SAPAPs

PSD-95/SAP90 is a member of membrane-associated guanylate kinases localized at postsynaptic density (PSD) in neuronal cells. Membrane-associated guanylate kinases are a family of signaling molecules expressed at various submembrane domains which have the PDZ (DHR) domains, the SH3 domain, and the guanylate kinase domain. PSD-95/SAP90 interacts withN-methyl-d-aspartate receptors 2A/B, Shaker-type potassium channels, and brain nitric oxide synthase through the PDZ (DHR) domains and clusters these molecules at synaptic junctions. However, neither the function of the SH3 domain or the guanylate kinase domain of PSD-95/SAP90, nor the protein interacting with these domains has been identified. We have isolated here a novel protein family consisting of at least four members which specifically interact with PSD-95/SAP90 and its related proteins through the guanylate kinase domain, and named these proteins SAPAPs (SAP90/PSD-95-AssociatedProteins). SAPAPs are specifically expressed in neuronal cells and enriched in the PSD fraction. SAPAPs induce the enrichment of PSD-95/SAP90 to the plasma membrane in transfected cells. Thus, SAPAPs may have a potential activity to maintain the structure of PSD by concentrating its components to the membrane area. PSD-95/SAP90 is a member of membrane-associated guanylate kinases localized at postsynaptic density (PSD) in neuronal cells. Membrane-associated guanylate kinases are a family of signaling molecules expressed at various submembrane domains which have the PDZ (DHR) domains, the SH3 domain, and the guanylate kinase domain. PSD-95/SAP90 interacts withN-methyl-d-aspartate receptors 2A/B, Shaker-type potassium channels, and brain nitric oxide synthase through the PDZ (DHR) domains and clusters these molecules at synaptic junctions. However, neither the function of the SH3 domain or the guanylate kinase domain of PSD-95/SAP90, nor the protein interacting with these domains has been identified. We have isolated here a novel protein family consisting of at least four members which specifically interact with PSD-95/SAP90 and its related proteins through the guanylate kinase domain, and named these proteins SAPAPs (SAP90/PSD-95-AssociatedProteins). SAPAPs are specifically expressed in neuronal cells and enriched in the PSD fraction. SAPAPs induce the enrichment of PSD-95/SAP90 to the plasma membrane in transfected cells. Thus, SAPAPs may have a potential activity to maintain the structure of PSD by concentrating its components to the membrane area. The PSD 1The abbreviations used are: PSD, postsynaptic density; NMDA, N-methyl-d-aspartate; MAGUK, membrane-associated guanylate kinase; SAPAP, SAP90/PSD-95-associated protein; GST, glutathione S-transferase; MBP, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; PSRP1, PSD-95/SAP90-related protein 1; kb, kilobase pair(s). 1The abbreviations used are: PSD, postsynaptic density; NMDA, N-methyl-d-aspartate; MAGUK, membrane-associated guanylate kinase; SAPAP, SAP90/PSD-95-associated protein; GST, glutathione S-transferase; MBP, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; PSRP1, PSD-95/SAP90-related protein 1; kb, kilobase pair(s). is a dense thickening of postsynaptic submembranous cytoskeleton observed in electron microscopy. Since PSD has a characteristic structure and is contiguous to the presynaptic active zone, where neurotransmitter release occurs, PSD is proposed to have several functions, such as the stabilization of synaptic junctions, the concentration and regulation of neurotransmitter receptors, and the induction of the transcriptions in response to the synaptic transmission (reviewed in Ref. 1Kennedy M.B. Curr. Opin. Neurobiol. 1993; 3: 732-737Google Scholar). Many studies have revealed several components of PSD, including fodrin (2Carlin R.K. Bartelt D.C. Siekevitz P. J. Cell Biol. 1983; 96: 443-448Google Scholar), tubulin (3Walters B.B. Matus A.I. Nature. 1975; 257: 496-498Google Scholar), actin (4Kelly P.T. Cotman C.W. J. Cell Biol. 1978; 79: 173-183Google Scholar), calmodulin (5Carlin R.K. Grab D.J. Siekevitz P. J. Cell Biol. 1981; 89: 449-455Google Scholar), Ca2+/calmodulin-dependent protein kinase II (6Kennedy M.B. Bennett M.K. Erondu N.E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7357-7361Google Scholar), and PSD-95/SAP90 (7Cho K. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Google 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: 4580-4583Google Scholar, 9Hunt C.A. Schenker L.J. Kennedy M.B. J. Neurosci. 