The Dual PDZ Domain from Postsynaptic Density Protein 95 Forms a Scaffold with Peptide Ligand
Nazahiyah Ahmad RodzliMichael P. Lockhart‐CairnsColin LevyJ. R. ChipperfieldLouise E. BirdClair BaldockStephen M. Prince
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Abstract:
PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms scaffolding interactions with partner proteins, including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/disks large/zona occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the ligand association and small-angle x-ray scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95.Keywords:
PDZ domain
Guanylate kinase
Postsynaptic density
Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile α motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membranevia the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions. Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile α motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membranevia the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions. postsynaptic density synapse-associated protein PSD-95/Dlg-A/ZO-1 guanylate kinase N-methyl-d-aspartate SAP90/PSD-95-associated protein synaptic scaffolding molecule glutathione S-transferase sterile α motif plekstrin homology synaptic plasma membrane mitogen-activated protein kinase Chinese hamster ovary Synaptic junctions are interneuronal cell-cell junctions differentiated for neurotransmission. Neurotransmitters are released from the synaptic vesicles into the synaptic cleft and bind to the receptors accumulated at the postsynapse, opening the ion channels and generating the second messengers involved in synaptic plasticity (reviewed in Ref. 1Hammond C. Cellular and Molecular Neurobiology. 1st Ed. Academic Press, Inc., San Diego, CA1996: 20-40Google Scholar). The components required for this process are organized at the synaptic junctions to play specific roles in felicitous orders. Several scaffolding proteins are reported to be involved in the assembly of components of synaptic junctions (reviewed in Refs. 2Kennedy M.B. Curr. Opin. Neurobiol. 1993; 3: 732-737Crossref PubMed Scopus (129) Google Scholar, 3Garner C. Kindler S. Trends Cell Biol. 1996; 19: 429-433Abstract Full Text PDF Scopus (63) Google Scholar, 4Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 5Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 6Hata Y. Nakanishi H. Takai Y. Neurosci. Res. 1998; 32: 1-7Crossref PubMed Scopus (74) Google Scholar). Postsynaptic density (PSD)1-95/synapse-associated protein (SAP) 90 has three PSD-95/Dlg-A/ZO-1 (PDZ) domain, one Src homology 3 domain, and one guanylate kinase (GK) domain (7Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 8Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauer U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar). The PDZ domain is a protein-interacting module (reviewed in Refs. 9Ponting C.P. Phillips C. Trends Biochem. Sci. 1995; 20: 102-103Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 10Kennedy M.B. Trends Biochem Sci. 1995; 20: 350Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 11Fanning A.S. Anderson J.M. Curr. Biol. 1996; 6: 1385-1388Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 12Sheng M. Neuron. 1996; 17: 575-578Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), and PSD-95/SAP90 binds the C termini of N-methyl-d-aspartate (NMDA) receptors, K+ channels, neuroligins, synGAP, and CRIPT through distinct PDZ domains to assemble these components at the synaptic junctions (13Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1631) Google Scholar, 14Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar, 15Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Sudhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (612) Google Scholar, 16Kim J.H. Liao D. Lau L.-F. Huganir R.L. Neuron. 1998; 20: 683-691Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 17Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 18Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg R.J. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). PSD-95/SAP90 interacts with SAP90/PSD-95-associated protein (SAPAP) (also called GK-associated protein and hDLG-associated protein) (19Kim E. Naisbitt S. Hsueh Y.-P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (434) Google Scholar, 20Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 21Satoh K. Yanai H. Senda T. Kohu K. Nakamura T. Okumura N. Matsumine A. Kobayashi S. Toyoshima K. Akiyama T. Genes Cells. 1997; 2: 415-424Crossref PubMed Scopus (112) Google Scholar), and a recently identified protein, BEGAIN (brain-enriched guanylate kinase-interacting protein) via the GK domain (22Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Glutamate receptor-interacting protein has seven PDZ domains and binds α amino-3-hydroxy-5-methyl-4-isoxazaole propionic acid receptorsvia the fourth and fifth PDZ domains (23Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (758) Google Scholar). The ligands for other PDZ domains of glutamate receptor-interacting protein have not been so far reported. Synaptic scaffolding molecule (S-SCAM) was originally identified as a SAPAP-interacting protein (24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). We have first reported that S-SCAM has one GK, two WW, and five PDZ domains (24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The GK domain of S-SCAM is shorter than that of PSD-95/SAP90. The WW domain is a protein-interacting module binding a proline-rich sequence (25Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (489) Google Scholar). The recent version of simple modular architecture research tool recognizes an additional PDZ domain at the N terminus of S-SCAM (26Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (3029) Google Scholar). We number these PDZ domains consecutively from 0 to 5 (PDZ0, -1, -2, -3, -4, and -5) to keep consistency with the first report. Among six PDZ domains, PDZ1 and -5 bind to the C termini of neuroligin and NMDA receptors, respectively (24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Because S-SCAM has more PDZ domains than PSD-95/SAP90, it may integrate more components of synaptic junctions. Based on this assumption, we have performed a yeast two-hybrid screening using the PDZ domains of S-SCAM to obtain a novel neuronal molecule. Eventually, this molecule binds not only to S-SCAM but also to PSD-95/SAP90. We have named this protein MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). Rat brain yeast two-hybrid library was constructed using pVP16 vector and screened (27Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). Rat brain cDNA libraries were screened with the [α-32P]dCTP-labeled random-primed probes (27Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). Various expression vectors were constructed by conventional molecular biology techniques and polymerase chain reaction method using pBTM116, pBTM116–2, pCMV Myc, pCMV Myc2, pClneo Myc, pGex5X-3 (Amersham Pharmacia Biotech), pGex4T-1 (Amersham Pharmacia Biotech), and pGexKG. pBTM116–2, pCMV Myc, and pClneo Myc were constructed from pBTM116, pCMV5, and pClneo, respectively (24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). pCMV Myc2 was constructed by ligating aattcgagatctcgggtaccacgcgtatcgatatcgcggccg/ctagcggccgcgatatcgatacgcgtggtacccgagatctcg into EcoRl/Xbal sites of pCMV Myc. pCMV PSD-95, pCMV S-SCAM, and pClneo Myc S-SCAM-1, -2, -3, and -4 were described previously (20Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar,24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). pBTM116 S-SCAM-13, -14, and -16 and pClneo Myc S-SCAM-9, -10, and -11 contain the amino acid residues 413–1114, 413–1277, 728–1277, 603–1023, 906–1277, and 772–1277 of S-SCAM, respectively. pBTM116 MAGUIN-9, pGex4T-1 MAGUIN-1, pGex5X-3 MAGUIN-12, pCMV Myc MAGUIN-1f, and pClneo Myc MAGUIN-n, -m, and -c contain the amino acid residues 846–1032, 716–858, 846–1032, 1–1032, 1–469, 458–858, and 568–1032 of MAGUIN-1, respectively. pGex5X-3 MAGUIN-16 and pCMV Myc MAGUIN-2f contain the amino acid residues 568–896 and 1–896 of MAGUIN-2, respectively. pCMV Myc PSD-95-1, -5, and -6 contain the amino acid residues 1–724, 1–407, and 432–724 of PSD-95/SAP90, respectively. Rabbit polyclonal antibodies were raised against the products of pGex4T-1 MAGUIN-1 and pGex5X-3 MAGUIN-16. The monoclonal anti-Myc-tag antibody, 9E10, was obtained from American Type Culture Collection. The anti-γ-aminobutyric acid A receptor antibody was purchased from Chemicon. The anti-NMDAR1 antibody was a generous gift of Dr. Nils Brose (Max Planck Institute). The anti-S-SCAM antibody was described (24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). COS cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum under 10% CO2 at 37 °C and transfected with DEAE-dextran (20Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). COS cells of two 10-cm plates were homogenized in 0.5 ml of 20 mm Tris/HCl, pH 7.4, containing 100 mm NaCl and 1% (w/v) Triton X-100 and centrifuged at 100,000 × gfor 30 min. The supernatant was used as a COS cell extract. 