Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Mar 15;330(Pt 3):1461–1467. doi: 10.1042/bj3301461

Characterization of the kainate-binding domain of the glutamate receptor GluR-6 subunit.

K Keinänen 1, A Jouppila 1, A Kuusinen 1
PMCID: PMC1219296  PMID: 9494120

Abstract

Recombinant fragments of the kainate-selective glutamate recepto subunit GluR-6 were expressed in insect cells and analysed for [3H]kainate binding activity in order to characterize the structural determinants responsible for ligand recognition. Deletion of the N-terminal approximately 400 amino-acid-residue segment and the C-terminal approximately 90 residues resulted in a membrane-bound core fragment which displayed pharmacologically native-like [3H]kainate binding properties. Further replacement of the membrane-embedded segments M1-M3 by a hydrophilic linker peptide gave rise to a soluble polypeptide which was accumulated in the culture medium. When bound to chelating Sepharose beads via a C-terminal histidine tag, the soluble fragment showed low-affinity binding of [3H]kainate, which was displaced in a concentration-dependent manner by unlabelled domoic acid, L-glutamate and 6-cyano-7-nitroquinoxaline-2,3-dione. Our results indicate that the kainate-binding site is formed exclusively by the two discontinuous extracellular segments (S1 and S2) which are homologous to bacterial amino-acid-binding proteins. Ligand binding characteristics of soluble S1-S2 chimaeras between the GluR-6 and GluR-D subunits showed that, whereas both S1 and S2 segments contribute to agonist-selectivity, the N-terminal one-third of the GluR-D S2 segment is sufficient to confer alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-binding capacity to the chimaeric ligand-binding domain.

