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• W1969863013 abstract "The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase ) domain. It binds to theN-methyl-d-aspartate glutamate receptor NR2 subunit or to the Shaker-type K+ channel, Kv1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mΔ) and/or PDZ2 (PDZ2mΔ) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Kv1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mΔ) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (PSD-95:1mΔ-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mΔ was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Kv1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity. The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase ) domain. It binds to theN-methyl-d-aspartate glutamate receptor NR2 subunit or to the Shaker-type K+ channel, Kv1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mΔ) and/or PDZ2 (PDZ2mΔ) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Kv1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mΔ) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (PSD-95:1mΔ-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mΔ was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Kv1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity. postsynaptic density synapse-associated protein membrane-associated guanylate kinase N-methyl-d-aspartate PSD-95/discs large/ZO-1 guanylate kinase glutathioneS-transferase phosphate-buffered saline Src homolgy-3 As viewed by an electron microscope, dense thickenings are visible beneath the excitatory postsynaptic membranes in the central nervous system (1Ziff E.B. Neuron. 1997; 19: 1163-1174Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). More than 30 proteins have been identified that associate with these specialized submembraneous structures, referred to as postsynaptic density (PSD)1(2Walikonis R.S. Jensen O.N. Mann M. Provance Jr., D.W. Mercer J.A. Kennedy M.B. J. Neurosci. 2000; 20: 4069-4080Crossref PubMed Google Scholar). Many of these proteins in the PSD fraction are insoluble in mild detergents such as Triton X-100 (3Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). Therefore, the PSD is considered to be a tight aggregation of associated proteins that regulate synaptic transmission (4Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 5Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 6Sheng M. Pak D.T.S. Annu. Rev. Physiol. 2000; 62: 755-778Crossref PubMed Scopus (307) Google Scholar). PSD-95/SAP90, a main component of the PSD fraction (3Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 7Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauner U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar), is a member of the membrane-associated guanylate kinase (MAGUK) superfamily, which has multiple protein-protein interaction domains. MAGUK proteins, localized mainly beneath the membrane at cell-cell interaction sites, have been implicated as central scaffolds for the protein assembly of specialized membrane domains such as PSDs in neurons, tight junctions in epithelia, and neuromuscular junctions (8Willott E. Balda M.S. Fanning A.S. Jameson B. Van Itallie C. Anderson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7834-7838Crossref PubMed Scopus (425) Google Scholar, 9Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 10Tejedor F.J. Bokhari A. Rogero O. Gorczyca M. Zhang J. Kim E. Sheng M. Budnik V. J. Neurosci. 1997; 17: 152-159Crossref PubMed Google Scholar, 11Lahey T. Gorczyca M. Jia X.X. Budnik V. Neuron. 1994; 13: 823-835Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Strong support for the role of PSD-95 as a central organizer is found in its ion channel clustering activity. When PSD-95 is expressed heterologously together with the Shaker-type K+ channel (Kv1.4) or NMDA glutamate receptors, the expressed proteins become colocalized in plaque-like clusters (12Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar, 13Kim E. Cho K.