Synaptic Targeting of PSD-Zip45 (Homer 1c) and Its Involvement in the Synaptic Accumulation of F-actin
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m210802200
ISSN1083-351X
AutoresShinichi Usui, Daijiro Konno, Kei Hori, Hisato Maruoka, Shigeo Okabe, Takashi Fujikado, Yasuo Tano, Kenji Sobue,
Tópico(s)Retinal Development and Disorders
ResumoPSD-Zip45/Homer1c, which contains an enabled/VASP homology 1 (EVH1) domain and leucine zipper motifs, is a postsynaptic density (PSD) scaffold protein that interacts with metabotropic glutamate receptors and the shank family. We studied the molecular mechanism underlying the synaptic targeting of PSD-Zip45 in cultured hippocampal neurons. The EVH1 domain and the extreme C-terminal leucine zipper motif were molecular determinants for its synaptic targeting. The overexpression of the mutant of the EVH1 domain or deletion of the extreme C-terminal leucine zipper motif markedly suppressed the synaptic localization of endogenous shank but not PSD-95 or GKAP. In contrast, an overexpressed GKAP mutant lacking shank binding activity had no effect on the synaptic localization of shank. Actin depolymerization by latrunculin A reduced the synaptic localization of PSD-Zip45, shank, and F-actin but not of PSD-95 or GKAP. Overexpression of PSD-Zip45 enhanced the accumulation of synaptic F-actin. Additionally, overexpression of PSD-Zip45 and an isoform of shank induced synaptic enlargement in association with the further accumulation of synaptic F-actin. The EVH1 domain and extreme C-terminal leucine zipper motif of PSD-Zip45 were also critical for these events. Thus, these data suggest that the PSD-Zip45-shank and PSD-95-GKAP complexes form different synaptic compartments, and PSD-Zip45 alone or PSD-Zip45-shank is involved in the synaptic accumulation of F-actin. PSD-Zip45/Homer1c, which contains an enabled/VASP homology 1 (EVH1) domain and leucine zipper motifs, is a postsynaptic density (PSD) scaffold protein that interacts with metabotropic glutamate receptors and the shank family. We studied the molecular mechanism underlying the synaptic targeting of PSD-Zip45 in cultured hippocampal neurons. The EVH1 domain and the extreme C-terminal leucine zipper motif were molecular determinants for its synaptic targeting. The overexpression of the mutant of the EVH1 domain or deletion of the extreme C-terminal leucine zipper motif markedly suppressed the synaptic localization of endogenous shank but not PSD-95 or GKAP. In contrast, an overexpressed GKAP mutant lacking shank binding activity had no effect on the synaptic localization of shank. Actin depolymerization by latrunculin A reduced the synaptic localization of PSD-Zip45, shank, and F-actin but not of PSD-95 or GKAP. Overexpression of PSD-Zip45 enhanced the accumulation of synaptic F-actin. Additionally, overexpression of PSD-Zip45 and an isoform of shank induced synaptic enlargement in association with the further accumulation of synaptic F-actin. The EVH1 domain and extreme C-terminal leucine zipper motif of PSD-Zip45 were also critical for these events. Thus, these data suggest that the PSD-Zip45-shank and PSD-95-GKAP complexes form different synaptic compartments, and PSD-Zip45 alone or PSD-Zip45-shank is involved in the synaptic accumulation of F-actin. postsynaptic density enabled/VASP homology 1 metabotropic glutamate receptors green fluorescent protein wild type glutathione S-transferase bovine serum albumin phosphate-buffered saline γ-aminobutyric acid, type A N-methyl-d-aspartic acid As revealed by electron microscopy, the postsynaptic density (PSD)1 is a dense structure of the excitatory synapses in the central nervous system (1Palay S.L. Exp. Cell Res. 1958; 5: 275-293Google Scholar, 2Carlin R.K. Siekevitz P. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3517-3521Crossref PubMed Scopus (198) Google Scholar). Recently, numerous studies (3Kim J.H. Huganir R.L. Curr. Opin. Cell Biol. 1999; 11: 248-254Crossref PubMed Scopus (114) Google Scholar, 4Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (652) Google Scholar, 5Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1036) Google Scholar) have reported on the identification of PSD scaffold proteins and their involvement in synaptic function. We isolated PSD-Zip45, a novel member of the Homer family, using a PSD-specific monoclonal antibody. This protein contains an enabled/VASP homology 1 (EVH1) domain at its N- and C-terminal leucine zipper motifs (6Sun J. Tadokoro S. Imanaka T. Murakami S.D. Nakamura M. Kashiwada K. Ko J. Nishida W. Sobue K. FEBS Lett. 1998; 437: 304-308Crossref PubMed Scopus (65) Google Scholar, 7Tadokoro S. Tachibana T. Imanaka T. Nishida W. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1999; 93: 1540-1544Google Scholar). The same protein was independently reported as Homer 1c (8Xiao B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar) and vesl-1L (9Kato A. Ozawa F. Saitoh Y. Fukazawa Y. Sugiyama H. Inokuchi K. J. Biol. Chem. 1998; 273: 23969-23975Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). The EVH1 domain interacts with the PPXXF motif found in group 1 metabotropic glutamate receptors (mGluRs) (7Tadokoro S. Tachibana T. Imanaka T. Nishida W. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1999; 93: 1540-1544Google Scholar, 8Xiao B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 10Brakeman P.R. Lanahan A.A. O'Brien R. Roche K. Barnes C.A. Huganir R.L. Worley P.F. Nature. 1997; 386: 284-288Crossref PubMed Scopus (922) Google Scholar), shank family members (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 12Lim S. Naisbitt S. Yoon J. Hwang J.I. Suh P.G. Sheng M. Kim E. J. Biol. Chem. 1999; 274: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), inositol triphosphate receptors, and ryanodine receptors (13Tu J.C. Xiao B. Yuan J.P. Lanahan A.A. Leoffert K. Li M. Linden D.J. Worley P.F. Neuron. 1998; 21: 717-726Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). The extreme C-terminal leucine zipper motif is involved in self-multimerization (7Tadokoro S. Tachibana T. Imanaka T. Nishida W. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1999; 93: 1540-1544Google Scholar). We recently analyzed the dynamic behavior of PSD-Zip45 within dendritic spines using time-lapse confocal microscopy, and we demonstrated its high steady-state turnover rate and assembly/disassembly dynamics, which depend on Ca2+ signals from different sources (14Okabe S. Urushido T. Konno D. Okado H. Sobue K. J. Neurosci. 2001; 21: 9561-9571Crossref PubMed Google Scholar).The first-discovered PSD scaffold protein, PSD-95 (SAP90), interacts with the NR2 subunits of the NMDA receptor (15Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 16Kistner 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, 17Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1619) Google Scholar) and other PSD scaffold proteins such as GKAP (18Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (429) Google Scholar, 19Kawashima N. Takamiya K. Sun J. Kitabatake A. Sobue K. FEBS Lett. 1997; 418: 301-304Crossref PubMed Scopus (22) Google Scholar), signaling molecules, and cell adhesion molecules (4Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (652) Google Scholar, 20Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 21Sheng M. Lee S.H. Nat. Neurosci. 2000; 3: 633-635Crossref PubMed Scopus (44) Google Scholar). The shank family of proteins, which are composed of multiple protein-protein interaction domains, were isolated as GKAP-binding proteins (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar) and have also been called ProSAP (22Boeckers T.M. Kreutz M.R. Winter C. Zuschratter W. Smalla K.H. Sanmarti-Vila. Lydia Wex H. Langnaese K. Bockmann J. Garner C.C. Gundelfinger E.D. J. Neurosci. 