1996; 16: 1380-1388Google Scholar). However, there still remain many components to be identified and the mechanism how the individual components are assembled to form PSD is unknown. PSD-95/SAP90, one of the components of PSD, has a characteristic molecular structure composed of three PDZ (DHR) domains, one SH3 domain, and one guanylate kinase domain. Three isoforms, SAP97, PSD-93/chapsyn, and SAP102, have been reported and all isoforms keep the same molecular structure (10Lue R.A. Marfatia S.M. Branton D. Husain-Chishti A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 11Mueller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar, 12Brenman 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: 757-767Google Scholar, 13Kim E. Cho K-O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Google Scholar, 14Mueller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S. Lau L-F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Google Scholar). Recently, many studies have revealed the function of the PDZ (DHR) domains. The PDZ (DHR) domain is responsible for protein-protein interactions and identified in various proteins (reviewed in Ref. 15Ponting C.P. Phillips C. Trends Biochem. Sci. 1995; 20: 102-103Google Scholar). PSD-95/SAP90 and its isoforms interact with NMDA receptors, Shaker-type potassium channels, and brain nitric oxide synthase through the PDZ (DHR) domains to induce the clustering of these molecules at PSD (12Brenman 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: 757-767Google Scholar, 13Kim E. Cho K-O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Google Scholar, 14Mueller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S. Lau L-F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Google Scholar, 16Kornau H. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Google Scholar, 17Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Google Scholar, 18Niethammer M. Kim E. Sheng M. J. Neruosci. 1996; 16: 2157-2163Google Scholar, 19Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Google Scholar, 20Cabral J.H.M. Petrosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chisti A.H. Liddington R.C. Nature. 1996; 382: 649-652Google Scholar). Thus, PSD-95/SAP90 is important for the concentration of receptors and channels at PSD. Another line of evidence for the importance of PSD-95/SAP90 at synaptic junctions comes from the recent findings about MAGUKs (reviewed in Ref. 21Anderson J.M. Curr. Biol. 1996; 8: 382-384Google Scholar). MAGUKs are a family of proteins expressed at various submembrane domains. They include Drosophiladiscs-large tumor suppressor gene (dlg-A) (22Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Google Scholar), nematodelin-2 (23Hoskins R. Hajnal A.F. Harp S.A. Kim S.K. Development. 1996; 22: 97-111Google Scholar), palmitoylated erythrocyte membrane protein (p55) (24Ruff P. Speicher D.W. Husan-Chishti A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6595-6599Google Scholar), ZO-1 (25Itoh M. Nagafuchi S. Yonemura R. Kitani-Yasuda T. Tsukita S. Tsukita S. J. Cell Biol. 1993; 121: 491-502Google Scholar, 26Willott E. Balda M.S. Fanning A.S. Jameson B. Itallie C.V. Anderson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7834-7838Google Scholar), ZO-2 (27Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Google Scholar), CASK (28Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Google Scholar), PSD-95/SAP90 (7Cho K. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Google 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: 4580-4583Google Scholar), and the isoforms of PSD-95/SAP90 (10Lue R.A. Marfatia S.M. Branton D. Husain-Chishti A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 11Mueller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar, 12Brenman 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: 757-767Google Scholar, 13Kim E. Cho K-O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Google Scholar, 14Mueller B.M. Kistner U. Kindler S. Chung W.J. Kuhlendahl S. Fenster S. Lau L-F. Veh R.W. Huganir R.L. Gundelfinger E.D. Garner C.C. Neuron. 1996; 17: 255-265Google Scholar). All the members of MAGUKs keep the same molecular structures as that of PSD-95/SAP90. They have the PDZ (DHR), the SH3, and the guanylate kinase domains. Based on the characteristic molecular structure, MAGUKs are considered to play important roles in maintaining the structures of submembrane domains and to be involved in signaling at these domains. Indeed, genetic evidence indicates that MAGUKs are essential for maintenance of the structures of cell junctions. The product of dlg-A is expressed at separate junctions and neuromuscular junctions, and the mutations of this gene lead to neoplastic overgrowth of imaginal discs and morphological changes of synaptic bouton structures (22Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Google Scholar, 29Lahey T. Gorczyca M. Jia X-X. Budnik V. Neuron. 1994; 13: 823-835Google Scholar).Lin-2 expressed at Pn.p cells in nematode is involved in the localization of let-23, a receptor for lin-3, and essential for vulval induction (23Hoskins R. Hajnal A.F. Harp S.A. Kim S.K. Development. 