Binding of neuroligins to PSD-95

Science

Volumn 277, Issue 5331, 1997, Pages 1511-1515

  Irie, M ^a   Hata, Y ^a,b   Takeuchi, M ^a   Ichtchenko, K ^b   Toyoda, A ^a   Hirao, K ^a   Takai, Y ^a,c   Rosahl, T W ^b   Sudhof T C ^b  

^a JAPAN SCIENCE AND TECHNOLOGY AGENCY   (Japan)

^b HOWARD HUGHES MEDICAL INSTITUTE   (United States)

^c OSAKA UNIVERSITY MEDICAL SCHOOL   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

ION CHANNEL; MEMBRANE PROTEIN; N METHYL DEXTRO ASPARTIC ACID RECEPTOR; NERVE CELL ADHESION MOLECULE; NERVE PROTEIN; NEUROLIGIN DERIVATIVE; POTASSIUM CHANNEL; POTASSIUM ION; UNCLASSIFIED DRUG;

ANIMAL CELL; ARTICLE; CELL ADHESION; EMBRYO; GENE EXPRESSION; HUMAN; HUMAN CELL; NEUROTRANSMISSION; NONHUMAN; PRIORITY JOURNAL; PROTEIN BINDING; RECEPTOR BINDING; SYNAPTIC TRANSMISSION;

AMINO ACID SEQUENCE; ANIMALS; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CELL LINE; CELL MEMBRANE; COS CELLS; CYTOPLASM; GUANYLATE KINASE; INTERCELLULAR JUNCTIONS; INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS; MEMBRANE PROTEINS; MICE; MOLECULAR SEQUENCE DATA; NERVE TISSUE PROTEINS; NEURONS; NUCLEOSIDE-PHOSPHATE KINASE; POTASSIUM CHANNELS; PROSENCEPHALON; RECEPTORS, N-METHYL-D-ASPARTATE; RECOMBINANT FUSION PROTEINS; TUMOR SUPPRESSOR PROTEINS;

EID: 0030770840     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5331.1511     Document Type: Article

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22. 32P signals were quantified by Phosphorlmager detection and are expressed as ratio to GAPDH.
23. A rat brain cDNA library in the yeast-two hybrid prey vector pVP16 [A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74, 205 (1993)] was screened as described [S. Fields and O. Song, Nature 340, 245 (1994)] (14) with the bait vector pBTM116-NL2-1 (8). Of 16 prey clones isolated, pPrey500 encodes residues 1 to 287, and pPrey514 encodes residues 308 to 425 of PSD-95 (1). β-Galactosidase assays were corrected for protein concentration [M. D. Rose, F. Winston, P. Hieter, Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990); Y. Hata and T. C. Südhof, J. Biol. Chem. 270, 13022 (1995)].
24. A rat brain cDNA library in the yeast-two hybrid prey vector pVP16 [A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74, 205 (1993)] was screened as described [S. Fields and O. Song, Nature 340, 245 (1994)] (14) with the bait vector pBTM116-NL2-1 (8). Of 16 prey clones isolated, pPrey500 encodes residues 1 to 287, and pPrey514 encodes residues 308 to 425 of PSD-95 (1). β-Galactosidase assays were corrected for protein concentration [M. D. Rose, F. Winston, P. Hieter, Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990); Y. Hata and T. C. Südhof, J. Biol. Chem. 270, 13022 (1995)].
25. A rat brain cDNA library in the yeast-two hybrid prey vector pVP16 [A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74, 205 (1993)] was screened as described [S. Fields and O. Song, Nature 340, 245 (1994)] (14) with the bait vector pBTM116-NL2-1 (8). Of 16 prey clones isolated, pPrey500 encodes residues 1 to 287, and pPrey514 encodes residues 308 to 425 of PSD-95 (1). β-Galactosidase assays were corrected for protein concentration [M. D. Rose, F. Winston, P. Hieter, Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990); Y. Hata and T. C. Südhof, J. Biol. Chem. 270, 13022 (1995)].
26. A rat brain cDNA library in the yeast-two hybrid prey vector pVP16 [A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74, 205 (1993)] was screened as described [S. Fields and O. Song, Nature 340, 245 (1994)] (14) with the bait vector pBTM116-NL2-1 (8). Of 16 prey clones isolated, pPrey500 encodes residues 1 to 287, and pPrey514 encodes residues 308 to 425 of PSD-95 (1). β-Galactosidase assays were corrected for protein concentration [M. D. Rose, F. Winston, P. Hieter, Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990); Y. Hata and T. C. Südhof, J. Biol. Chem. 270, 13022 (1995)].
27. + channel isoform. The different prey vectors are from rat except for ZO-1 (mouse) and dlg-A (Drosophila) and encode the following residues; PSD-95, pVP16PSD-95-2 = 1-431; pVP16SAP90-5 = 69-150; pVP16SAP90-6 = 160-245. SAP102, PVP16SAP102-1 = 1-519. PSD-93, pVP16PSD-93-1 = 1-539. ZO1, pVP16ZO1-1 = 1-500. dlg-A, pVP16dlg-1 = 1-598. Nitric oxide synthase, PVP16NOS = 1-101.
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30. 2, 0.2 M NaCl, and 0.5% NP-40 and eluted sequentially with 0.3 ml of the wash buffer containing 0.5 M NaCl and with 0.5 ml of 20 mM Hepes-NaOH (pH 8.0), 0.1 M NaCl, 10 mM EGTA, and 0.5% NP-40. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis and by immunoblotting with antibodies to PSD-95 (polyclonal antiserum L667 and two independent monoclonal antibodies) and to neuroligin 1 (polyclonal antiserum L067). To exclude the possibility that PSD-95 directly interacts with neurexin 1β, extracts from COS cells transfected with full-length PSD-95 were affinity chromatographed on immobilized neurexin 1β in the absence and presence of recombinant neuroligin.
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35. Abbreviations for amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
36. We thank A. Roth, E. Borowicz, and I. Leznicki for excellent technical assistance; M. B. Kennedy (CalTech) for monoclonal antibodies to PSD-95; S. Nakanishi (Kyoto) for NMDA receptor cDNA clones; and M. Missler, M. S. Brown, and J. L. Goldstein for advice. Partially supported by grants from the NIH (RO1-MH52804), the Perot Family Foundation, and the ERATO (Japan Science and Technology Co.). Y.H. was supported by a postdoctoral fellowship from the HFSP, and T.W.R. from the DFG.