Dual-requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity

Science

Volumn 282, Issue 5392, 1998, Pages 1321-1324

  Feng, Guoping ^a   Tintrup, Hartmut ^a   Kirsch, Joachim ^b   Nichol, Mia C ^a   Kuhse, Jochen ^b   Betz, Heinrich ^b   Sanes, Joshua R ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b MAX PLANCK INSTITUTE FOR BRAIN RESEARCH   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

GEPHYRIN; GLYCINE RECEPTOR; MOLYBDENUM COMPLEX;

ANIMAL TISSUE; ARABIDOPSIS; ARTICLE; DROSOPHILA; ENZYME ACTIVITY; ESCHERICHIA COLI; GENE TARGETING; MOLECULAR CLONING; MOUSE; NEUROTRANSMISSION; NONHUMAN; PRIORITY JOURNAL; SYNAPTIC TRANSMISSION;

EID: 0032514872     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5392.1321     Document Type: Article

References (39)

1. M. H. Aprison, in Glycine Neurotransmission, O. P. Ottoson and J. Storm-Mathisen, Eds. (Wiley, New York, 1990), pp. 1-24; P. Jonas, J. Bischofberger, J. Sandkühler, Science 281, 419 (1998).
2. M. H. Aprison, in Glycine Neurotransmission, O. P. Ottoson and J. Storm-Mathisen, Eds. (Wiley, New York, 1990), pp. 1-24; P. Jonas, J. Bischofberger, J. Sandkühler, Science 281, 419 (1998).
3. F. Pfeiffer, D. Graham, H. Betz, J. Biol. Chem. 257, 9389 (1982).
4. G. Meyer, J. Kirsch, H. Betz, D. Langosch, Neuron 15, 563 (1995); J. Kirsch et al., J. Biol. Chem. 266, 22242 (1991); J. Kirsch and H. Betz, J. Neurosci. 15, 4148 (1995).
5. G. Meyer, J. Kirsch, H. Betz, D. Langosch, Neuron 15, 563 (1995); J. Kirsch et al., J. Biol. Chem. 266, 22242 (1991); J. Kirsch and H. Betz, J. Neurosci. 15, 4148 (1995).
6. G. Meyer, J. Kirsch, H. Betz, D. Langosch, Neuron 15, 563 (1995); J. Kirsch et al., J. Biol. Chem. 266, 22242 (1991); J. Kirsch and H. Betz, J. Neurosci. 15, 4148 (1995).
7. A. Triller, F. Cluzeaud, F. Pfeiffer, H. Betz, H. Korn, J. Cell Biol. 101, 683 (1985); J. Kirsch and H. Betz, Brain Res. 621, 301 (1993); A. J. Todd, R. C. Spike, D. Chong, M. Neilson, Eur. J. Neurosci. 7, 1 (1995).
8. A. Triller, F. Cluzeaud, F. Pfeiffer, H. Betz, H. Korn, J. Cell Biol. 101, 683 (1985); J. Kirsch and H. Betz, Brain Res. 621, 301 (1993); A. J. Todd, R. C. Spike, D. Chong, M. Neilson, Eur. J. Neurosci. 7, 1 (1995).
9. A. Triller, F. Cluzeaud, F. Pfeiffer, H. Betz, H. Korn, J. Cell Biol. 101, 683 (1985); J. Kirsch and H. Betz, Brain Res. 621, 301 (1993); A. J. Todd, R. C. Spike, D. Chong, M. Neilson, Eur. J. Neurosci. 7, 1 (1995).
10. J. Kirsch, J. Kuhse, H. Betz, Mol. Cell. Neurosci. 6, 450 (1995); J. Kirsch, I. Wolters, A. Triller, H. Betz, Nature 366, 745 (1993).
11. J. Kirsch, J. Kuhse, H. Betz, Mol. Cell. Neurosci. 6, 450 (1995); J. Kirsch, I. Wolters, A. Triller, H. Betz, Nature 366, 745 (1993).
12. J. Kirsch, G. Meyer, H. Betz, Mol. Cell. Neurosci. 8, 93 (1996); C. Bechade and A. Triller, Semin. Cell Dev. Biol. 7, 717 (1996).
13. J. Kirsch, G. Meyer, H. Betz, Mol. Cell. Neurosci. 8, 93 (1996); C. Bechade and A. Triller, Semin. Cell Dev. Biol. 7, 717 (1996).
14. P. Prior et al., Neuron 8, 1161 (1992).
15. K. V. Rajagopalan, Biochem. Soc. Trans. 25, 757 (1997); K. P. Kamdar et al., ibid., p. 778; B. Stallmeyer, A. Nerlich, J. Schiemann, H. Brinkmann, R. R. Mendel, Plant J. 8, 751 (1995).
