Dual Requirement for Gephyrin in Glycine Receptor Clustering and Molybdoenzyme Activity
10.1126/science.282.5392.1321
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.

Bibliography

Feng, G., Tintrup, H., Kirsch, J., Nichol, M. C., Kuhse, J., Betz, H., & Sanes, J. R. (1998). Dual Requirement for Gephyrin in Glycine Receptor Clustering and Molybdoenzyme Activity. Science, 282(5392), 1321–1324.

Authors 7
  1. Guoping Feng (first)
  2. Hartmut Tintrup (additional)
  3. Joachim Kirsch (additional)
  4. Mia C. Nichol (additional)
  5. Jochen Kuhse (additional)
  6. Heinrich Betz (additional)
  7. Joshua R. Sanes (additional)
References 43 Referenced 360
  1. M. H. Aprison in Glycine Neurotransmission O. P. Ottoson and J. Storm-Mathisen Eds. (Wiley New York 1990) pp. 1–24;
  2. 10.1126/science.281.5375.419
  3. Pfeiffer F., Graham D., Betz H., J. Biol. Chem. 257, 9389 (1982). (10.1016/S0021-9258(18)34082-1) / J. Biol. Chem. by Pfeiffer F. (1982)
  4. Meyer G., Kirsch J., Betz H., Langosch D., Neuron 15, 563 (1995); (10.1016/0896-6273(95)90145-0) / Neuron by Meyer G. (1995)
  5. Kirsch J., et al., J. Biol. Chem. 266, 22242 (1991); (10.1016/S0021-9258(18)54560-9) / J. Biol. Chem. by Kirsch J. (1991)
  6. Kirsch J., Betz H., J. Neurosci. 15, 4148 (1995). (10.1523/JNEUROSCI.15-06-04148.1995) / J. Neurosci. by Kirsch J. (1995)
  7. Triller A., Cluzeaud F., Pfeiffer F., Betz H., Korn H., J. Cell Biol. 101, 683 (1985); (10.1083/jcb.101.2.683) / J. Cell Biol. by Triller A. (1985)
  8. Kirsch J., Betz H., Brain Res. 621, 301 (1993); (10.1016/0006-8993(93)90120-C) / Brain Res. by Kirsch J. (1993)
  9. Todd A. J., Spike R. C., Chong D., Neilson M., Eur. J. Neurosci. 7, 1 (1995). (10.1111/j.1460-9568.1995.tb01014.x) / Eur. J. Neurosci. by Todd A. J. (1995)
  10. Kirsch J., Kuhse J., Betz H., Mol. Cell. Neurosci. 6, 450 (1995); (10.1006/mcne.1995.1033) / Mol. Cell. Neurosci. by Kirsch J. (1995)
  11. Kirsch J., Wolters I., Triller A., Betz H., Nature 366, 745 (1993). (10.1038/366745a0) / Nature by Kirsch J. (1993)
  12. Kirsch J., Meyer G., Betz H., Mol. Cell. Neurosci. 8, 93 (1996); (10.1006/mcne.1996.0048) / Mol. Cell. Neurosci. by Kirsch J. (1996)
  13. Bechade C., Triller A., Semin. Cell Dev. Biol. 7, 717 (1996). (10.1006/scdb.1996.0088) / Semin. Cell Dev. Biol. by Bechade C. (1996)
  14. P. Prior et al. Neuron 8 1161 (1992). (10.1016/0896-6273(92)90136-2)
  15. Rajagopalan K. V., Biochem. Soc. Trans. 25, 757 (1997); (10.1042/bst0250757) / Biochem. Soc. Trans. by Rajagopalan K. V. (1997)
  16. ; K. P. Kamdar et al. ibid. p. 778; B. Stallmeyer
  17. Nerlich A., Schiemann J., Brinkmann H., Mendel R. R., Plant J. 8, 751 (1995). (10.1046/j.1365-313X.1995.08050751.x) / Plant J. by Nerlich A. (1995)
  18. Ramming M., Betz H., Kirsch J., FEBS Lett. 405, 137 (1997). (10.1016/S0014-5793(97)00164-6) / FEBS Lett. by Ramming M. (1997)
  19. The targeting vector contained a 5-kb Eco RI/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 transfected into R1 ES cells [
  20. 10.1073/pnas.90.18.8424
  21. ] 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)].
  22. 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.
  23. Sheng M., Wyszynski M., Bioessays 19, 847 (1997). (10.1002/bies.950191004) / Bioessays by Sheng M. (1997)
