Abstract
The ionotropic glutamate receptors (iGluRs) are postsynaptic ion channels involved in excitatory neurotransmission. The iGluRs can be classified according to their specific agonists into N-methyl-D-aspartate (NMDA), α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. Each of these classes contains several homologous subunits that assemble in a subtype-specific manner into oligomeric (tetra/pentameric) complexes. The iGluRs are integral membrane proteins for which there is as yet no structure available. However, some structural features of the subunits, including the primary structure and its modifications, topology and domain organisation, have been solved. Extensive biochemical and biophysical characterisation has been so far hampered by the lack of sufficient amounts of homogeneous material. In this study, the production and purification of an AMPA-type glutamate
receptor was investigated. Recombinant GluRD receptors were expressed in
Spodoptera frugiperda Sf21 insect cells and purified by affinity chromatography.
The purified receptor preparation contained over 2000 pmol of high-affinity (Kd
52 nM) binding sites/mg protein and exhibited a single 110 kDa band on silver-stained SDS-PAGE. A yield of 50-100 µg of purified receptor was obtained from one litre of Sf21 suspension culture.
In addition, the ligand binding sites of an AMPA receptor subunit GluRD and
a kainate receptor subunit GluR6 were studied. Two discontinuous segments, S1 and S2, which show sequence similarity to bacterial amino acid binding proteins, were expressed as soluble secreted fusion proteins. The S1S2 fragment was shown to comprise the ligand-binding domain in glutamate receptors as it reproduced the ligand binding characteristics of an intact receptor. A role for the N-terminal third of the S2 segment in AMPA-selectivity was identified by studying chimaeric GluRD/GluR6 S1S2 fragments.
Moreover, the entire extracellular domain (XS1S2) and an N-terminal ~400
residue segment (X) were expressed in High Five insect cells as soluble affinitytagged recombinant proteins in order to study their structure and properties. The Nterminal X domain was shown not to contribute to ligand binding, but was suggested to participate in the oligomerisation of the extracellular domain as a hydrodynamic analysis of the domains showed dimerisation of the XS1S2 ectodomain but not of the S1S2 domain.
receptor was investigated. Recombinant GluRD receptors were expressed in
Spodoptera frugiperda Sf21 insect cells and purified by affinity chromatography.
The purified receptor preparation contained over 2000 pmol of high-affinity (Kd
52 nM) binding sites/mg protein and exhibited a single 110 kDa band on silver-stained SDS-PAGE. A yield of 50-100 µg of purified receptor was obtained from one litre of Sf21 suspension culture.
In addition, the ligand binding sites of an AMPA receptor subunit GluRD and
a kainate receptor subunit GluR6 were studied. Two discontinuous segments, S1 and S2, which show sequence similarity to bacterial amino acid binding proteins, were expressed as soluble secreted fusion proteins. The S1S2 fragment was shown to comprise the ligand-binding domain in glutamate receptors as it reproduced the ligand binding characteristics of an intact receptor. A role for the N-terminal third of the S2 segment in AMPA-selectivity was identified by studying chimaeric GluRD/GluR6 S1S2 fragments.
Moreover, the entire extracellular domain (XS1S2) and an N-terminal ~400
residue segment (X) were expressed in High Five insect cells as soluble affinitytagged recombinant proteins in order to study their structure and properties. The Nterminal X domain was shown not to contribute to ligand binding, but was suggested to participate in the oligomerisation of the extracellular domain as a hydrodynamic analysis of the domains showed dimerisation of the XS1S2 ectodomain but not of the S1S2 domain.
| Original language | English |
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| Qualification | Doctor Degree |
| Awarding Institution |
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| Supervisors/Advisors |
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| Award date | 24 Mar 2000 |
| Place of Publication | Helsinki |
| Publisher | |
| Print ISBNs | 951-45-9235-2 |
| Electronic ISBNs | 951-45-9156-9 |
| Publication status | Published - 2000 |
| MoE publication type | G5 Doctoral dissertation (article) |