-O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar), whereas when the Kv1.4 channel, the NMDA receptor, or PSD-95 is expressed individually, the proteins are localized diffusely throughout the cellular membranes or the cytosol. PSD-95 contains three N-terminal PDZ domains, a central Src homology-3 (SH3) domain, and a C-terminal guanylate kinase (GK)-like domain (Fig.1A) (3Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 14Anderson J.M. Curr. Biol. 1996; 6: 382-384Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). SAP97/hdlg (15Müller 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, 16Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Crossref PubMed Scopus (344) Google Scholar), chapsyn-110/PSD-93 (13Kim E. Cho K.-O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar,17Brenman J.E. Christopherson K.S. Craven S.E. McGee A.W. Bredt D.S. J. Neurosci. 1996; 16: 7407-7415Crossref PubMed Google Scholar), and SAP102 (18Müller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S.D. Lau L.-F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) also share this domain organization. Among the three PDZ domains of PSD-95, PDZ1 and PDZ2 have about 52% homology to each other and interact with the NR2 subunits of the NMDA receptor and the Kv1.4, as well as others, via the -S/TXV sequence motif located at the extreme C terminus (12Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar, 19Niethammer M. Kim E. Sheng M. J. Neurosci. 1996; 16: 2157-2163Crossref PubMed Google Scholar, 20Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1631) Google Scholar, 21O'Brien R.J. Lau L.-F. Huganir R.L. Curr. Opin. Neurobiol. 1998; 8: 364-369Crossref PubMed Scopus (242) Google Scholar). Additionally, PDZ1 interacts with the kainate-binding glutamate receptor, GluR6, via its C-terminal sequence, ETMA (22Garcia E.P. Mehta S. Blair L.A.C. Wells D.G. Shang J. Fukushima T. Fallon J.R. Garner C.C. Marshall J. Neuron. 1998; 21: 727-739Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). On the other hand, the third PDZ domain (PDZ3) has about 42 and 35% sequence homology to PDZ1 and PDZ2, respectively, and is clearly distinguished from the other two domains by its different sets of binding partners, such as neuroligin, CRIPT (23Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Südhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (612) Google Scholar, 24Niethammer 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), and the β1-adrenergic receptor (25Hu L.A. Tang Y. Miller W.E. Cong M. Lau A.G. Lefkowitz R.J. Hall R.A. J. Biol. Chem. 2000; 275: 38659-38666Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Structure studies of PDZ2 and PDZ3 revealed that both domains consist of a similar six-stranded, anti-parallel β-barrel flanked by two α-helices, forming a ligand binding pocket between βB and αB, with the exception that PDZ1 and PDZ2 have longer variable loops connecting βB and βC than that of PDZ3 (Fig. 1B) (26Tochio H. Hung F. Li M. Bredt D.S. Zhang M. J. Mol. Biol. 2000; 295: 225-237Crossref PubMed Scopus (92) Google Scholar,27Doyle D.A. 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). Although no binding partner for the SH3 domain has been identified except for a kainate receptor subunit in vitro (22Garcia E.P. Mehta S. Blair L.A.C. Wells D.G. Shang J. Fukushima T. Fallon J.R. Garner C.C. Marshall J. Neuron. 1998; 21: 727-739Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), the GK-like domain binds to the GK domain-associated proteins (GKAP/SAPAP/DAP) (28Kim 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, 29Naisbitt S. Kim E. Weinberg R.J. Rao A. Yang F.-C. Craig A.M. Sheng M. J. Neurosci. 1997; 17: 5687-5696Crossref PubMed Google Scholar, 30Takeuchi 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, 31Satoh 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), BEGAIN (32Deguchi 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), MAP1A (33Brenman 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), etc., which interact with other postsynaptic proteins. Extensive studies have revealed that a palmitoylated pair of cysteines in the N-terminal region and PDZ1 or PDZ2 of PSD-95 are essential for membrane targeting (34Topinka J.R. Bredt D.S. Neuron. 1998; 20: 125-134Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) and for clustering of Kv1.4 and NR2B in COS cells (35Hsueh Y.-P. Kim E. Sheng M. Neuron. 1997; 18: 803-814Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 36El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-171Crossref PubMed Scopus (242) Google Scholar), as well as for synaptic targeting of PSD-95 in hippocampal cultures (37Craven S.E. El-Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) as shown by deletion analyses of PSD-95. Furthermore, previously reported differences in the biochemical properties between PDZ1 and PDZ2 include the following: (i) PSD-95 PDZ2 exhibits a higher affinity for the target peptides corresponding to the C-terminal sequences of Kv1.4 and NR2B than PDZ1 (24Niethammer 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); (ii) a single PDZ protein, neuronal nitric oxide synthase, interacts only with PDZ2 via the PDZ-PDZ interaction (38Brenman J.E. Chao D.S. 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, 39Christopherson K.S. Hillier B.J. Lim W.A. Bredt D.S. J. Biol. Chem. 1999; 274: 27467-27473Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar); and (iii) only PDZ1 binds to the kainate-type GluR6 (22Garcia E.P. Mehta S. Blair L.A.C. Wells D.G. Shang J. Fukushima T. Fallon J.R. Garner C.C. Marshall J. Neuron. 1998; 21: 727-739Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). However, the intramolecular interactions between the SH3 and GK-like domains in the full-length PSD-95 are also required for the clustering of Kv1.4 (40McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 41Shin H. Hsueh Y.-P. Yang F.-C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar). In addition, the binding of the GK-like domain to MAP1A is stimulated by peptides corresponding to the C-terminal sequences of Kv1.4, NR2B, and CRIPT (33Brenman 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). Therefore, to understand the molecular mechanisms of PSD-95 and ion channel clustering, the roles of each domain should also be assessed using full-length PSD-95 molecules. Because PDZ1 and PDZ2 in PSD-95 are fundamental to the clustering of Kv1.4 or NR2B, we analyzed the functional differences between PDZ1 and PDZ2 in the channel clustering. Here, we introduced mutations and deletions into PDZ1 and/or PDZ2 of the full-length PSD-95 based on the reported structure and also constructed a series of PDZ1-PDZ2 inversion mutants, so that the mutated domains were impaired in channel binding but the overall structures of each PDZ domain and the full-length PSD-95 remained more or less intact. These mutants were further evaluated for their Kv1.4 clustering activity in COS-1 cells. The results show that PDZ2 is a key domain for Kv1.4 clustering and that the ligand binding function of PDZ, within the second domain, is essential for developing plaque-like clusters. In addition, the introduced mutations and loop deletions increased the sensitivity to trypsin, suggesting the compact molecular packing of PSD-95. These results imply the importance of the intramolecular interactions of PSD-95 for ion channel clustering. The PSD-95, NR2B, and Kv1.4 cDNAs were acquired from mouse whole brain by reverse transcriptase-mediated PCR using appropriate oligonucleotides as primers. The isolated PSD-95 and NR2B cDNAs, and also Kv1.4 cDNA, were subcloned into theNheI-NotI and XhoI-EcoRI sites of the mammalian expression vector pcDNA3.1 (Invitrogen), respectively. The obtained cDNAs contained several mutations resulting in amino acid substitutions L73F, V496M, L805I, and T1043A in NR2B and E126G, T395P, L465P, and D636E in Kv1.4, compared with the published cDNAs (42Mori H. Mishina M. Neuropharmacology. 1995; 34: 1219-1237Crossref PubMed Scopus (588) Google Scholar, 43Wymore R.S. Korenberg J.R. Kinoshita K.D. Aiyar J. Coyne C. Chen X.N. Hustad C.M. Copeland N.G. Gutman G.A. Jenkins N.A. Chandy K.G. Genomics. 