZ-SH3-GK (green circles) and
PDZ-SH3-GK(sw) (red squares) indicate no significant change in
α helical character. E, Sedfit analysis of sedimentation
velocity scans of WT PDZ-SH3-GK (green solid line) and
PDZ-SH3-GKsw (red dashed line). Note that the estimated
molecular weights provided are based upon the assumption that the protein
exists in a globular conformation.In the studies presented here, we utilized the dlgsw
mutant allele to explore the role of the intramolecular interaction in
regulating complex assembly and furthermore, to assess the in vivo,
physiological significance of this interaction for Dlg function in neuroblast
asymmetric cell division. We find that disruption of the SH3-GK intramolecular
interaction results in unregulated complex assembly. Such a loss in regulated
complex assembly results in loss of Dlg function in the process of asymmetric
cell division as assessed by localization of cell fate determinants in a
dividing neuroblast and alignment of the mitotic spindle along the
apical-basal cortical polarity axis, ultimately resulting in defects in
neurogenesis. Thus, these data contribute to our understanding of how the
SH3-GK intramolecular interaction regulates MAGUK function in formation and
regulation of membrane specializations. 相似文献
18.
Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex 总被引:10,自引:0,他引:10
Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved. 相似文献
19.
Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization. 相似文献
20.
Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins. 总被引:7,自引:0,他引:7
Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization. 相似文献