0.5-ml aliquots of the COS cell extract were incubated with 200 pmol of various GST constructs fixed on 20 μl of glutathione-Sepharose 4B beads. After the beads were washed with 50 mm Tris/HCl, pH 7.4, containing 100 mm NaCl and 1% (w/v) Triton X-100, proteins on the beads were detected with the immunoblottings using the appropriate antibodies. The crude synaptosomal fraction was prepared from four rat brains as described (24Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The fraction was homogenized in 16 ml of 50 mm Hepes/NaOH, pH 8.0, containing 100 mm NaCl, 5 mm EDTA, 1% (w/v) deoxycholic acid, and 1% (w/v) Nonidet P-40 and centrifuged at 100,000 × g for 30 min to collect the supernatant. 4-ml aliquots of the supernatant were incubated with the anti-PSD-95/SAP90 antibody, the anti-S-SCAM antibody, or the preimmune serum fixed on 20 μl of protein G-Sepharose Fast Flow beads. After the beads were washed four times with 50 mm NaOH/Hepes, pH 8.0, containing 100 mm NaCl, and 1% (w/v) Triton X-100, proteins on the beads were detected with the immunoblottings using the appropriate antibodies. CHO cells were transfected with various eukaryote expression vectors using TransFast Transfection Reagent (Promega). After 48 h, the cells were collected and homogenized in 300 μl of 20 mm Hepes/NaOH, pH 7.4, by sonication. 80 μl of the homogenate was kept for the analysis, and the remaining samples were centrifuged at 100,000 ×g for 30 min to separate the supernatant and the pellet. The pellet was homogenized in 220 μl of 20 mm Hepes/NaOH, pH 7.4, containing 1% (w/v) Triton X-100 and centrifuged at 10,000 × g for 10 min to separate the supernatant and the pellet. Other procedures, including subcellular fractionation of rat brain, primary cultures of rat hippocampal neurons, immunocytostaining, SDS-polyacrylamide gel electrophoresis, and protein determination, were performed as described (20Takeuchi M. Hata Y. Hirao K. Toyoda A. Irie M. Takai Y. J. Biol. Chem. 1997; 272: 11943-11951Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Northern and Western blottings were performed using multiple tissue Northern blots (CLONTECH) and ECL reagents (Amersham Pharmacia Biotech), respectively. We performed the yeast two-hybrid screening using baits containing the PDZ domains of S-SCAM (pBTM116 S-SCAM-13, -14, and -16). We obtained 34 positive independent clones from 5 × 106 clones of a rat brain yeast two-hybrid library. The sequences of 22 clones were novel, and we performed Northern blot analysis for these clones. The messages of two clones (pPrey 4233 and pPrey 4514) were detected only in the brain. During the study, the sequence of human brain-specific angiogenesis inhibitor-1 was reported, and pPrey 4514 turned out to be rat brain-specific angiogenesis inhibitor-1. We obtained the presumptive full-length coding sequences of pPrey 4233 through the conventional hybridization screening using a rat brain cDNA library and polymerase chain reaction using rat brain cDNAs as templates. Two isoforms were detected and named MAGUIN-1 and -2. MAGUIN-1 had 1032 amino acids and was composed of one sterile α motif (SAM) domain, one PDZ domain, and one plekstrin homology (PH) domain (Fig.1 A). MAGUIN-1 had Thr-His-Val at the C terminus, which corresponded to the PDZ domain-binding motif. pPrey 4233 contained the C-terminal 319 residues of MAGUIN-1 (amino acids 714–1032, underlined in Fig. 1 A). MAGUIN-2 contained the N-terminal 895 amino acids of MAGUIN-1 and is terminated with Ser as the last (896th) residue, lacking the PDZ domain-binding motif (Fig. 1 B). The SAM domain of MAGUINs is about 30% homologous to those of Caenorhabditis elegans R01H10.8 and yeast byr2 and about 15% homologous to that of yeastSTE50 (Fig. 2 A) (28Ponting C.P. Protein Sci. 1995; 4: 1928-1930Crossref PubMed Scopus (142) Google Scholar, 29Schultz J. Ponting C.P. Hofman K. Bork P. Protein Sci. 1997; 6: 249-253Crossref PubMed Scopus (270) Google Scholar, 30Wang Y. Xu H.P. Riggs M. Rodgers L. Wigler M. Mol. Cell. Biol. 1991; 11: 3554-3563Crossref PubMed Scopus (150) Google Scholar, 31Ramezani R.M. Xu G. Hollenberg C.P. Mol. Gen. Genet. 1992; 236: 145-154Crossref PubMed Scopus (41) Google Scholar). The PDZ domain of MAGUINs is about 40% homologous to that ofC. elegans R01H10.8 (Fig. 2 B). The homology with the PDZ domain of PSD-95/SAP90 or CASK is about 15%, and among others the residues of the first α helix are rather well conserved (Fig.2 B) (32Doyle A.D. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 33Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (292) Google Scholar). The PH domain of MAGUINs is about 20% homologous to those of C. elegans R01H10.8, human dynamin, and rsec7 (Fig. 2 C) (34van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (591) Google Scholar, 35Telemenakis I. Benseler F. Stenius K. Sudhof T.C. Brose N. Eur. J. Cell Biol. 1997; 74: 143-149PubMed Google Scholar). This region of MAGUINs is about 55% homologous to human KIAA0403 (Fig. 2 C). Although the complete coding sequence of KIAA0403 is not reported, it may be a human isoform of MAGUINs. Because C. elegans R01H10.8 has a similar molecular structure, it may be a C. eleganshomologue of MAGUINs.Figure 2Sequence alignment of each domain of MAGUINs. Residues are shown in single-letter codes. The residues conserved among four, three, and two proteins are indicated on thegreen, orange, and light or dark blue backgrounds, respectively. A, alignment of the SAM domains of MAGUINs, C. elegans R01H10.8, yeastbyr2, and STE50. B, alignment of the PDZ domain of MAGUINs with the PDZ domain of C. elegans R01H10.8, the PDZ domain of CASK, and the third PDZ domain of PSD-95/SAP90. The amino acids of α helixes and β sheets are indicated in boxes.C, alignment of the PH domains of MAGUINs, C. elegans R01H10.8, KIAA0403, dynamin, and rsec7.View Large Image Figure ViewerDownload (PPT) To confirm the interaction of MAGUIN-1 with S-SCAM, the extract of COS cells expressing S-SCAM was incubated with either GST-MAGUIN-12 containing the C terminus of MAGUIN-1 or GST-MAGUIN-16 containing the C terminus of MAGUIN-2. The C terminus of MAGUIN-1 interacted with S-SCAM, whereas the C terminus of MAGUIN-2 did not (Fig.3 A). MAGUIN-1 was coimmunoprecipitated with S-SCAM from the rat crude synaptosomal fraction (Fig. 3, B and C). To determine the interacting region of S-SCAM with MAGUIN-1, various Myc-tagged constructs of S-SCAM were incubated with GST-MAGUIN-12 containing the C terminus of MAGUIN-1 (Fig.4 A). Myc-S-SCAM-1, -4, -11, and -10 interacted, whereas Myc-S-SCAM-2, -3, and -9 did not (Fig.4 B), suggesting that the fourth and fifth PDZ domains were involved in the interaction. From the reverse yeast two-hybrid screening using pBTM116 MAGUIN-9 as a bait, the PDZ domains of PSD-95/SAP90, PSD93/chapsyn110, and SAP97 were obtained (36Brenman J.E. Chao D. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar, 37Kim E. Cho K.