1994; 20: 191-202Crossref PubMed Scopus (32) Google Scholar). The c-Myc tag (EQKLISEEDL) was inserted between amino acid residues 27 and 28 of the NR2B using oligonucleotide-directed mutagenesis. FLAG-tagged PSD-95 was constructed by inverse PCR using primers encoding the FLAG epitope (DYKDDDDK) and the stop codon. The cDNA was introduced into theNheI and EcoRI sites of pcDNA3.1. TheKpnI, SacII, and AflII sites were introduced before the PDZ1 gene, between thePDZ1 and PDZ2 genes, and after thePDZ2 gene, respectively, to construct a series of PDZ1-PDZ2 inverted PSD-95 mutants. Mutant PDZ domains PDZ1Δ, PDZ2Δ, PDZ1mΔ, and PDZ2mΔ (Fig. 1B) were constructed by a general mutagenesis method using PCR. To construct the GST-fused PDZ domains, PDZ1, PDZ2, PDZ1Δ, PDZ2Δ, PDZ1mΔ, and PDZ2mΔ were PCR-amplified and subcloned between the XhoI and EcoRI sites of pGEX-4T3 (Amersham Biosciences, Inc.). GST-fused PDZ domains were expressed in Escherichia coli BL21 strains. Bacteria were harvested and lysed in PBS containing 1% Triton X-100, 1 mm EDTA, 10 mm MgCl2, 5 mm dithiothreitol, 10% glycerol, 100 μg/ml lysozyme, 100 μg/ml DNase I, and protease inhibitors. Lysates were incubated at 4 °C for 1 h and pelleted at 15,000 rpm at 4 °C for 10 min. The bacterial supernatants were incubated with a 200-μl bed volume of glutathione-Sepharose beads (Amersham Biosciences, Inc.) at 4 °C for 1 h and washed three times with PBS containing 1% Triton X-100 and 10% glycerol. The bound proteins were eluted with 10 mm glutathione and dialyzed against binding buffer (25 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm MgCl2, 10% glycerol, and 1% Triton X-100) at 4 °C. COS-1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and penicillin/streptomycin. Before transfection, COS-1 cells were seeded on serum-coated cover glasses for clustering assays or in 100-mm plates for GST pull-down and immunoprecipitation assays and were cultured overnight. For clustering assays, cells were transfected by the calcium phosphate precipitation method. The calcium phosphate-DNA mixtures were added within 24 h of seeding and were left for 18–24 h. The cells were shocked with 10% dimethyl sulfoxide (Me2SO) and were incubated at 37 °C for additional 24 h. For GST pull-down and immunoprecipitation assays, COS-1 cells were transfected with LipofectAMINE reagent (Invitrogen) according to the manufacturer's procedures. For the GST pull-down assay, NR2B- or Kv1.4-expressing COS cells were lysed in binding buffer 2 days after transfection. The cell lysates were mixed with beads previously bound with either the GST or GST-fused PDZ domain (∼30 μg) at 4 °C for 2 h and were washed four times with PBS containing 1% Triton X-100 and 10% glycerol. The bound proteins were eluted with SDS sample buffer and analyzed by immunoblotting or silver staining. For the immunoprecipitation assay, cells expressing Kv1.4 or each PSD-95 protein were solubilized separately in radioimmune precipitation buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS) containing protease inhibitors. The solubilized supernatants of Kv1.4 and each PSD-95 mutant were mixed and incubated at 4 °C for 2 h. Then, monoclonal anti-Kv1.4 antibodies (Upstate Biotechnology) or polyclonal anti-FLAG epitope antibodies (Zymed Laboratories Inc.) (∼2 μg) were added to the mixtures, which were further incubated overnight at 4 °C. Protein G-Sepharose resin (Amersham Biosciences, Inc.) was added, and the mixtures were incubated for 1 h. The immunoprecipitates were washed, eluted with SDS sample buffer, and analyzed by immunoblotting. Transfected COS-cells expressing each PSD-95 protein were homogenized, and the postnuclear supernatants were further centrifuged at 100,000 ×g for 30 min. The obtained membrane suspensions (∼1.7 mg/ml protein) were treated with trypsin (∼28 μg/ml) at 37 °C for 5, 60, and 120 min. Twenty μl of each reaction were mixed with SDS sample buffer, fractionated by SDS-PAGE, and analyzed by immunoblotting with a monoclonal anti-PSD-95 antibody (M16) (Transduction Laboratories) with an epitope in the region of 353–504. An anti-FLAG antibody and a monoclonal anti-PSD-95 antibody (7E3-1B8) (Affinity Bioreagents, Inc.) were also used, although the 7E3 antibody did not recognize