1999; 19: 6506-6518Crossref PubMed Google Scholar) and synamon (23Yao I. Hata Y. Hirao K. Deguchi M. Ide N. Takeuchi M. Takai Y. J. Biol. Chem. 1999; 274: 27463-27466Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). One of the shank family proteins was originally reported as cortactin-binding protein 1, CortBP1 (24Du Y. Weed S.A. Xiong W.C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (215) Google Scholar). The PDZ and the SH3 domain of shank family members interact with the extreme C terminus of GKAP (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar) and the α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor-interacting protein GRIP (25Sheng M. Kim E. J. Cell Sci. 2000; 113: 1851-1856Crossref PubMed Google Scholar), respectively. The proline-rich region of shank includes binding sites for the Homer proteins (26Tu J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar) and the actin-binding protein cortactin (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Tu et al. (26Tu J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar) demonstrated the macromolecular coupling of the mGluR-Homer and NMDA receptor-PSD-95-GKAP complexes by shank family members in vitro. Taking these observations together, Xiao et al.(27Xiao B. Tu J.C. Worley P.F. Curr. Opin. Neurobiol. 2000; 10: 370-374Crossref PubMed Scopus (365) Google Scholar) proposed that shank-dependent protein-protein interactions in the postsynaptic protein lattice link together different classes of glutamate receptors and further couple them to the actin cytoskeleton and the Ca2+ sequestering machinery. However, these protein-protein interactions were identified in a piecemeal fashion in vitro. The molecular organization and dynamic interactions of a postsynaptic protein lattice in vivo therefore remain largely unknown. Here we focused on the molecular mechanism underlying the synaptic targeting of PSD-Zip45 and its effect on other PSD scaffold proteins. We demonstrated that both the EVH1 domain and C-terminal leucine zipper motif of PSD-Zip45 are critical for its synaptic targeting and that PSD-Zip45/shank and PSD-95/GKAP form different compartments in the synapse. In addition, the synaptic localization of shank is PSD-Zip45-dependent, and their interaction is involved in the enhanced accumulation of synaptic F-actin, and overexpressing these molecules induces synaptic enlargement in association with the further accumulation of synaptic F-actin.DISCUSSIONRecent studies (31Craven S.E. El-Husseini A.E. Bredt D.S. Neuron. 1999; 22: 497-509Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 37Arnold D.B. Clapham D.E. Neuron. 1999; 23: 149-157Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) have demonstrated the synaptic targeting of some PSD scaffold proteins. The first example was the synaptic targeting of PSD-95, in which the N-terminal palmitoylation, the first two PDZ domains, and/or a C-terminal targeting motif are critical. Naisbitt et al. (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar) reported that the overexpression of GKAP variant, which lacks the PDZ-binding sequence, caused a marked reduction in the levels of the endogenous shank family members in the synapse. Sala et al. (36Sala C. Piëch V. Wilson N.R. Passafaro M. Liu G. Sheng M. Neuron. 2001; 31: 115-130Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar) also identified the molecular determinants of the shank family required for their synaptic targeting as their PDZ domain and the flanking sequences and concluded that an interaction between the PDZ domain of shank 1B and the C terminus of GKAP was important for the synaptic localization of shank 1B. Angoet al. (38Ango F. Pin J.P. Tu J.C. Xiao B. Worley P.F. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 8710-8716Crossref PubMed Google Scholar) analyzed the distribution of exogenous type 5 mGluR (mGluR5) and Homer proteins using cerebellar granule cells in which mGluR5 and Homer 1 proteins were absent, and their findings suggested that the synaptic targeting of mGluR5 is Homer protein-dependent. However, they did not address the molecular determinants of the Homer proteins. We recently reported (14Okabe S. Urushido T. Konno D. Okado H. Sobue K. J. Neurosci. 