-O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 38Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Crossref PubMed Google Scholar), suggesting that MAGUIN-1 interacted with not only S-SCAM but also PSD-95/SAP90 and its isoforms. MAGUIN-1 was coimmunoprecipitated with PSD-95/SAP90 from the rat crude synaptosomal fraction (Fig.5 A). The interaction of MAGUIN-1 with the PDZ domains of PSD-95/SAP90 was confirmed using Myc-tagged constructs of PSD-95/SAP90 and the GST construct of MAGUIN-1 (Fig. 5 B).Figure 4MAGUIN-1-interacting domain of S-SCAM. A, schematic description of various Myc-tagged constructs of S-SCAM. The letter (a–g) to the leftof each construct corresponds to the letter of eachlane in panel B. B, the extract of COS cells expressing various Myc-tagged constructs of S-SCAM were incubated with GST-MAGUIN-12 fixed on the glutathione beads, and the proteins attached to the beads were detected with the anti-Myc antibody. Lane a, pClneo Myc S-SCAM-1; lane b, pClneo Myc S-SCAM-2;lane c, pClneo Myc S-SCAM-3; lane d, pClneo Myc S-SCAM-4; lane e, pClneo Myc S-SCAM-9; lane f, pClneo Myc S-SCAM-10; lane g, pClneo Myc S-SCAM-11.View Large Image Figure ViewerDownload (PPT)Figure 5Interaction of MAGUIN-1 with PSD-95/SAP90. A, coimmunoprecipitation of MAGUIN-1 with PSD-95/SAP90. The Triton X-100 extract of the rat crude synaptosomal fraction was incubated with either the anti-PSD-95/SAP90 serum or the preimmune serum on protein G-Sepharose beads, and the proteins attached to the beads were immunoblotted with the anti-MAGUIN antibody.Lane 1, the original Triton X-100 extract of rat crude synaptosome before the incubation; lane 2, with the preimmune serum; lane 3, with the anti-PSD-95/SAP90 antibody. B, interaction of MAGUIN-1 with the PDZ domains of PSD-95/SAP90. The extract of COS cells expressing various Myc-tagged PSD-95/SAP90 was incubated with GST-MAGUIN-12 (the C-terminal construct of MAGUIN-1) or GST-MAGUIN-16 (the C-terminal construct of MAGUIN-2) fixed on the glutathione beads, and the proteins attached to the beads were detected with the anti-Myc antibody. Lanes 1–3, the full-length of PSD-95/SAP90; lanes 4–6, the PDZ domains of PSD-95/SAP90; lanes 7-9, the Src homology 3 and GK domains of PSD-95/SAP90. Lanes 1, 4, and7, the original samples before the incubation; lanes 2, 5, and 8, after the incubation with GST-MAGUIN-12; lanes 3, 6, and 9, after the incubation with GST-MAGUIN-16.View Large Image Figure ViewerDownload (PPT) Northern blot analysis revealed 4.4- and 5.4-kilobase pair messages only in brain (Fig. 6). The two messages with different sizes may reflect differential polyadenylation. No message was detected in heart, spleen, lung, liver, kidney, skeletal muscles, or testis. In the subcellular fractionation of rat brain, MAGUINs were detected mainly in the synaptic plasma membrane (SPM) and PSD fractions (Fig.7 A). Two bands with different molecular sizes may represent protein degradation, post-translational modifications, or alternative splicing isoforms. In rat hippocampal neurons, MAGUINs were distributed in the cell body and the neurites and colocalized with NMDAR1 (Fig. 7 B).Figure 7Subcellular localization of MAGUINs. A, Western blot analysis of the subcellular fractions of rat brain. Equal aliquots of the subcellular fractions of rat brain (25 μg of protein each) were immunoblotted with the anti-MAGUIN antibody.Lane 1, the homogenate fraction; lane 2, the nuclear pellet fraction; lane 3, the crude synaptosomal fraction; lane 4, the synaptosomal cytosol fraction;lane 5, the crude synaptosomal pellet fraction; lane 6, the crude synaptic vesicle fraction; lane 7, the lysed synaptosomal membrane fraction; lane 8, the SPM fraction; lane 9, the 0.5% (w/v) Triton X-100-soluble fraction of the SPM; lane 10, the 0.5% (w/v) Triton X-100-insoluble fraction of the SPM; lane 11, the 1% (w/v) Triton X-100-soluble fraction of the SPM; lane 12, the 1% (w/v) Triton X-100-insoluble fraction of the SPM. B, expression of MAGUINs in rat primary cultured hippocampal neurons. Rat hippocampal neurons were immunostained with the polyclonal mouse anti-MAGUIN antibody. The scale bar indicates 10 μm.View Large Image Figure ViewerDownload (PPT) The PH domain is known to interact with the phospholipid membrane (reviewed in Refs. 39Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar and40Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar). To test whether MAGUINs associate with the plasma membrane through the PH domain, various Myc-tagged constructs of MAGUINs were transfected in CHO cells (Fig.8 A). Full-length MAGUIN-1 and -2 and the construct containing the PH domain with the C-terminal stretch were localized at the plasma membrane (Fig. 8 B,1f, 2f, and m). The construct containing the SAM and PDZ domains was distributed in the cytosol (Fig.8 B, n). The construct containing the C-terminal region of MAGUIN-1 was localized at the plasma membrane, although it lacked the PH domain (Fig. 8 B, c). The similar results were obtained in the subcellular fractionation of CHO cells transfected with these constructs. Full-length MAGUIN-1 was recovered in the membrane fraction (Fig.9 A). The N-terminal construct containing the SAM and PDZ domains was distributed more in the cytosol than in the membrane fraction (Fig. 9 B). The PH domain with the C-terminal stretch was recovered in the membrane fraction (Fig.9 C). The C-terminal construct was recovered mainly in the membrane fraction with a smaller amount in the cytosol (Fig.9 D). Because the construct containing only the PH domain was not expressed, we could not determine whether the PH domain was directly involved in the membrane association of MAGUIN-1. However, these findings suggest that MAGUIN-1 associates with the plasma membrane through the region containing the PH domain and the C-terminal stretch of the PH domain. The N-terminal, PH domain, and C-terminal constructs were Triton X-100-soluble, whereas the full-length construct of MAGUIN-1 was Triton X-100-insoluble (Fig. 9), suggesting that the whole structure of MAGUIN-1 is necessary for the interaction with the Triton X-100-insoluble structures.Figure 9Subcellular localization of MAGUIN-1 in CHO cells. CHO cells were transfected with various Myc-tagged constructs of MAGUIN-1. The homogenates of the cells were subfractionated into the cytosol, membrane, Triton X-100-soluble, and Triton X-100-insoluble fractions. Comparable amounts of fractions were immunoblotted with the monoclonal anti-Myc antibody. Lanes 1, homogenate; lanes 2, cytosol; lanes 3, membrane; lanes 4, Triton X-100-soluble; lanes 5, Triton X-100-insoluble. A, pCMV Myc MAGUIN-1f (the full length of MAGUIN-1). B, pClneo Myc MAGUIN-n (the SAM and PDZ domains). C, pClneo Myc MAGUIN-m (the PH domain with the C-terminal stretch). D, pClneo Myc MAGUIN-c (the C-terminal region of MAGUIN-1).View Large Image Figure ViewerDownload (PPT) In the last set of experiments, we tested whether MAGUIN-1 affected the subcellular localization of PSD-95/SAP90 and S-SCAM in the transfected cells. PSD-95/SAP90 and S-SCAM were distributed in the Triton X-100-soluble fraction in CHO cells (Fig.10, A and B). MAGUIN-1 and -2 were distributed in the Triton X-100-insoluble fraction (Fig. 9 A and data not shown). PSD-95/SAP90 and S-SCAM were recruited into the Triton X-100-insoluble fraction, when coexpressed with MAGUIN-1 (Fig. 10, C and D). In contrast, PSD-95/SAP90 and S-SCAM remained in the Triton X-100-soluble fraction, when coexpressed with MAGUIN-2 (data not shown). In this paper, we have identified a novel ligand for S-SCAM and named it MAGUIN-1. We have also reported its short isoform, MAGUIN-2. MAGUIN-1 binds to PSD-95/SAP90 as well as to S-SCAM. MAGUINs have a unique combination of protein modules including SAM, PDZ, and PH domains. SAM domain is proposed to mediate protein binding or DNA binding (reviewed in Refs. 28Ponting C.P. Protein Sci. 1995; 4: 1928-1930Crossref PubMed Scopus (142) Google Scholar and 29Schultz J. Ponting C.P. Hofman K. Bork P. Protein Sci. 1997; 6: 249-253Crossref PubMed Scopus (270) Google Scholar). The subcellular localization of MAGUINs precludes a DNA binding role, but some protein may interact with MAGUINs via the SAM domain. The PDZ domain is a well known protein module that binds to the C terminus of other proteins (reviewed in Refs. 2Kennedy M.B. Curr. Opin. Neurobiol. 1993; 3: 732-737Crossref PubMed Scopus (129) Google Scholar, 3Garner C. Kindler S. Trends Cell Biol. 1996; 19: 429-433Abstract Full Text PDF Scopus (63) Google Scholar, 4Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 5Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 6Hata Y. Nakanishi H. Takai Y. Neurosci. Res. 1998; 32: 1-7Crossref PubMed Scopus (74) Google Scholar). The study using the peptide library revealed the existence of two classes of the PDZ domains (41Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chisti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). Class I PDZ domains, such as those of PSD-95/SAP90, select the peptides containing Glu-(Ser/Thr)-Xaa-(Val/Ile) (Xaa is any amino acid) at the C terminus, whereas class II PDZ domains, such as that of CASK, select the peptides with hydrophobic or aromatic side chains at the C-terminal three residues (41Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chisti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). The PDZ domains are also reported to interact with other PDZ domains (36Brenman J.E. Chao D. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar, 42Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The PDZ domain is composed of two α helixes and six β sheets, and the second α helix and second β sheet provide a carboxyl-binding loop (32Doyle A.D. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 33Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (292) Google Scholar). The residues of this loop of MAGUINs are diverged from those of PSD-95/SAP90 or CASK, suggesting that the ligand for the PDZ domain of MAGUINs has the C-terminal residues different from either group 1 or group 2 consensus motif (41Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chisti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). No interacting protein was obtained from the yeast two-hybrid screening using the PDZ domain of MAGUINs as a bait (data not shown), and whether the PDZ domain of MAGUINs functions as a protein-interacting module needs to be investigated. The PH domain binds to inositol phosphates and phosphoinositides and regulates the membrane association of many signaling proteins (reviewed in Refs. 39Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar and 40Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar). We could not determine whether the PH domain was required for the membrane association, but we observed that MAGUIN-1 binds to the plasma membrane through the region containing the PH domain and the C-terminal region. This finding suggests that the PH domain can support the membrane attachment, as well as the C-terminal region. The C-terminal stretch of the PH domain of β-adrenergic receptor kinase is reported to bind the βγ subunits of heterotrimeric GTP-binding proteins (Gβγ) (43Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar). The C-terminal stretch of the PH domain of MAGUINs is diverged from that of β-adrenergic receptor kinase, and the interaction of MAGUIN-1 with Gβγ is not detected (data not shown). However, because the C-terminal region of MAGUIN-1 also mediates the membrane association, this region also has a binding activity for lipid or some membrane protein. MAGUIN-1 is a common ligand for PSD-95/SAP90 and S-SCAM, which are both neuronal multiple PDZ domain-containing proteins. PSD-95/SAP90 and S-SCAM bind NMDA receptors, K+ channels, and neuroligin through distinct PDZ domains and assemble these molecules at synaptic junctions. PSD-95/SAP90 further interacts with neuronal nitric-oxide synthase, synGAP, and CRIPT (16Kim J.H. Liao D. Lau L.-F. Huganir R.L. Neuron. 1998; 20: 683-691Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 17Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 18Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg R.J. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 36Brenman J.E. Chao D. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar). synGAP regulates the activity of a small GTP-binding protein, Ras (16Kim J.H. Liao D. Lau L.-F. Huganir R.L. Neuron. 1998; 20: 683-691Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 17Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). During this study, aDrosophila gene that genetically interacts with kinase suppressor of ras (ksr) has been reported and namedconnector enhancer of ksr (cnk) (44Therrien M. Wong A.M. Rubin G.M. Cell. 1998; 95: 343-353Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar).cnk functions in the Ras/mitogen-activated protein kinase (MAPK) pathway, and the product of cnk physically binds Raf kinase. CNK has a molecular structure similar to that of MAGUINs. Therefore, MAGUIN-1 may also bind Raf kinase and links it to PSD-95/SAP90 and S-SCAM. The yeast two-hybrid screening using the GK domain of PSD-95/SAP90 revealed SPA-1-like protein besides SAPAP and BEGAIN (22Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). SPA-1 is a GAP protein for Rap1 (45Kurachi H. Wada Y. Tsukamoto N. Maeda M. Kubota H. Hattori M. Iwaki K. Minato N. J. Biol. Chem. 1997; 272: 28081-28088Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and Rap1 plays roles in the MAPK pathway (46York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (762) Google Scholar). We have not confirmed the interaction of SPA-1-like protein with PSD-95/SAP90 using other methods and have not tested whether it has a GAP activity. However, these findings suggest a model that the components implicated in the Ras/MAPK pathway are assembled through the complex of PSD-95/SAP90 and MAGUIN-1. This model is interesting, because the Ras/MAPK pathway is suggested to be implicated in the synaptic plasticity (reviewed in Ref. 47Finkbeiner S. Greenberg M.E. Neuron. 1996; 16: 233-236Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). We are now testing this model. MAGUIN-1 is Triton X-100-insoluble and recruits PSD-95/SAP90 and S-SCAM into the Triton X-100-insoluble fraction. SAPAP has a similar activity for PSD-95/SAP90 and S-SCAM (22Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 2K. Hirao, unpublished observation. These findings suggest that PSD-95/SAP90 and S-SCAM are connected to the Triton X-100-insoluble structures via the PDZ domain by MAGUIN-1 and via the GK domain by SAPAP. MAGUIN-2 does not bind to PSD-95/SAP90 or S-SCAM. MAGUIN-2 may compete with MAGUIN-1 for the same ligands, such as Raf kinase, and switch off these ligands from the network around PSD-95/SAP90 and S-SCAM. C. elegans R01H10.8 also has a structure similar to that of MAGUINs and may be a homologue of MAGUINs (28Ponting C.P. Protein Sci. 1995; 4: 1928-1930Crossref PubMed Scopus (142) Google Scholar). C. eleganshas a putative S-SCAM homologue, K01A.6. Analysis of the mutants of R01H10.8 and K01A.6 may enlighten the physiological function of MAGUINs and the significance of the interaction of MAGUIN-1 with S-SCAM. We thank Nils Brose (Max Planck Institute) for the monoclonal anti-NMDAR1 antibody.
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Membrane-associated guanylate kinases (MAGUKs) are the major family of scaffolding proteins at the postsynaptic density. The PSD-MAGUK subfamily, which includes PSD-95, PSD-93, SAP97, and SAP102, is well accepted to be primarily involved in the synaptic anchoring of numerous proteins, including N-methyl-D-aspartate receptors (NMDARs). Notably, the synaptic targeting of NMDARs depends on the binding of the PDZ ligand on the GluN2B subunit to MAGUK PDZ domains, as disruption of this interaction dramatically decreases NMDAR surface and synaptic expression. We recently reported a secondary interaction between SAP102 and GluN2B, in addition to the PDZ interaction. Here, we identify two critical residues on GluN2B responsible for the non-PDZ binding to SAP102. Strikingly, either mutation of these critical residues or knockdown of endogenous SAP102 can rescue the defective surface expression and synaptic localization of PDZ binding-deficient GluN2B. These data reveal an unexpected, nonscaffolding role for SAP102 in the synaptic clearance of GluN2B-containing NMDARs.
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Guanylate kinase
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Abstract Membrane‐associated guanylate kinase‐interacting protein (MAGUIN) has been identified as a protein binding postsynaptic density (PSD)‐95 and synaptic scaffolding molecule (S‐SCAM). MAGUIN has one sterile α motif, one conserved region in connector enhancer of ksr (Cnk) (CRIC), one PSD‐95/Dlg‐A/ZO‐1 (PDZ) and one pleckstrin homology (PH) domain. There are two isoforms, MAGUIN‐1 and ‐2. MAGUIN‐1 binds the PDZ domains of PSD‐95 and S‐SCAM by the C‐terminus, whereas MAGUIN‐2 does not bind to PSD‐95 or S‐SCAM. Here, we have determined that MAGUIN‐2 is also localized at synapses and that the synaptic localization of MAGUIN depends on the pleckstrin homology domain. The overexpressed C‐terminal PDZ‐binding region inhibits the synaptic targeting of PSD‐95. Furthermore, the synaptic targeting of MAGUIN does not require N‐methyl‐ d ‐aspartate (NMDA) receptor activity. These findings suggest that MAGUIN‐1 and ‐2 are recruited to synapses by the PH domain and that MAGUIN‐1 subsequently interacts with PSD‐95 at synapses.