PDZ2Δ mutant because of the epitope deletion. Both antibodies gave similar patterns in which the bands larger than ∼60 kDa were more sensitive to trypsin than those of the wild-type. For the limited digestion of PSD-95, Pronase, papain, and V8 were also examined. Because of the many digestion sites, we could not detect useful limited fragments under the conditions employed. Two days after transfection, the cells grown on coverslips were washed with ice-chilled PBS containing 1 mmMgCl2 and 1 mm CaCl2 and were fixed with the same buffer containing 4% paraformaldehyde and 0.1% Triton X-100 for 30 min at 4 °C. The cells were then washed with PBS containing 0.1% Triton X-100 and were incubated with the same buffer containing 50 mm glycine to quench the reactions. Nonspecific protein binding was blocked by an incubation with blocking buffer (PBS containing 3% bovine serum albumin and 0.1% Triton X-100) for 1 h at room temperature. Primary antibodies were then added to the blocking buffers, and the mixture was incubated for 3 h at room temperature or overnight at 4 °C. The samples were washed with blocking buffer, labeled with fluorescein isothiocyanate-conjugated goat anti-rabbit and Texas Red-conjugated goat anti-mouse antibodies diluted in blocking buffer for 1 h at room temperature, and washed again three times with PBS containing 0.1% Triton X-100. Finally, the coverslips were mounted on slides, and images were obtained with a Zeiss LSM Pascal laser scanning confocal microscope system. The cells shown in Fig. 5, c andf, were observed with an Olympus BX50 microscope. Clustering assays were repeated 4–5 times. For each assay, the number of cells that formed plaque-like clusters was counted among 50–100 cells coexpressing mutant PSD-95 and Kv1.4. The clusters in COS cells were judged referring to the published patterns (12Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar, 28Kim 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, 35Hsueh Y.-P. Kim E. Sheng M. Neuron. 1997; 18: 803-814Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 36El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-171Crossref PubMed Scopus (242) Google Scholar, 41Shin H. Hsueh Y.-P. Yang F.-C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar) using the following criteria: (i) PSD-95 and Kv1.4 were colocalized; (ii) the localization patterns between the individually expressed PSD-95 or Kv1.4 and the coexpressed PSD-95 and Kv1.4 were different; (iii) the clustered area of PSD-95 and Kv1.4 was larger (>∼1 μm) than that of just a colocalized dot; (iv) perinuclear signals were not included; (v) fine scattered puncta, as shown in Ref. 41Shin H. Hsueh Y.-P. Yang F.-C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar, were not included; (vi) multiple plaque-like protein patches were observed in a cell. The clustering efficiencies were calculated as cluster-formed cells/coexpressed cells, and the results from repeated experiments were averaged. The PDZ1, PDZ2, and PDZ3 domains exhibit distinct ligand specificities, and yet they form similar structures. As compared with PDZ3, PDZ1 and PDZ2 have an additional six-residue insertion in a variable loop connecting βB and βC (Fig. 1B). To disrupt the interactions of PDZ1 and PDZ2 with the Shaker-type K+ channel, Kv1.4, without affecting the overall structure, we first deleted these six residues in the βB/βC loop of PDZ1 (PDZ1Δ) and PDZ2 (PDZ2Δ) to make them resemble PDZ3. Although the interactions of the PDZ domains and NR2B were significantly impaired by the deletions (Fig. 1C), these constructs still exhibited weak but detectable levels of interactions in vitro (Fig.2B). Therefore, Ser-78 and Ala-80 in the βB of PDZ1, and Ser-173 and Ala-175 in the βB of PDZ2 were further mutated to asparagine and valine, the corresponding residues of PDZ3, respectively (Fig. 1B), because βB is part of a ligand binding pocket and the corresponding N326 in PDZ3 is important for its specific binding to CRIPT (24Niethammer 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). Each wild-type or mutant PDZ domain, fused to glutathione