1996; 22: 97-111Google Scholar). In the context of the general concept about MAGUKs, PSD-95/SAP90 is assumed to play pivotal roles in signaling at synaptic junctions, not only in the clustering of receptors, which may be mediated by domains other than the PDZ (DHR) domains. In contrast to the accumulation of information about the PDZ (DHR) domains, our knowledge about the SH3 domain and the guanylate kinase domain of PSD-95/SAP90 is limited. The SH3 domain is also responsible for protein-protein interactions and identified in a wide variety of proteins. However, the molecules interacting with the SH3 domain of PSD-95/SAP90 have not been identified. The guanylate kinase domain, is similar to an enzyme guanylate kinase, has the binding activity for GMP and GDP, but does not show kinase activity (30Kistner U. Garner C.C. Linial M. FEBS Lett. 1995; 359: 159-163Google Scholar). The mutations in this domain of dlg-A cause the abnormalities of imaginal discs (22Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Google Scholar). Thus, this domain should also have some essential role. To clarify the function of PSD-95/SAP90 and to identify novel components of PSD, we have tried to identify by use of the yeast two-hybrid method the molecules which interact with the region of PSD-95/SAP90 containing the SH3 domain and the guanylate kinase domain, and are localized at PSD. We have obtained novel proteins which are homologous to each other. We named them SAPAPs (SAP90/PSD-95-AssociatedProteins). SAPAPs function to induce the translocation of PSD-95/SAP90 from the cytosol to the plasma membrane and may be a new family of proteins involved in signaling at PSD. The bait vector, pBTM116 PSD-95-3, was constructed by subcloning the insert encoding amino acid residues 430–724 of PSD-95/SAP90 intoEcoRI/SalI sites of pBTM116 (31Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Google Scholar). The yeast two-hybrid library constructed from adult rat brain cDNA was screened using pBTM116 PSD95-3 as a bait, as described previously (28Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Google Scholar). β-Galactosidase assays were performed as described previously (28Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Google Scholar). To obtain full-length clones, rat brain cDNA libraries in λ ZAP II (Stratagene) were screened using the inserts from pPrey1201, pPrey1305, pPrey1310, and pPrey1377 as described previously (28Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Google Scholar). For pPrey1201, 1 × 106clones were screened and 25 positive clones were obtained. For pPrey1305, 2 × 106 clones were screened and 10 positive clones were obtained. For pPrey1310, 1 × 106clones were screened and positive clones were obtained. For 1 × 106 clones were screened and positive clones were obtained. at least clones for each were was performed by the method using an and bait and were constructed in J.E. Biochem. 1991; Scholar), of and using molecular J. T. Scholar). was constructed from from as described previously Y. Südhof T.C. J. Biol. Chem. 1995; Scholar). and maltose-binding protein the amino acid and of rat and of rat 430–724 of rat and of rat PSD-95/SAP90. bait the amino pBTM116 of rat pBTM116 of rat pBTM116 of pBTM116 of pBTM116 of rat pBTM116 of pBTM116 of rat pBTM116 of rat pBTM116 of rat pBTM116 of pBTM116 of rat and pBTM116 of rat PSD-95/SAP90. the full-length of the full-length of PSD-95/SAP90 and amino acid residues of PSD-95/SAP90. was constructed by the the of was constructed by the encoding the full-length of sites of that was expressed with amino as the The of rat and were obtained by and the were proteins and proteins were expressed and as described previously (28Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Google Scholar). cells were transfected using the method as described previously Y. Südhof T.C. J. Biol. Chem. 1995; Scholar). cells were transfected with the method using cells transfected with were in 1 25 1 10 and using of each The were at × for at 1 of the was with of various proteins on the for 2 at The were by with four and through and with the the The interactions of PSD-95/SAP90 and SAPAPs were by use of on the and as described previously U. H. J. S. E. R. C. H. M. 1991; Scholar). The were on a by the and the containing of and were on the to the of about amino acid residues of amino acid residues of The was with and various of various in of were the at a of The binding were as the the 10 to the and the at the All were performed at 25 were using were the proteins of and using The used for each the amino acid of of of and of The amino acid residues used for the were for each and each was to be for each The PSD-95/SAP90 was the protein containing the full-length of PSD-95/SAP90 using or The NMDA receptors and various were from The the was obtained from were obtained from on the were on and in essential with the was with essential with J.E. Proc. Natl. Acad. Sci. U. S. A. Scholar), 1 1 and of cells and cells were performed using a cells transfected with various were in 10 10 and using The were at × for at of cytosol and membrane were through and with or of rat was performed as described previously D.J. Matus A.I. Scholar, K. Y. Südhof T.C. J. Biol. Chem. 1995; Scholar). and protein were performed using as described previously (28Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Google Scholar). were and with to the use of the yeast two-hybrid we for the proteins which with the region of PSD-95/SAP90 containing the SH3 domain and the guanylate kinase domain. × with a rat brain cDNA library were positive clones were obtained and clones of them were clones pPrey1305, pPrey1310, and similar but not the same which to proteins. The clones and not show to proteins. We on pPrey1201, pPrey1305, pPrey1310, and the using the inserts from these clones only in brain and named the proteins to To the structures of rat brain cDNA libraries were screened to obtain cDNA clones for each using the insert from the as each The of the of and with the for and were by M. 15: Scholar). The of at to the We identified the in the cDNA of by with of other The of SAPAPs at the amino acid and not show and to of and and of and amino The amino acid of to proteins a region or a All SAPAPs domains in the region and the revealed to proteins. pPrey1201, pPrey1305, pPrey1310, and pPrey1377 amino acid residues of of of and of Based on the the from these four clones, the domain of SAPAPs essential for the with the region of PSD-95/SAP90 used as the bait be to be in the region of about amino acid residues in the 1 To the clones of SAPAPs were full-length clones, we constructed using these clones and the of SAPAPs in cells. The were and using for each The and the proteins with of about and the the protein with of about in cells transfected with each not The of the proteins in the transfected cells were from the on the However, these also the proteins with of or in brain not clones of to are to be the full-length clones of and SAPAPs may show on to We by the yeast two-hybrid method SAPAPs interact with MAGUKs other than PSD-95/SAP90. bait which the SH3 domain and the guanylate kinase domain of various MAGUKs were and for interactions with clones All clones of SAPAPs pPrey1305, pPrey1310, and with bait containing the SH3 domain and the guanylate kinase domain of not only PSD-95/SAP90, but also SAP97, PSD-95/SAP90-related protein 1 and The clones not interact with the bait containing the SH3 domain and the guanylate kinase domain of or is a novel of PSD-95/SAP90, which is considered to be an of these MAGUKs, PSD-95/SAP90, SAP97, PSRP1, form a related family from and All SAPAPs specifically with members of this related of SAPAPs with various of not containing the SH3 and guanylate kinase × × not containing the guanylate kinase containing the SH3 clones with the and the bait pBTM116 in the were on the and in pBTM116 pBTM116 pBTM116 pBTM116 pBTM116 pBTM116 and pBTM116 the SH3 domain and the guanylate kinase domain of various pBTM116 pBTM116 pBTM116 pBTM116 and pBTM116 the guanylate kinase domain of various pBTM116 the SH3 domain of PSD-95/SAP90. the of The activity protein in the yeast was in the with not not in a new clones with the and the bait pBTM116 in the were on the and in pBTM116 pBTM116 pBTM116 pBTM116 pBTM116 pBTM116 and pBTM116 the SH3 domain and the guanylate kinase domain of various pBTM116 pBTM116 pBTM116 pBTM116 and pBTM116 the guanylate kinase domain of various pBTM116 the SH3 domain of PSD-95/SAP90. the of The activity protein in the yeast was in the with To which region of PSD-95/SAP90 is for with we the interactions of the clones and bait containing various of PSD-95/SAP90 and its related proteins. The clones of SAPAPs with the bait containing only the guanylate kinase domains of PSD-95/SAP90, SAP97, PSRP1, and but not with the bait containing the SH3 domain of PSD-95/SAP90 the guanylate kinase domains of PSD-95/SAP90 and its related proteins are for with We also the interactions the bait containing the SH3 domain or the guanylate kinase domain of PSD-95/SAP90 and the containing full-length The bait containing the SH3 domain not interact with the containing full-length in contrast to the bait containing the guanylate kinase domain that the SH3 domain of PSD-95/SAP90 is not involved in the with To the of PSD-95/SAP90 and SAPAPs in the of cells with the at the were with various proteins on The proteins were by by with the the was to containing full-length PSD-95/SAP90 and containing the SH3 domain and the guanylate kinase domain was not to or containing three PDZ (DHR) domains To SAPAPs interact with PSD-95/SAP90, was proteins containing the potential PSD-95/SAP90 interacting region of SAPAPs or were on the through the protein containing the guanylate kinase domain of PSD-95/SAP90 was on the and the for the were The of PSD-95/SAP90 for the protein of was to be × from the a × and × The of PSD-95/SAP90 for the protein of was to be × from the a × and × The protein of not interact with SAPAPs not the protein containing three PDZ (DHR) domains nor the protein containing the SH3 domain with SAPAPs not using the region of and in rat of In rat with a was The of this was unknown. rat including and not show using the region of a and three only in brain of and and only in not In using the the was only in rat brain using and similar not To SAPAPs are neuronal we these proteins in from rat using The in the the cell and the of the was with an of protein of the in the and cell the in the in and cell were considered to be was similar to that of PSD-95/SAP90 not The cells were not with the not the same were obtained using the and not of the of rat brain that proteins with were enriched in the The of the synaptic the membrane and the membrane that was enriched in in which PSD-95/SAP90 and NMDA receptors were also PSD was by of was enriched in PSD using and the same not In the of we expressed SAPAPs and PSD-95/SAP90 in various in cells to SAPAPs with PSD-95/SAP90 in cells. PSD-95/SAP90 was expressed in PSD-95/SAP90 was in the cytosol the other was at the plasma membrane in cells of PSD-95/SAP90 with the translocation of PSD-95/SAP90 from the cytosol to the plasma membrane where was However, the encoding only three PDZ (DHR) domains of PSD-95/SAP90 was the of the product of this was not by SAPAPs the same on the translocation of PSD-95/SAP90 not The translocation of PSD-95/SAP90 on the of SAPAPs was also through of transfected cells as described was in the membrane of the transfected was expressed and was with PSD-95/SAP90 PSD-95/SAP90 was in the cytosol in the transfected was expressed but was in the membrane was with The protein containing only the PDZ (DHR) domains of PSD-95/SAP90 was in the cytosol with were obtained using other SAPAPs than not In this we have reported four novel proteins which interact with the guanylate kinase domain of PSD-95/SAP90. We have named these proteins We have each and the of SAPAPs in cells. The of SAPAPs in cells transfected with containing the of the clones of SAPAPs are the same as in these clones are considered to be full-length We have that SAPAPs interact with PSD-95/SAP90 on the of the of SAPAPs interact with PSD-95/SAP90 in with of about 2 × these are in a SAPAPs are expressed in and enriched in the PSD in which PSD-95/SAP90 are SAPAPs also show interactions with PSD-95/SAP90 in cells and induce the translocation of PSD-95/SAP90 from the cytosol to the plasma and in the yeast two-hybrid for PSD-95/SAP90, SAP97, and are obtained SAPAPs are about homologous to each other at the amino acid The amino in the region are the are also observed in the region and in the SAPAPs show to proteins and the not the of Since the clones obtained from the yeast two-hybrid various of we the region of region is in the region and about amino acid which not The domains are the domains in the and and The domains are considered to interact with the SH3 domain. the domains in the of and have the of the of the proteins which interact with the SH3 domain of the amino acid of other domains not to the of the domains reported to interact with the SH3 domain R. D. Cell. 1995; 80: Scholar, A.B. J.E. L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). is at these domains function for the with other The to identify the region of PSD-95/SAP90 has been performed using the yeast two-hybrid The clones of SAPAPs interact with the bait containing the guanylate kinase domain of PSD-95/SAP90 and not the bait containing the SH3 domain. Thus, the guanylate kinase domain is for the The bait containing the SH3 domain does not interact with the containing the full-length of The of this bait is PSD-95/SAP90 is obtained from the yeast two-hybrid using this bait SAPAPs are obtained from the yeast two-hybrid using the SH3 domain of PSD-95/SAP90 as a are not considered to be the interacting proteins with the SH3 domain of PSD-95/SAP90. expressed in cells not interact with the protein containing only three PDZ (DHR) domains of PSD-95/SAP90. SAPAPs not interact with the product of the containing only three PDZ (DHR) domains in the transfected cells the guanylate kinase domain of PSD-95/SAP90 is to be the interacting domain with SAPAPs are expressed in brain and enriched in the PSD fraction. However, in rat the by the are in the cell and the of and not show enrichment at synaptic junctions. is the same as that observed for PSD-95/SAP90. The the enrichment of SAPAPs and PSD-95/SAP90 at synaptic junctions is not observed in rat cells is not The of the synaptic junctions may some such as and which are not in the of cells. the of SAPAPs are may function as molecules to the interactions of PSD-95/SAP90 and the molecules at the plasma SAPAPs induce the enrichment of PSD-95/SAP90 at the plasma membrane in cells. However, that SAPAPs have on the clustering of NMDA receptor by PSD-95/SAP90, studies are not all the domains of SAPAPs have the for the interacting domains with the SH3 domains. However, there is a that some of the domains of SAPAPs may interact with molecules and SAPAPs may as an PSD-95/SAP90 and these We D. of at for vector, S. for cDNA of NMDA receptor S. Tsukita for cDNA of S. Hollenberg of for the of the yeast two-hybrid and H. for used in this were by Y. H. in the of T. C. Südhof of at use of these