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Guanylate kinase
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PDZ domain
Guanylate kinase
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PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms scaffolding interactions with partner proteins, including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/disks large/zona occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the ligand association and small-angle x-ray scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95.
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단백질-단백질 결합은 수용체 단백질, 효소, 세포 골격 단백질의 세포내 위치 결정 및 기능 조절에 중요한 역할을 한다. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) 도메인을 가진 단백질들은 시냅스 가소성, 신경세포 성장과 분화뿐만 아니라 많은 질병의 병태생리에 중요하게 관여하는 scaffold 단백질로 작용한다. Multi-PDZ domain protein 1 (MUPP1)은 13개 PDZ 도메인을 가지는 단백질로서 세포막 수용체 군집화, 신호전달 복합체 구성, 세포 골격 조정에 대한 매개 역할을 하는 것으로 알려지고 있지만 MUPP1의 세포 내 기능은 아직 명확히 밝혀지지 않았다. 본 연구에서 MUPP1의 아미노 말단 PDZ 도메인과 결합하는 새로운 단백질을 규명하기 위하여 효모 two-hybrid 방법을 이용하였고 Wdpcp (전에 Fritz로 알려짐)이 MUPP1과 결합하는 것을 확인하였다. Wdpcp는 planar cell polarity (PCP) effector로서 세포 이동과 섬모형성에 관여하는 것으로 알려져 있다. Wdpcp는 MUPP1의 첫 번째 PDZ 도메인과 결합하지만, 다른 PDZ 도메인과는 결합하지 않았다. 또한 MUPP1와 Wdpcp의 결합에서 Wdpcp의 C-말단부위가 결합에 필수적임을 효모 two-hybrid 방법으로 확인하였다. 이러한 단백질간 결합은 glutathione S-transferase (GST) pull-down assay, 공동면역침강, HEK-293T 세포에서의 발현위치를 통하여 추가적으로 확인하였다. 이러한 결과들은, MUPP1과 Wdpcp 결합은 세포내 액틴 다이내믹스(dynamics)와 세포이동 조절에 역할을 할 가능성을 시사한다. Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.
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PDZ domain
Guanylate kinase
Postsynaptic density
Linker
Protein kinase domain
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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. 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. Communication and adhesion between cells is mediated by specialized regions of the plasma membrane. For example, in excitatory synapses in the brain, the postsynaptic membrane contains an actin-rich cytoskeletal region known as the postsynaptic density (1Sheng M. Pak D.T. Annu. Rev. Physiol. 2000; 62: 755-778Crossref PubMed Scopus (307) Google Scholar). Analogous structures are present at sites of cell-cell contact, including the junctions between epithelial cells, which are important for signaling and the formation of physical barriers (2Tepass U. Tanentzapf G. Ward R. Fehon R. Annu. Rev. Genet. 2001; 35: 747-784Crossref PubMed Scopus (420) Google Scholar, 3Matter K. Balda M.S. Nat. Rev. Mol. Cell Biol. 2003; 4: 225-236Crossref PubMed Scopus (720) Google Scholar). The establishment and function of these important structures is regulated by a large number of proteins that serve to organize receptors and downstream signaling proteins and to anchor signaling complexes at specific membrane locations. Membrane-associated guanylate kinases (MAGUKs) 2The abbreviations used are: MAGUK, membrane associated guanylate kinase; GukH, GukHolder; GK, guanylate kinase; SH3, Src homology 3; Discs Large, ZO-1; Dlg, Discs Large; GKAP, GK-associated protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HA, hemagglutinin; GFP, green fluorescent protein; MAP, mitogen-activated protein. are scaffolding proteins that regulate the formation and function of membrane specializations, such as synapses and tight junctions (3Matter K. Balda M.S. Nat. Rev. Mol. Cell Biol. 2003; 4: 225-236Crossref PubMed Scopus (720) Google Scholar, 4Montgomery J.M. Zamorano P.L. Garner C.C. Cell. Mol. Life Sci. 2004; 61: 911-929Crossref PubMed Scopus (102) Google Scholar). MAGUKs have a unique domain architecture that is typified by one or three PDZ domains, an SH3 domain, a variable HOOK sequence, and a region with homology to the enzyme guanylate kinase (GK) that lacks enzymatic activity but instead acts as a protein interaction domain. The SH3 and GK domains form an intramolecular interaction in the MAGUK PSD-95, which is thought to be a common feature of MAGUK proteins (5McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 6Shin H. Hsueh Y.P. Yang F.C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar). One of the best studied MAGUK proteins is the Drosophila tumor suppressor Discs Large (Dlg). Dlg plays a role in the formation and function of diverse polarized cellular structures, including epithelial junctions (7Hough C.D. Woods D.F. Park S. Bryant P.J. Genes Dev. 1997; 11: 3242-3253Crossref PubMed Scopus (124) Google Scholar), stem cell cortical domains (8Peng C.Y. Manning L. Albertson R. Doe C.Q. Nature. 2000; 408: 596-600Crossref PubMed Scopus (287) Google Scholar, 9Ohshiro T. Yagami T. Zhang C. Matsuzaki F. Nature. 2000; 408: 593-596Crossref PubMed Scopus (271) Google Scholar), and neuronal synapses (10Budnik V. Koh Y.H. Guan B. Hartmann B. Hough C. Woods D. Gorczyca M. Neuron. 1996; 17: 627-640Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). In the neuromuscular synapse, Dlg is present at high levels at both pre- and postsynaptic sites (11Lahey T. Gorczyca M. Jia X.X. Budnik V. Neuron. 1994; 13: 823-835Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Dlg is thought to function at these sites by clustering ion channels and organizing signal transduction pathways. The intramolecular interaction between the SH3 and GK domains is important for MAGUK function. All genetically identified mutations in the SH3 and GK regions of dlg and the related Caenorhabditis elegans lin-2 gene disrupt the intramolecular interaction (5McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). However, the exact role of the intramolecular interaction in MAGUK function has remained obscure. The crystal structure of the PSD-95 SH3-GK revealed that the two domains interact through a unique mechanism in which a two-stranded β-sheet is composed of strands that emerge from the SH3-HOOK and GK domains (12McGee A.W. Dakoji S.R. Olsen O. Bredt D.S. Lim W.A. Prehoda K.E. Mol. Cell. 2001; 8: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 13Tavares G.A. Panepucci E.H. Brunger A.T. Mol. Cell. 2001; 8: 1313-1325Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The nature of this interaction is such that movements of the domains relative to one another could create functionally distinct conformations that result from hinge movements about the linking strands. However, how the distribution among these conformations might be modulated and what their functions are has remained unclear. One function of the SH3-GK intramolecular interaction may be to regulate the assembly of MAGUK complexes. For example, GK-associated protein (GKAP) binds to a fragment of the MAGUK SAP-97 containing only the GK domain but fails to bind to the SH3-GK, indicating competition between the intra- and intermolecular interactions (14Wu H. Reissner C. Kuhlendahl S. Coblentz B. Reuver S. Kindler S. Gundelfinger E.D. Garner C.C. EMBO J. 2000; 19: 5740-5751Crossref PubMed Scopus (94) Google Scholar). A secondary intramolecular event with an NH2-terminal L27 domain rescues the interaction with GKAP in the full-length protein. However, the mechanism by which this interaction may be regulated in the context of the full-length protein is unknown. Not all SH3-GK ligands compete against the intramolecular interaction, indicating that multiple binding surfaces are utilized by ligands of this unique domain. Here we have analyzed how binding of Dlg to the synaptic protein GukHolder (GukH) is regulated by complex interdomain interactions within Dlg that involve transitions in the SH3-GK intramolecular interaction. GukH was first identified in a yeast two-hybrid screen as a binding partner for the Dlg GK domain (15Mathew D. Gramates L.S. Packard M. Thomas U. Bilder D. Perrimon N. Gorczyca M. Budnik V. Curr. Biol. 2002; 12: 531-539Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and mammalian homologues have been identified (16Katoh M. Int. J. Oncol. 2004; 24: 1033-1038PubMed Google Scholar, 17Sharma S. Ang S.L. Shaw M. Mackey D.A. Gecz J. McAvoy J.W. Craig J.E. Hum. Mol. Genet. 