  24. Simon E. S., Mov. Disord. 12, 221 (1997). (10.1002/mds.870120213) / Mov. Disord. by Simon E. S. (1997)
  25. 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
  26. For sulfite oxidase assay livers were homogenized and centrifuged at 16 000 g for 20 min. Supernatants were desalted on Sephadex G-25 (Pharmacia). Activity in the flow-through was determined as described [
  27. Johnson J. L., et al., J. Inherit. Metab. Dis. 14, 932 (1991)]. (10.1007/BF01800477) / J. Inherit. Metab. Dis. by Johnson J. L. (1991)
  28. Nakamura M., Yamazaki I., J. Biochem. 92, 1279 (1972). (10.1093/oxfordjournals.jbchem.a134046) / J. Biochem. by Nakamura M. (1972)
  29. 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 16 000 g 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 [
  30. Watts R. W. E., Watts J. E. M., Seegmiller J. E., J. Lab. Clin. Med. 66, 688 (1965)]. / J. Lab. Clin. Med. by Watts R. W. E. (1965)
  31. Lactate dehydrogenase activity was determined as described [
  32. Yoshida A., Freese E., Methods Enzymol. 41, 304 (1975); (10.1016/S0076-6879(75)41069-2) / Methods Enzymol. by Yoshida A. (1975)
  33. ]. Protein was assayed with the Bradford reagent (Bio-Rad).
  34. Rupar C. A., et al., Neuropediatrics 27, 299 (1996). (10.1055/s-2007-973798) / Neuropediatrics by Rupar C. A. (1996)
  35. Nishimaru H., Iizuka M., Ozaki S., Kudo N., J. Physiol. (London) 497, 131 (1996). (10.1113/jphysiol.1996.sp021755) / J. Physiol. (London) by Nishimaru H. (1996)
  36. Wu W.-L., Ziskind-Conhaim L., Sweet M. A., J. Neurosci. 12, 3935 (1992). (10.1523/JNEUROSCI.12-10-03935.1992) / J. Neurosci. by Wu W.-L. (1992)
  37. Gautam M., et al., Nature 377, 232 (1995); (10.1038/377232a0) / Nature by Gautam M. (1995)
  38. Gautum M., et al., Cell 85, 525 (1996); (10.1016/S0092-8674(00)81253-2) / Cell by Gautum M. (1996)
  39. ; T. M. DeChiara et al. ibid. p. 501; J. R. Sanes and J. W. Lichtman Annu. Rev. Neurosci. in press.
  40. Brune W., et al., Am. J. Hum. Genet. 58, 959 (1996); / Am. J. Hum. Genet. by Brune W. (1996)
  41. Andrew M., Owen M. J., Br. J. Psychiatry 170, 106 (1997). (10.1192/bjp.170.2.106) / Br. J. Psychiatry by Andrew M. (1997)
  42. Pfeiffer F., Simler R., Grenningloh G., Betz H., Proc. Natl. Acad. Sci. U.S.A. 81, 7224 (1984). (10.1073/pnas.81.22.7224) / Proc. Natl. Acad. Sci. U.S.A. by Pfeiffer F. (1984)
  43. Supported by grants from NIH (J.R.S.) the Jane Coffin Childs Memorial Fund for Medical Research (G.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.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:44 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 12:30 a.m.)
Indexed 9 hours, 31 minutes ago (Aug. 30, 2025, 12:41 p.m.)
Issued 26 years, 9 months ago (Nov. 13, 1998)
Published 26 years, 9 months ago (Nov. 13, 1998)
Published Print 26 years, 9 months ago (Nov. 13, 1998)
Funders 0

None

@article{Feng_1998, title={Dual Requirement for Gephyrin in Glycine Receptor Clustering and Molybdoenzyme Activity}, volume={282}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.282.5392.1321}, DOI={10.1126/science.282.5392.1321}, number={5392}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Feng, Guoping and Tintrup, Hartmut and Kirsch, Joachim and Nichol, Mia C. and Kuhse, Jochen and Betz, Heinrich and Sanes, Joshua R.}, year={1998}, month=nov, pages={1321–1324} }