16. K. V. Rajagopalan, Biochem. Soc. Trans. 25, 757 (1997); K. P. Kamdar et al., ibid., p. 778; B. Stallmeyer, A. Nerlich, J. Schiemann, H. Brinkmann, R. R. Mendel, Plant J. 8, 751 (1995).
17. K. V. Rajagopalan, Biochem. Soc. Trans. 25, 757 (1997); K. P. Kamdar et al., ibid., p. 778; B. Stallmeyer, A. Nerlich, J. Schiemann, H. Brinkmann, R. R. Mendel, Plant J. 8, 751 (1995).
18. M. Ramming, H. Betz, J. Kirsch, FEBS Lett. 405, 137 (1997).
19. The targeting vector contained a 5-kb Eco Rl/Sac I genomic fragment in which a phosphoglycerate kinase-neo cassette replaced a Pst I/Eag I fragment that encoded the putative promoter region of the murine gephyrin gene as well as the first exon, which included the initiator methionine (9). The construct was transferted into R1 ES cells [A. Nagy, J. Rossant, R. Nagy, W. Abramow-Newerly, J. C. Roder, Proc. Natl. Acad. Sci. U.S.A. 90, 8424 (1993)], and 20 of 200 clones screened were identified as homologous recombinants. Homozygotes derived from two independently derived ES cell clones showed identical phenotypes. For Southern (DNA) analysis, a 0.6-kb probe external to the short arm (Fig. 1B) was hybridized to Eco RV-digested genomic DNA. For Northern analysis, 10-μg aliquots of polyadenylated RNA from brain were separated in a 1% agarose gel, and a blot was probed successively with cDNAs encoding mouse gephyrin, sulfite oxidase, or GlyR α1 subunit. For Western analysis, brain protein extracts were fractionated on 6% or 10% SDS polyacrylamide gels, and immunoblots were probed with mouse monoclonal antibodies directed against the COOH-terminus of gephyrin (Transduction Labs) or GlyR subunits [monoclonal antibody (mAb) 4a; (24)].
20. Spinal cords and brains were cross-sectioned at 7 μm in a cryostat. Sections were fixed with 4% paraformaldehyde and stained with antibodies specific for GlyR α1 subunit [mAb 2b; (24)], gephyrin [mAbs 5a and 7a; (24)], SV2 (gift of K. Buckley, Harvard University), the PSD-95/SAP-90 family (Upstate Biotechnology), the glutamate receptor GluR1 subunit (Upstate Biotechnology), or synaptophysin (gift of A. Czernik, Rockefeller University). Other sections were stained with hematoxylin and eosin.
21. M. Sheng and M. Wyszynski, Bioessays 19, 847 (1997).
22. E. S. Simon, Mov. Disord. 12, 221 (1997).
23. J. L. Johnson and S. K. Wadman, in The Metabolic Basis of Inherited Diseases, C. R. Scrivner, A. L. Beaudef, W. S. Sly, D. Valle, Eds. (McGraw-Hill, New York, 1995), pp. 2271-2283
24. For sulfite oxidase assay, livers were homogenized and centrifuged at 16,000g for 20 min. Supernatants were desalted on Sephadex G-25 (Pharmacia). Activity in the flow-through was determined as described [J. L. Johnson et al., J. Inherit. Metab. Dis. 14, 932 (1991)].
25. M. Nakamura and I. Yamazaki, J. Biochem. 92, 1279 (1972).