2006; 15: 1972-1983Crossref PubMed Scopus (39) Google Scholar). GukH colocalizes with Dlg at synaptic borders, and the interaction of the two proteins appears to be required for proper localization of the tumor suppressor Scribble. GukH also colocalizes with Dlg in neuroblasts, precursors of the Drosophila central nervous system (18Albertson R. Doe C.Q. Nat. Cell Biol. 2003; 5: 166-170Crossref PubMed Scopus (203) Google Scholar). The interaction of GukH with Dlg is mediated by an ∼300-residue region at the GukH COOH terminus that contains no known domains. We find that GukH binding to Dlg is actively regulated by the SH3-GK intramolecular interaction. GukH binds to a composite site formed by not only the GK domain but the SH3 domain as well. However, the intramolecular interaction between the two domains competes against GukH binding. Binding is rescued by a PDZ domain directly NH2-terminal to the SH3 domain, a common feature of MAGUK proteins. The complex interdomain interactions in Dlg cause GukH-Dlg- and PDZ-bound complexes to be mutually exclusive in a cellular context. These results have implications for the types of complexes that are formed by Dlg and therefore its role in regulating the formation and function of membrane specializations, such as epithelial junctions and synapses. Molecular Cloning, Protein Expression, and Purification—DNA encoding full-length Drosophila, the PE isoform of Dlg (which contains the "I3" exon), and the GukH Dlg binding domain fragment from the PA isoform (1494–1788) were cloned from an embryonic cDNA library. For the PDZ, PDZ-SH3, SH3, GK, HOOK-GK, SH3-GK, linker-SH3-GK, and PDZ-SH3-GK fragments of Dlg, amino acids 481–571, 481–691, 581–681, 764–960, 675–960, 598–960, 560–960, and 474–960 were used, respectively. The sequence of all inserts was verified by DNA sequencing. For expression of glutathione S-transferase (GST) fusions, cDNAs were ligated into pGEX 4T-1, whereas the pET-19b derivative pBH was used for hexahistidine tags. The pBH vector encodes for a tobacco etch virus protease site following the hexahistidine tag to allow for removal of the tag. All proteins were expressed in the Escherichia coli strain BL21(DE3). Hexahistidine fusion proteins were purified using nickel-nitrilotri-acetic acid resin and standard protocols. Unless otherwise noted, nickel-nitrilotriacetic acid purification was followed by incubation with tobacco etch virus protease to remove the histidine tag (after cleavage, the protein contains an extra glycine and serine residue on the NH2 terminus). Ion exchange chromatography was used to further purify proteins if necessary. Purity was established using SDS-PAGE and/or MALDI-TOF mass spectrometry. Binding Assays—For yeast two hybrid, the vectors pGADT7 and pGBKT7 were from Clontech (Palo Alto, CA). GukH-(1494–1788) was cloned into the Gal4 activation domain vector pGADT7, whereas all of the DLG constructs were cloned into DNA binding domain vector pGBKT7. The Saccharomyces cerevisiae HF7c strain was transformed with pGADT7 and pGBKT7 plasmids and plated on SD medium (-Leu/-Trp/-His, +20 mm 3-aminotriazole (3-AT; Sigma)). Colonies were tested for β-galactosidase activity using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) as substrate. For qualitative "pulldown" assays, E. coli cell lysates containing the GST fusion protein of interest were incubated with glutathione-agarose beads and washed with binding buffer (10 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, 0.5% Triton X-100). Potential interacting proteins were added to a concentration of 10 μm and incubated with the beads at room temperature for 15 min. The reactions were washed three times with binding buffer to remove unbound proteins. Bound proteins were eluted with SDS loading buffer and analyzed by staining with Coomassie Blue and/or Western blotting using an anti-hexahistidine antibody (Qiagen). For quantitative binding measurements, a peptide with the sequence from the last ten residues of CRIPT and an NH2-terminal rhodamine was synthesized by Fmoc solid phase peptide synthesis. A sulfonyl chloride rhodamine derivative (Molecular Probes L-20) was used after the addition of the final Fmoc amino acid. Following cleavage from the solid support, the peptide was purified by reverse-phase high pressure liquid chromatography and its sequence verified by MALDI-TOF mass spectrometry. A series of solutions were prepared with increasing concentrations of the appropriate Dlg fragment and a concentration of 100 nm rhodamine-labeled peptide. The anisotropy of each solution was measured using an ISS PC1 fluorometer. The Kd of interactions was determined by nonlinear fitting of the data to a bimolecular binding equation. Cell Culture and Immunoprecipitation—We transiently transfected Drosophila S2 cells grown in Schneider's insect medium supplemented with 10% fetal bovine serum with expression vectors for full-length Dlg and/or the GFP-CRIPT and hemagglutinin (HA)-GukH Dlg binding domains using 1 μg of total DNA, which resulted in an efficiency of ∼30%. We induced protein expression after 24 h using 0.5 mm copper sulfate and collected the cells after an additional 24 h of growth. To collect the cells, we centrifuged them for 5 min at 1000 × g and washed the resulting pellet with ice-cold phosphate-buffered saline twice. For immunoprecipitation experiments, extracts were prepared by incubation with lysis buffer (150 mm NaCl, 1% Nonidet P-40, 50 mm Tris, pH 8.0, 1 mm phenylmethylsulfonyl fluoride) for 30 min on ice. Cell lysate was precleared by gently mixing with protein A-Sepharose beads (Amersham Biosciences) at 4 °C for 1 h. The beads were then removed by centrifugation at 12,000 × g for 20 s. The proteins were immunoprecipitated by incubating anti-HA, anti-GFP, or anti-His with precleared lysate at 4 °C for 1 h. Protein A-Sepharose beads were added to the mixture and incubated at 4 °C for 1 h with rotation. The pellets were collected at 12,000 × g for 20 s and washed three times with lysis buffer and once with phosphate-buffered saline. The final pellets were suspended in protein loading buffer and analyzed by SDS-PAGE followed by western. For immunostaining, Dlg localization was detected with an anti-Dlg antibody and Cy3-labeled secondary antibody (endogenous Dlg was below the level of detection for immunostaining). After labeling, the cells were imaged by confocal microscopy on a Nikon Eclipse TE2000-U microscope with a Photometrics CoolSNAP fx CCD camera. Images were analyzed with the ImageJ software (NIH), and cells were binned into cortical or cytoplasmic localization based upon the pixel intensity distribution across the cell. Cells having cortical signal intensity 2× or greater than the cytoplasmic pool were scored as cortically localized. Results are reported from two independent experiments. The Third Dlg PDZ Domain Modulates the Interaction of Dlg with GukH—The interaction of Dlg with GukH has been shown to occur through the Dlg GK domain (15Mathew D. Gramates L.S. Packard M. Thomas U. Bilder D. Perrimon N. Gorczyca M. Budnik V. Curr. Biol. 2002; 12: 531-539Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). While analyzing fragments of Dlg for their ability to bind to GukH, we found that, although the GK is able to bind GukH, a fragment that also includes the SH3 domain does not bind (Fig. 1A). This result indicates that the intramolecular interaction between the SH3 and GK domains competes with GukH binding. However, elements NH2-terminal to the SH3-GK control the ability of Dlg to interact with GukH, as larger fragments of Dlg, including the full-length protein, are able to bind GukH. We analyzed several Dlg fragments to identify the minimal components necessary for regulation of the Dlg-GukH complex assembly using a GST fusion of the GukH Dlg binding domain (Fig. 1B). A fragment containing the third PDZ domain along with the short linker that connects it to the SH3-GK module are necessary and sufficient to allow GukH binding (Fig. 1, A and C). As shown in Fig. 1D, the PDZ-SH3-GK architecture is highly conserved among MAGUK proteins with the number of residues linking the PDZ and SH3 domains ranging from 5 to 40. The functional linkage between the Dlg PDZ and SH3-GK domains and the conserved architecture of these domains in MAGUK proteins suggests that the PDZ domain is an integral component of a larger PDZ-SH3-GK module. GukH Binds to a Composite Site on Dlg Formed by Both the SH3 and GK Domains—In experiments using purified components, we noticed that a proteolytic fragment of the PDZ-SH3-GK also interacts with GukH (Fig. 1C, starred band). This proteolytic fragment corresponds to a COOH-terminal truncation that lacks the GK domain (based on the molecular weight of the fragment and the presence of the NH2-terminal His tag), indicating that one or more additional GukH binding sites exist outside of the GK domain. We tested both the Dlg PDZ and SH3 domains for the ability to bind GukH. Consistent with a GukH binding site in Dlg outside of the GK domain, purified fragments of Dlg that lack the GK but contain the SH3 domain are able to bind GukH (Fig. 2A). However, the third PDZ domain is not required for GukH binding in this context. This interaction is qualitatively weaker than with the PDZ-SH3-GK (Fig. 2B), consistent with the SH3 domain being only one part of a larger interaction surface that includes the GK domain. As the region of GukH that binds to Dlg contains several proline-rich sequences (Fig. 2C) and SH3 domains bind to a consensus sequence of PXXP (19Zarrinpar A. Bhattacharyya R.P. Lim W.A. Science's STKE 2003. 