2001; 21: 9561-9571Crossref PubMed Google Scholar) that PSD-Zip45 that is exogenously expressed in hippocampal neurons is targeted to the synapses and that its targeting is dynamically regulated by Ca2+ influxes from different sources. Here, we studied the molecular determinants required for the synaptic targeting of PSD-Zip45 and the effects of PSD-Zip45 variants on the synaptic localization of other PSD scaffold proteins and F-actin.Synaptic Targeting of PSD-Zip45 and Compartmentalization of Major PSD Scaffold ProteinsExogenous PSD-Zip45 showed a synaptic localization with other major PSD scaffold proteins, including PSD-95, GKAP, and shank (Fig. 1). We identified both the EVH1 domain and the extreme C-terminal leucine zipper motif of PSD-Zip45 as the molecular determinants for its synaptic targeting (Fig. 2). Furthermore, overexpression of PSD-Zip45G89A or PSD-Zip45ΔZipB markedly suppressed the synaptic localization of endogenous shank, but not of PSD-95 or GKAP, in a dominant-negative fashion (Fig. 3). These results suggest that the synaptic localization of PSD-Zip45 is a prerequisite for that of shank, and both of PSD-Zip45G89A and PSD-Zip45ΔZipB cannot recruit shank to the synapses because neither of them is targeted to the synapses. It has been reported that GKAP interacts with the shank family via its PDZ domain and that overexpressed GKAP-L694A, which does not bind shank, suppresses the synaptic localization of shank (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). In contrast, we demonstrated here that whereas GKAP-WT and GKAP-L694A were targeted precisely to the synapses, overexpressed GKAP-WT and GKAP-L694A did not affect the synaptic localization of endogenous shank (Fig. 4). Thus, there is a discrepant result regarding the interaction between GKAP and shank within the synapses. The molecular length of GKAP, clone2–2A, used by us is longer than that by Naisbitt et al. (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). We also constructed GKAP-L694A, a mutant of clone2-2A, that lacked the shank binding activity. Naisbitt et al. (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar) used a C-terminal deleted variant of GKAP lacking the shank binding activity. Furthermore, we introduced the plasmids into cultured neurons by the microinjection method, whereas Naisbitt et al. (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar) used the transfection method with calcium phosphate. However, it remains unknown whether these different constructs and expression methods might produce the above discrepant result. Our present results indicate that the synaptic localization of shank depends on that of PSD-Zip45 but not of GKAP. It might be inferred from these data that PSD-Zip45-shank and PSD-95-GKAP interactions form different compartments in the synapses. The currently accepted model for a postsynaptic protein lattice is that the shank family members provide molecular bridges between NMDA receptor-PSD-95-GKAP, mGluR-PSD-Zip45, and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor-GRIP complexes, the actin cytoskeleton, and the Ca2+-sequestering machinery (25Sheng M. Kim E. J. Cell Sci. 2000; 113: 1851-1856Crossref PubMed Google Scholar, 27Xiao B. Tu J.C. Worley P.F. Curr. Opin. Neurobiol. 2000; 10: 370-374Crossref PubMed Scopus (365) Google Scholar, 39Ehlers M.D. Curr. Biol. 1999; 9: R848-R850Abstract Full Text Full Text PDF PubMed Google Scholar). Almost all of these interactions, however, have been demonstrated by in vitro assays. We reported previously (14Okabe S. Urushido T. Konno D. Okado H. Sobue K. J. Neurosci. 2001; 21: 9561-9571Crossref PubMed Google Scholar) that although PSD-95 clusters in the synapses of living hippocampal neurons are less dynamic, PSD-Zip45 shows a highly dynamic and rapid redistribution that is regulated by Ca2+ influxes from different Ca2+ sources. Combining our results, we suggest that PSD-95-GKAP and PSD-Zip45-shank complexes form different compartments in the synapse with respect to their interactions and dynamics.Linkages between PSD-Zip45, Shank, and the Actin Cytoskeleton in the SynapseBecause the shank family interacts in vitro with cortactin through a cortactin-binding motif in the proline-rich region of the shank proteins (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 24Du Y. Weed S.A. Xiong W.C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (215) Google Scholar), it has been postulated that the actin cytoskeleton is involved in the synaptic localization of PSD-Zip45 and shank. In support of this idea, our recent studies (14Okabe S. Urushido T. Konno D. Okado H. Sobue K. J. Neurosci. 