26. Xanthine dehydrogenase assay was modified from D. A. Parks, T. K. Williams, and J. S. Beckman [Am. J. Physiol. 254 768 (1988)]. Intestines were homogenized in 50 mM potassium phosphate (pH 7.0), 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM EDTA. After centrifugation at 16,000g for 20 min, 200-μl aliquots of supernatant were incubated with 100 μl of 500 μM nicotinamide adenine dinucleotide, 100 μl of 50 μM xanthine, and 600 μl of 50 mM potassium phosphate (pH 7.8). Formation of uric acid from xanthine was monitored at 295 nm wavelength in the presence or absence of 5 μM allopurinol, an inhibitor of xanthine dehydrogenase [ R. W. E. Watts, J. E. M. Watts, J. E. Seegmiller, J. Lab. Clin. Med. 66, 688 (1965)].
27. Xanthine dehydrogenase assay was modified from D. A. Parks, T. K. Williams, and J. S. Beckman [Am. J. Physiol. 254 768 (1988)]. Intestines were homogenized in 50 mM potassium phosphate (pH 7.0), 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM EDTA. After centrifugation at 16,000g for 20 min, 200-μl aliquots of supernatant were incubated with 100 μl of 500 μM nicotinamide adenine dinucleotide, 100 μl of 50 μM xanthine, and 600 μl of 50 mM potassium phosphate (pH 7.8). Formation of uric acid from xanthine was monitored at 295 nm wavelength in the presence or absence of 5 μM allopurinol, an inhibitor of xanthine dehydrogenase [ R. W. E. Watts, J. E. M. Watts, J. E. Seegmiller, J. Lab. Clin. Med. 66, 688 (1965)].
28. Lactate dehydrogenase activity was determined as described [A. Yoshida and E. Freese, Methods Enzymol. 41, 304 (1975)]. Protein was assayed with the Bradford reagent (Bio-Rad).
29. C. A. Rupar et al., Neuropediatrics 27, 299 (1996).
30. H. Nishimaru, M. Iizuka, S. Ozaki, N. Kudo, J. Physiol. (London) 497, 131 (1996).
31. W.-L. Wu, L. Ziskind-Conhaim, M. A. Sweet, J. Neurosci. 12, 3935 (1992).
32. M. Gautam et al., Nature 377, 232 (1995); M. Gautum et al., Cell 85, 525 (1996); T. M. DeChiara et al., ibid., p. 501; J. R. Sanes and J. W. Lichtman, Annu. Rev. Neurosci., in press.
33. M. Gautam et al., Nature 377, 232 (1995); M. Gautum et al., Cell 85, 525 (1996); T. M. DeChiara et al., ibid., p. 501; J. R. Sanes and J. W. Lichtman, Annu. Rev. Neurosci., in press.
34. M. Gautam et al., Nature 377, 232 (1995); M. Gautum et al., Cell 85, 525 (1996); T. M. DeChiara et al., ibid., p. 501; J. R. Sanes and J. W. Lichtman, Annu. Rev. Neurosci., in press.
35. M. Gautam et al., Nature 377, 232 (1995); M. Gautum et al., Cell 85, 525 (1996); T. M. DeChiara et al., ibid., p. 501; J. R. Sanes and J. W. Lichtman, Annu. Rev. Neurosci., in press.
36. W. Brune et al., Am. J. Hum. Genet. 58, 959 (1996); M. Andrew and M. J. Owen, Br. J. Psychiatry 170, 106 (1997).
37. W. Brune et al., Am. J. Hum. Genet. 58, 959 (1996); M. Andrew and M. J. Owen, Br. J. Psychiatry 170, 106 (1997).
38. F. Pfeiffer, R. Simler, G. Grenningloh, H. Betz, Proc. Natl. Acad. Sci. U.S.A. 81, 7224 (1984).
39. Supported by grants from NIH (J.R.S.), the Jane Coffin Childs Memorial Fund for Medical Research (C.F.), the McKnight Foundation (J.R.S.) the Deutsche Forschungsgemeinschaft, BMBF, and Fonds der Chemischen Industrie (H.B.). We thank I. Bartuik, C. Borgmeyer, J. Cunningham, J. Gross, and S. Weng for assistance. We dedicate this paper to the memory of Rolf Westkamp.