2003; : RE8Google Scholar), we tested these sequences for their ability to bind the Dlg SH3 domain (using an SH3 domain that lacked the "HOOK" segment that links the SH3 and GK domains). Although MAGUK SH3s deviate from canonical SH3 domains (12McGee A.W. Dakoji S.R. Olsen O. Bredt D.S. Lim W.A. Prehoda K.E. Mol. Cell. 2001; 8: 1291-1301Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), we found that each of the GukH proline-rich segments is able to bind to the Dlg SH3 domain (Fig. 2D). Similar to the interaction of GukH with the Dlg GK domain, the binding site on the SH3 domain is obscured when the GK domain is present (Fig. 2E), presumably because of the intramolecular interaction between the two domains. Another similarity between the two binding sites is that GukH binding is rescued by the presence of the third PDZ domain (Fig. 2E). Binding of proline-rich sequences requires the presence of specific proline residues as mutation of the PXXP to AXXA completely disrupts binding (Fig. 2D). We therefore conclude that GukH binds to a composite binding site formed by the SH3 and GK domains and that this binding site is obscured by the intramolecular interaction between the two domains. The presence of three SH3 ligand sequences in GukH indicates that each GukH may bind multiple Dlg proteins. Modulation of GukH Binding by COOH-terminal PDZ Ligands—The interplay between the Dlg PDZ and SH3-GK modules appears to be a mechanism for communication between the PDZ and SH3-GK binding sites. PDZ domains are common protein interaction domains that bind short, COOH-terminal sequences present in target proteins (20Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar), although binding to internal motifs can also occur (21Penkert R.R. DiVittorio H.M. Prehoda K.E. Nat. Struct. Mol. Biol. 2004; 11: 1122-1127Crossref PubMed Scopus (107) Google Scholar). The third PDZ domain from Dlg or its mammalian homologues has been shown to bind two COOH-terminal sequences, one from CRIPT (cysteine-rich interactor of PDZ; sequence DTKNYKQTSV-COOH) and Drosophila neuroligin (sequence KRVHIQEISV-COOH) (22Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg T.M. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 23Lise M.F. El-Husseini A. Cell Mol. Life Sci. 2006; 63: 1833-1849Crossref PubMed Scopus (136) Google Scholar). CRIPT binds to microtubules providing a link between MAGUK scaffolding proteins and the cytoskeleton (24Passafaro M. Sala C. Niethammer M. Sheng M. Nat. Neurosci. 1999; 2: 1063-1069Crossref PubMed Scopus (97) Google Scholar), whereas neuroligin is a membrane protein involved in synapse formation (23Lise M.F. El-Husseini A. Cell Mol. Life Sci. 2006; 63: 1833-1849Crossref PubMed Scopus (136) Google Scholar). We synthesized peptides containing the last ten residues of CRIPT and neuroligin and used these peptides to probe the coupling of PDZ and SH3-GK ligand binding activity. Although the PDZ-SH3-GK fragment is able to bind GukH, the addition of the CRIPT or neuroligin peptide lowers the affinity for GukH in a dose-dependent fashion, whereas an unrelated PDZ ligand peptide has no effect (Fig. 3A). These results indicate that PDZ ligand binding influences the GukH (SH3-GK) binding site. The communication between the Dlg PDZ and SH3-GK domains must have energetic consequences. To explore the coupling between the two, we measured the affinity of the CRIPT and neuroligin peptides for the isolated PDZ domain and the entire PDZ-SH3-GK fragment using the fluorescence anisotropy of NH2-terminal tetramethylrhodamine (Fig. 3B). Although the affinity of CRIPT for the isolated PDZ domain is 14 μm, which is typical for interactions of isolated PDZ domains with COOH-terminal ligands, the affinity of CRIPT for the PDZ-SH3-GK fragment is 0.8 μm, representing an ∼15-fold difference in affinity. No binding was observed to the SH3-GK (data not shown). The difference between PDZ-SH3-GK and PDZ binding is similar for neuroligin (Kd = 71.5 and ∼900 μm, respectively), although the affinities are significantly weaker. The difference between the affinity for the two fragments is consistent with the coupling of PDZ ligands and GukH binding sites. However, as the affinity for PDZ-SH3-GK is higher than for PDZ alone, the data excludes a simple model in which the PDZ ligands compete with an intramolecular interaction formed with the PDZ domain. Interdomain PDZ-SH3-GK Interactions Lead to Mutually Exclusive Cellular Complexes—How might the complex interdomain interactions in the Dlg PDZ-SH3-GK module affect the complexes that Dlg forms in a cellular context? The in vitro analysis of these proteins indicates that GukH-Dlg and PDZ ligand complexes are likely to exist as distinct complexes (Fig. 4A). To examine whether the CRIPT and GukH interactions affected the complexes that Dlg forms in a cellular context, we transfected Drosophila S2 cells with the Dlg binding domains from GukH and CRIPT. Immunoprecipitation of the Dlg ligands from extracts of these cells leads to coimmunoprecipitation of full-length Dlg (Fig. 4B). However, neither of the Dlg ligands is able to immunoprecipitate the other, consistent with a model in which GukH and PDZ ligands form mutually exclusive Dlg complexes. We also examined the effect of the GukH and CRIPT Dlg binding domains on Dlg localization in these cells. Immunofluorescence from an HA fusion of the GukH Dlg binding domain shows a large percentage of cells with cortical localization (Fig. 4C), whereas full-length Dlg is consistently found in the cytoplasm, excluded from the nucleus (Fig. 4D). Expression of GukH induces localization of Dlg at the cell cortex in a significant fraction of the transfected cells (Fig. 4E), indicating that GukH recruits Dlg to the cell cortex. Expression of a GFP fusion of the Dlg binding portion of CRIPT (which localizes to the cytoplasm) leads to a significant decrease in the fraction of cells with cortical Dlg localization (Fig. 4F), which we interpret as arising from competition between the CRIPT and GukH complexes of Dlg. Additionally, a Dlg mutant in which the PDZ domain no longer rescues the ability to bind GukH (DlgΔΔ; see below) is not significantly localized to the cortex by GukH. These results are also consistent with mutually exclusive PDZ ligand and GukH-Dlg complexes. Mechanism of PDZ-based Regulation of the SH3-GK Module—How does binding of COOH-terminal ligands to the PDZ domain alter the ability of SH3-GK to bind GukH? As the CRIPT and GukH binding sites are likely to be fairly distant from one another, competition through a direct steric mechanism is unlikely. One possible mechanism for PDZ regulation of the SH3-GK module is that the PDZ domain disrupts the intramolecular interaction between the SH3 and GK domains to expose the composite GukH binding site. In this model, the PDZ-SH3 would fail to interact with the GK. However, in an intermolecular assay, in which the PDZ-SH3 and HOOK-GK are separately expressed, we find that these two domains are able to bind one another and that this binding is not qualitatively altered by the presence of CRIPT peptide (Fig. 5A). This indicates that the SH3-GK intramolecular interaction is not qualitatively affected by the PDZ domain. The Dlg PDZ-SH3-GK module contains a conserved linker between the PDZ and SH3 domains (Fig. 5B). In the structure of the Dlg PDZ domain (25Morais Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (292) Google Scholar), a portion of the sequence following the PDZ domain forms a short helix that packs against the PDZ domain. The conservation and structure of this ∼40-residue sequence prompted us to examine the role of the linker in functionally coupling the PDZ and SH3-GK modules. To test the contribution of this sequence to the coupling between CRIPT and GukH binding, we constructed a series of PDZ-SH3-GK fragments with short deletions in the sequence. When either half of the linker is removed (Δ1 and Δ2), the PDZ domain no longer efficiently rescues binding of GukH to the composite SH3-GK binding site (Fig. 5C). Combining both deletions (ΔΔ) results in an even more severe effect. In addition, we find that the PDZ domain is unable to relieve GukH inhibition in trans. 3Y. Qian and K. E. Prehoda, unpublished observations. To determine whether the sequence of the linker is important or, alternatively, whether the spacing provided by the linker is only required, we replaced the deleted residues with glycine-serine repeats (Fig. 5C, GS). As this flexible linker is unable to restore GukH binding activity, the linker does not function solely to provide proper spacing between the domains. These results indicate that the covalent attachment of the PDZ and SH3 domains through a conserved linker is necessary for the regulation of the composite GukH binding site and that this regulation does not occur by disruption of the SH3-GK intramolecular interaction. We have demonstrated a set of interdomain interactions in the Drosophila tumor suppressor Dlg that leads to regulation of the ligand binding activity of these domains. The Dlg SH3-GK module, a defining feature of MAGUK proteins, forms a composite binding site for the synaptic protein GukH, but the intramolecular interaction between the SH3 and GK domains obscures this binding site. The third PDZ domain from Dlg relieves this inhibition, making use of the short linker that attaches it to the SH3 domain. Binding of CRIPT to the PDZ domain induces a change in the SH3-GK module that again obscures the GukH binding site, effectively leading to competition between CRIPT and GukH binding that influences the organization of Dlg-mediated protein complexes in a cellular context. The active scaffolding of Dlg complexes does not utilize disruption of the SH3-GK intramolecular interaction but requires a conserved, structured linker that attaches the PDZ and SH3 domains. Active Scaffolding of MAGUK Complexes—Cellular signaling relies on the formation of specific protein complexes, and scaffolding proteins play a central role in this process (26Vondriska T.M. Pass J.M. Ping P. J. Mol. Cell. Cardiol. 2004; 37: 391-397Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Although scaffolds are critical components of many signaling pathways, their exact function has remained unclear (27Ferrell Jr., J.E. Science's STKE 2000. 2000; : PE1Google Scholar). In particular, are scaffolds simple tethers that passively bind to their many ligands or is ligand binding actively regulated? The answers to these questions have significant implications for the types of scaffold-mediated complexes that are formed in cells. In the yeast mitogen-activated protein (MAP) kinase scaffold Ste5, heterologous protein interaction domains can functionally replace the native kinase recruitment modules, although at reduced levels (28Park S.H. Zarrinpar A. Lim W.A. Science. 2003; 299: 1061-1064Crossref PubMed Scopus (291) Google Scholar). As the heterologous domains are unlikely to participate in interactions that would lead to regulated binding of scaffold ligands, this suggests that certain scaffolds may function in a passive manner. However, the fact that domain-swapped Ste5 scaffolds do not function at wild-type levels leaves open the possibility that this scaffold also has characteristics of an active scaffold. Clearly, in the case of Dlg, however, the complexes that it forms are regulated by dynamic interdomain interactions. We are currently assessing how these interactions affect the diversity of Dlg complexes that might be formed in different cellular contexts. PDZ-based Regulation of MAGUK Complex Formation—How might the third PDZ domain from Dlg expose the composite GukH binding site within the SH3-GK module? Our data exclude a simple model in which the PDZ domain directly competes against the interaction between the SH3 and GK domains as the PDZ-SH3 and GK domains bind to one another in an intermolecular assay (Fig. 5A). We propose that the PDZ domain stabilizes the linker that connects it to the SH3 domain and that this linker interacts with the SH3 domain to induce an SH3-GK conformation that allows for GukH binding. The structure of the PSD-95 SH3-GK indicates that the SH3 ligand binding site is obscured in the closed conformation. The PDZ must therefore alter the position of residues that occupy the PXXP binding site to allow for GukH binding. Although there is no direct structural information on the position of the PDZ domain, based on the NH2 terminus of the SH3 domain, we can infer that the approximate position of the PDZ domain is likely to be in close proximity to the PXXP binding site. Binding of CRIPT would then return the SH3-GK to its basal conformation, which binds GukH with low affinity. This would explain why the affinity of CRIPT for the PDZ-SH3-GK fragment is higher than that for the PDZ alone. Such a supramolecular interaction induced by a PDZ ligand has been observed in the PDZ-regulated protease DegS (29Walsh N.P. Alba B.M. Bose B. Gross C.A. Sauer R.T. Cell. 2003; 113: 61-71Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 30Wilken C. Kitzing K. Kurzbauer R. Ehrmann M. Clausen T. Cell. 2004; 117: 483-494Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) (see below). The PDZ domain of the MAGUK PSD-93 has also been shown to be involved in the regulation of its SH3-GK module, although in this case, the PDZ domain negatively regulates ligand binding to SH3-GK. A fragment of PSD-93 containing the SH3 and GK domains binds to microtubule-associated protein 1A (MAP1A), but MAP1A fails to bind to full-length PSD-93 (31Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Crossref PubMed Google Scholar). Binding of MAP1A is restored by the presence of a COOH-terminal ligand for the third PSD-93 PDZ domain, although the other two PDZ domains appear to play a small role. In this system, the PDZ domain appears to repress binding to the SH3-GK and the PDZ ligand somehow restores binding. The distinct behavior of these two systems indicates that ligands can utilize the interdomain interactions in MAGUK proteins to achieve very different regulatory effects. Regulation of interactions that are modulated by the SH3-GK intramolecular interaction have also been shown to occur by PDZ-independent mechanisms. The GK domain from the Dlg homologue SAP97 binds to GKAP (32Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (434) Google Scholar). GKAP binding is inhibited by the SH3-GK intramolecular interaction (14Wu H. Reissner C. Kuhlendahl S. Coblentz B. Reuver S. Kindler S. Gundelfinger E.D. Garner C.C. EMBO J. 2000; 19: 5740-5751Crossref PubMed Scopus (94) Google Scholar). In this case, binding is rescued by an L27 domain present at the very NH2 terminus of the protein. However, how GKAP binding might be modulated in the context of the full-length protein is unknown. PDZ domains have been utilized in the regulation of diverse functions including the enzymatic activity of proteases. In the DegS protease, which is responsible for initiation of the misfolded protein proteolytic cascade in the periplasm of bacteria, a NH2-terminal PDZ domain regulates the protease domain activity using a mechanism that may be similar to PDZ regulation of the Dlg SH3-GK module. In DegS, the PDZ domain does not directly repress the protease domain. Instead, the protease is normally found in an inactive conformation (30Wilken C. Kitzing K. Kurzbauer R. Ehrmann M. Clausen T. Cell. 2004; 117: 483-494Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Activation occurs when a COOH-terminal ligand binds to the PDZ domain, which induces an interaction between a loop within the protease domain and PDZ ligand. This interaction causes a large change in the protease to an active conformation. Such an interaction with the ligand of the Dlg PDZ domain would be consistent with the higher affinity of the PDZ-SH3-GK module for this ligand. We thank C. Doe, A. McGee, and T. Stevens for critical reading of the manuscript. We thank W. Breyer for help at the inception of this project.
PDZ domain
Guanylate kinase
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PDZ domain
Guanylate kinase
Postsynaptic density
Pleckstrin homology domain
Protein kinase domain
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