S-transferase (GST), was examined for an interaction with Kv1.4 in vitro (Fig.1D). Neither PDZ1mΔ nor PDZ2mΔ showed detectable binding activity to Kv1.4. Thus, the βB/βC loops and the mutated residues play important roles in the specific binding of PDZ1 and PDZ2 to Kv1.4. The PDZ1Δ, PDZ2Δ, PDZ1mΔ, and/or PDZ2mΔ domains were then replaced with the wild-type domains of full-length PSD-95. Fig. 2A shows the mutants constructed in this study. PSD-95:1mΔ-2 was constructed by replacing PDZ1 of PSD-95 with PDZ1mΔ, whereas PDZ2 was replaced with PDZ2mΔ in PSD-95:1-2mΔ, and both PDZ domains were replaced in PSD-95:1mΔ-2mΔ (Fig.2A, middle). In addition, a FLAG epitope tag was attached to the C termini of the mutated PSD-95 proteins for their detection. The expression level of the FLAG-tagged PSD-95 was comparable with that of the untagged PSD-95. Hereafter, FLAG-tagged PSD-95 was used as the wild type. The expression levels of these mutants were also comparable with that of the wild-type PSD-95, except for that of PSD-95:1mΔ-2mΔ, which was lower. The ability of these mutant PSD-95 proteins to interact with Kv1.4in vitro were examined by coimmunoprecipitation assays. The mixture of Kv1.4 and the wild-type or mutant PSD-95 was immunoprecipitated with an anti-Kv1.4 antibody, and the coimmunoprecipitated PSD-95 was analyzed. In addition, the wild-type and mutant PSD-95 proteins were immunoprecipitated with anti-FLAG epitope antibodies, and the coimmunoprecipitated Kv1.4 was analyzed by immunoblotting. The interactions of PSD-95:1Δ-2, 1-2Δ, 1mΔ-2, and 1-2mΔ mutants with Kv1.4 were similar to those of the wild-type (Fig.2, B and C), and PSD-95:1Δ-2Δ interacted weakly. However, no Kv1.4 signal was detected in the PSD-95:1mΔ-2mΔ immunoprecipitate (Fig. 2C). Thus, PSD-95 was able to associate with Kv1.4 as long as either the PDZ1 or PDZ2 domain remained intact. The entire domain assemblies of the PSD-95 mutants were compared with that of the wild-type by limited trypsin digestion. Fig.3 shows the digestion patterns of the wild-type and mutant proteins in the membrane fractions prepared from COS-1 cells expressing each protein, as analyzed by immunoblotting. The full-length and ∼60 kDa bands of the mutants were much more sensitive to trypsin than those of the wild-type, indicating that the βB/βC loop deletion and/or the mutations in βB loosened the molecular packing of PSD-95. It is interesting that the only six-residue deletion in the variable loop and/or the two point mutations of single PDZ domain affect the entire molecular packing of full-length PSD-95. Although the details of entire domain assemblies of the PSD-95 mutants were not clear from this experiment, the patterns of the limited fragments are similar, suggesting that the trypsin-sensitive sites of mutants similar to those of the wild-type were exposed. In addition, because PSD-95:1mΔ-2, which showed wild-type clustering activity, and the other mutants exhibited similar trypsin sensitivity to one another, the deficient clustering activities are not due to the trypsin-sensitive structures. The clustering activities are described later in detail. The wild-type and mutant PSD-95 proteins were coexpressed with Kv1.4 in COS cells, and their cluster-forming activities were examined by indirect immunofluorescence double labeling of transfected cells. In Fig. 4,a and b, individually expressed PSD-95 and Kv1.4 show diffuse and reticular distributions in the transfected cells, respectively. Fig. 4, c1/c2, shows the cell cotransfected with the wild-type PSD-95 and Kv1.4, in which plaque-like protein clusters are notable, as described previously (12Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar, 28Kim 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, 35Hsueh Y.-P. Kim E. Sheng M. Neuron. 1997; 18: 803-814Abstract Full Text Full Text PDF PubMed Scopus (185) Google" @default.
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• W1969863013 title "Ligand Binding of the Second PDZ Domain Regulates Clustering of PSD-95 with the Kv1.4 Potassium Channel" @default.
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