2001; 21: 9561-9571Crossref PubMed Google Scholar) suggest that the synaptic localization of PSD-Zip45 is linked to the polymerization/depolymerization dynamics of the actin cytoskeleton. In this paper, we further compared the linkages between four PSD scaffold proteins and the actin cytoskeleton. Treating hippocampal neurons with the actin-depolymerizing reagent latrunculin A caused a decrease in the synaptic localization of F-actin, PSD-Zip45, and shank but not of PSD-95 or GKAP (Fig. 5). These results suggest that the synaptic organization of the actin cytoskeleton is closely associated with PSD-Zip45 and shank.The PSD-Zip45-shank complex may link to the actin cytoskeleton through the cortactin-shank interaction or another actin-binding protein that interacts with the PSD-Zip45-shank complex. The former possibility, however, seems to be unlikely because the synaptic localization of shank was quite different from that of cortactin (data not shown). Alternatively, there are several pieces of evidence that support the latter possibility. The N terminus of Cupidin/Homer2a has been reported to interact with F-actin in vitro (40Shiraishi Y. Mizutani A. Bito H. Fujisawa K. Narumiya S. Mikoshiba K. Furuichi T. J. Neurosci. 1999; 19: 8389-8400Crossref PubMed Google Scholar). We did not, however, detect a direct interaction between PSD-Zip45 and F-actin (data not shown). In contrast, we demonstrated that overexpression of PSD-Zip45WT enhances the synaptic accumulation of F-actin without enhancing that of shank (Fig. 6 A,a1–a3). Sala et al.(36Sala C. Piëch V. Wilson N.R. Passafaro M. Liu G. Sheng M. Neuron. 2001; 31: 115-130Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar) reported that overexpression of both shank 1B and Homer 1b induces synaptic enlargement in association with an increase in synaptic F-actin. Consistent with this, we found that both of overexpressed PSD-Zip45 and CortBP1 were highly concentrated in the enlarged synapses with F-actin (Fig. 6 A,d1–d3). This enhanced accumulation of synaptic F-actin was also observed in the normal-sized synapses in neurons overexpressing PSD-Zip45WT alone (Fig.6 A,a1 and a3). In contrast, overexpressed PSD-Zip45G89A and PSD-Zip45ΔZipB did not affect the synaptic localization of F-actin whether or not CortBP1 was also overexpressed (Fig. 6). These results indicate that the synaptic localization of PSD-Zip45 is a prerequisite for that of shank, and the complex formation between PSD-Zip45 and shank is critically linked to the enhanced accumulation of synaptic F-actin. Why did overexpressed PSD-Zip45 alone enhance synaptic accumulation of F-actin without the accumulation of shank? This may be due to the difference of endogenous shank and actin pools, in which free shank is more limited than F-actin. Actually, both of the overexpressed PSD-Zip45 and CortBP1 were highly concentrated in the synapses with the enhanced accumulation of F-actin. Our present results further indicate that increasing the levels of synaptically targeted PSD-Zip45 and shank through overexpression induces synaptic enlargement in association with the enhanced accumulation of synaptic F-actin. Future study will be required to elucidate the involvement of the PSD-Zip45-shank and actin interaction in the postsynaptic structure and function. As revealed by electron microscopy, the postsynaptic density (PSD)1 is a dense structure of the excitatory synapses in the central nervous system (1Palay S.L. Exp. Cell Res. 1958; 5: 275-293Google Scholar, 2Carlin R.K. Siekevitz P. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3517-3521Crossref PubMed Scopus (198) Google Scholar). Recently, numerous studies (3Kim J.H. Huganir R.L. Curr. Opin. Cell Biol. 1999; 11: 248-254Crossref PubMed Scopus (114) Google Scholar, 4Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (652) Google Scholar, 5Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1036) Google Scholar) have reported on the identification of PSD scaffold proteins and their involvement in synaptic function. We isolated PSD-Zip45, a novel member of the Homer family, using a PSD-specific monoclonal antibody. This protein contains an enabled/VASP homology 1 (EVH1) domain at its N- and C-terminal leucine zipper motifs (6Sun J. Tadokoro S. Imanaka T. Murakami S.D. Nakamura M. Kashiwada K. Ko J. Nishida W. Sobue K. FEBS Lett. 1998; 437: 304-308Crossref PubMed Scopus (65) Google Scholar, 7Tadokoro S. Tachibana T. Imanaka T. Nishida W. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1999; 93: 1540-1544Google Scholar). The same protein was independently reported as Homer 1c (8Xiao B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar) and vesl-1L (9Kato A. Ozawa F. Saitoh Y. Fukazawa Y. Sugiyama H. Inokuchi K. J. Biol. Chem. 1998; 273: 23969-23975Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). The EVH1 domain interacts with the PPXXF motif found in group 1 metabotropic glutamate receptors (mGluRs) (7Tadokoro S. Tachibana T. Imanaka T. Nishida W. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1999; 93: 1540-1544Google Scholar, 8Xiao B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 10Brakeman P.R. Lanahan A.A. O'Brien R. Roche K. Barnes C.A. Huganir R.L. Worley P.F. Nature. 1997; 386: 284-288Crossref PubMed Scopus (922) Google Scholar), shank family members (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 12Lim S. Naisbitt S. Yoon J. Hwang J.I. Suh P.G. Sheng M. Kim E. J. Biol. Chem. 1999; 274: 29510-29518Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), inositol triphosphate receptors, and ryanodine receptors (13Tu J.C. Xiao B. Yuan J.P. Lanahan A.A. Leoffert K. Li M. Linden D.J. Worley P.F. Neuron. 1998; 21: 717-726Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). The extreme C-terminal leucine zipper motif is involved in self-multimerization (7Tadokoro S. Tachibana T. Imanaka T. Nishida W. Sobue K. Proc. Natl. Acad. Sci. U. S. A. 1999; 93: 1540-1544Google Scholar). We recently analyzed the dynamic behavior of PSD-Zip45 within dendritic spines using time-lapse confocal microscopy, and we demonstrated its high steady-state turnover rate and assembly/disassembly dynamics, which depend on Ca2+ signals from different sources (14Okabe S. Urushido T. Konno D. Okado H. Sobue K. J. Neurosci. 2001; 21: 9561-9571Crossref PubMed Google Scholar). The first-discovered PSD scaffold protein, PSD-95 (SAP90), interacts with the NR2 subunits of the NMDA receptor (15Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 16Kistner 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, 17Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1619) Google Scholar) and other PSD scaffold proteins such as GKAP (18Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (429) Google Scholar, 19Kawashima N. Takamiya K. Sun J. Kitabatake A. Sobue K. FEBS Lett. 1997; 418: 301-304Crossref PubMed Scopus (22) Google Scholar), signaling molecules, and cell adhesion molecules (4Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (652) Google Scholar, 20Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 21Sheng M. Lee S.H. Nat. Neurosci. 2000; 3: 633-635Crossref PubMed Scopus (44) Google Scholar). The shank family of proteins, which are composed of multiple protein-protein interaction domains, were isolated as GKAP-binding proteins (11Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar) and have also been called ProSAP (22Boeckers T.M. Kreutz M.R. Winter C. Zuschratter W. Smalla K.H. Sanmarti-Vila. Lydia Wex H. Langnaese K. Bockmann J. Garner C.C. Gundelfinger E.D. J. 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Neurobiol. 2000; 10: 370-374Crossref PubMed Scopus (365) Google Scholar) proposed that shank-dependent protein-protein interactions in the postsynaptic protein lattice link together different classes of glutamate receptors and further couple them to the actin cytoskeleton and the Ca2+ sequestering machinery. However, these protein-protein interactions were identified in a piecemeal fashion in vitro. The molecular organization and dynamic interactions of a postsynaptic protein lattice in vivo therefore remain largely unknown. Here we focused on the molecular mechanism underlying the synaptic targeting of PSD-Z
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