Full Text

The Full Text of this article is available as a PDF (505.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arvola M., Keinänen K. Characterization of the ligand-binding domains of glutamate receptor (GluR)-B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins. J Biol Chem. 1996 Jun 28;271(26):15527–15532. doi: 10.1074/jbc.271.26.15527. [DOI] [PubMed] [Google Scholar]
  2. Bennett J. A., Dingledine R. Topology profile for a glutamate receptor: three transmembrane domains and a channel-lining reentrant membrane loop. Neuron. 1995 Feb;14(2):373–384. doi: 10.1016/0896-6273(95)90293-7. [DOI] [PubMed] [Google Scholar]
  3. Bruns R. F., Lawson-Wendling K., Pugsley T. A. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem. 1983 Jul 1;132(1):74–81. doi: 10.1016/0003-2697(83)90427-x. [DOI] [PubMed] [Google Scholar]
  4. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  5. Egebjerg J., Bettler B., Hermans-Borgmeyer I., Heinemann S. Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature. 1991 Jun 27;351(6329):745–748. doi: 10.1038/351745a0. [DOI] [PubMed] [Google Scholar]
  6. Gregor P., Mano I., Maoz I., McKeown M., Teichberg V. I. Molecular structure of the chick cerebellar kainate-binding subunit of a putative glutamate receptor. Nature. 1989 Dec 7;342(6250):689–692. doi: 10.1038/342689a0. [DOI] [PubMed] [Google Scholar]
  7. Hirai H., Kirsch J., Laube B., Betz H., Kuhse J. The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6031–6036. doi: 10.1073/pnas.93.12.6031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hollmann M., Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci. 1994;17:31–108. doi: 10.1146/annurev.ne.17.030194.000335. [DOI] [PubMed] [Google Scholar]
  9. Hollmann M., Maron C., Heinemann S. N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron. 1994 Dec;13(6):1331–1343. doi: 10.1016/0896-6273(94)90419-7. [DOI] [PubMed] [Google Scholar]
  10. Keinänen K., Wisden W., Sommer B., Werner P., Herb A., Verdoorn T. A., Sakmann B., Seeburg P. H. A family of AMPA-selective glutamate receptors. Science. 1990 Aug 3;249(4968):556–560. doi: 10.1126/science.2166337. [DOI] [PubMed] [Google Scholar]
  11. Kuryatov A., Laube B., Betz H., Kuhse J. Mutational analysis of the glycine-binding site of the NMDA receptor: structural similarity with bacterial amino acid-binding proteins. Neuron. 1994 Jun;12(6):1291–1300. doi: 10.1016/0896-6273(94)90445-6. [DOI] [PubMed] [Google Scholar]
  12. Kuusinen A., Arvola M., Keinänen K. Molecular dissection of the agonist binding site of an AMPA receptor. EMBO J. 1995 Dec 15;14(24):6327–6332. doi: 10.1002/j.1460-2075.1995.tb00323.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laukkanen M. L., Oker-Blom C., Keinänen K. Secretion of green fluorescent protein from recombinant baculovirus-infected insect cells. Biochem Biophys Res Commun. 1996 Sep 24;226(3):755–761. doi: 10.1006/bbrc.1996.1425. [DOI] [PubMed] [Google Scholar]
  14. Li F., Owens N., Verdoorn T. A. Functional effects of mutations in the putative agonist binding region of recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Mol Pharmacol. 1995 Jan;47(1):148–154. [PubMed] [Google Scholar]
  15. Luckow V. A., Lee S. C., Barry G. F., Olins P. O. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol. 1993 Aug;67(8):4566–4579. doi: 10.1128/jvi.67.8.4566-4579.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mano I., Lamed Y., Teichberg V. I. A venus flytrap mechanism for activation and desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. J Biol Chem. 1996 Jun 28;271(26):15299–15302. doi: 10.1074/jbc.271.26.15299. [DOI] [PubMed] [Google Scholar]
  17. Nakanishi N., Shneider N. A., Axel R. A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron. 1990 Nov;5(5):569–581. doi: 10.1016/0896-6273(90)90212-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Hara P. J., Sheppard P. O., Thøgersen H., Venezia D., Haldeman B. A., McGrane V., Houamed K. M., Thomsen C., Gilbert T. L., Mulvihill E. R. The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron. 1993 Jul;11(1):41–52. doi: 10.1016/0896-6273(93)90269-w. [DOI] [PubMed] [Google Scholar]
  19. Oh B. H., Kang C. H., De Bondt H., Kim S. H., Nikaido K., Joshi A. K., Ames G. F. The bacterial periplasmic histidine-binding protein. structure/function analysis of the ligand-binding site and comparison with related proteins. J Biol Chem. 1994 Feb 11;269(6):4135–4143. [PubMed] [Google Scholar]
  20. Oh B. H., Pandit J., Kang C. H., Nikaido K., Gokcen S., Ames G. F., Kim S. H. Three-dimensional structures of the periplasmic lysine/arginine/ornithine-binding protein with and without a ligand. J Biol Chem. 1993 May 25;268(15):11348–11355. [PubMed] [Google Scholar]
  21. Paas Y., Eisenstein M., Medevielle F., Teichberg V. I., Devillers-Thiéry A. Identification of the amino acid subsets accounting for the ligand binding specificity of a glutamate receptor. Neuron. 1996 Nov;17(5):979–990. doi: 10.1016/s0896-6273(00)80228-7. [DOI] [PubMed] [Google Scholar]
  22. Roche K. W., Raymond L. A., Blackstone C., Huganir R. L. Transmembrane topology of the glutamate receptor subunit GluR6. J Biol Chem. 1994 Apr 22;269(16):11679–11682. [PubMed] [Google Scholar]
  23. Seeburg P. H. The TINS/TiPS Lecture. The molecular biology of mammalian glutamate receptor channels. Trends Neurosci. 1993 Sep;16(9):359–365. doi: 10.1016/0166-2236(93)90093-2. [DOI] [PubMed] [Google Scholar]
  24. Sommer B., Burnashev N., Verdoorn T. A., Keinänen K., Sakmann B., Seeburg P. H. A glutamate receptor channel with high affinity for domoate and kainate. EMBO J. 1992 Apr;11(4):1651–1656. doi: 10.1002/j.1460-2075.1992.tb05211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stern-Bach Y., Bettler B., Hartley M., Sheppard P. O., O'Hara P. J., Heinemann S. F. Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron. 1994 Dec;13(6):1345–1357. doi: 10.1016/0896-6273(94)90420-0. [DOI] [PubMed] [Google Scholar]
  26. Taverna F. A., Hampson D. R. Properties of a recombinant kainate receptor expressed in baculovirus-infected insect cells. Eur J Pharmacol. 1994 Jan 15;266(2):181–186. doi: 10.1016/0922-4106(94)90108-2. [DOI] [PubMed] [Google Scholar]
  27. Taverna F. A., Wang L. Y., MacDonald J. F., Hampson D. R. A transmembrane model for an ionotropic glutamate receptor predicted on the basis of the location of asparagine-linked oligosaccharides. J Biol Chem. 1994 May 13;269(19):14159–14164. [PubMed] [Google Scholar]
  28. Uchino S., Nakajima-Iijima S., Okuda K., Mishina M., Kawamoto S. Analysis of the glycine binding domain of the NMDA receptor channel zeta 1 subunit. Neuroreport. 1997 Jan 20;8(2):445–449. doi: 10.1097/00001756-199701200-00014. [DOI] [PubMed] [Google Scholar]
  29. Uchino S., Sakimura K., Nagahari K., Mishina M. Mutations in a putative agonist binding region of the AMPA-selective glutamate receptor channel. FEBS Lett. 1992 Aug 24;308(3):253–257. doi: 10.1016/0014-5793(92)81286-u. [DOI] [PubMed] [Google Scholar]
  30. Wada K., Dechesne C. J., Shimasaki S., King R. G., Kusano K., Buonanno A., Hampson D. R., Banner C., Wenthold R. J., Nakatani Y. Sequence and expression of a frog brain complementary DNA encoding a kainate-binding protein. Nature. 1989 Dec 7;342(6250):684–689. doi: 10.1038/342684a0. [DOI] [PubMed] [Google Scholar]
  31. Wafford K. A., Kathoria M., Bain C. J., Marshall G., Le Bourdellès B., Kemp J. A., Whiting P. J. Identification of amino acids in the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine binding site. Mol Pharmacol. 1995 Feb;47(2):374–380. [PubMed] [Google Scholar]
  32. Wo Z. G., Oswald R. E. Transmembrane topology of two kainate receptor subunits revealed by N-glycosylation. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7154–7158. doi: 10.1073/pnas.91.15.7154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yao N., Trakhanov S., Quiocho F. A. Refined 1.89-A structure of the histidine-binding protein complexed with histidine and its relationship with many other active transport/chemosensory proteins. Biochemistry. 1994 Apr 26;33(16):4769–4779. doi: 10.1021/bi00182a004. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES