Subunit assembly and vesicle trafficking of AMPA receptors (AMPA-Rs) play central roles in synaptic plasticity. The molecular basis for AMPA-R function, trafficking, and biogenesis remains, however, poorly understood. Current data in the field suggest that AMPA-Rs have a dimer-of-dimers organization, asymmetric overall structure, and in the brain contain auxiliary stargazin/TARP subunits that modulate AMPA-R function. To obtain more insight into AMPA-R structure and assembly, which are critical for understanding synaptic plasticity, we propose to compare the 3D EM structure of the mature GluR2 tetramer with those of dimeric biosynthetic intermediates. We will DOX dependently express GluR2 subunits in stable HEK cell lines, purify the recombinant receptors and study their structures by single-particle electron microscopy.
In Aim 1 we will test whether normal subunit assembly requires the subunits to adopt a specific conformation and whether point mutations interfering with subunit assembly change the subunit conformation. First, we will work towards obtaining an improved 3D structure of a fully assembled AMPA-R by imaging tetramers formed by recombinant GluR2 flip and locked into the non-desensitized state. We will also determine the structure of immature GluR2 dimers. By comparing the 3D structures of the mature and immature AMPA-Rs, we will unveil the inter-domain contacts that drive the assembly of dimers into tetramers. Finally, we will study the 3D structures of dimer intermediates of mutant GluR2 subunits and compare those with the structures of wild-type GluR2, providing insight into how mutations may interfere with subunit assembly. Because in the brain auxiliary stargazin/TARP subunits are associated with AMPA-Rs, in Aim 2 we will investigate when stargazin binds to GluR2 during maturation and how stargazin modulates trafficking and subunit assembly of GluR2. We have established stable HEK cell lines that constitutively express stargazin but GluR2 only in the presence of DOX. We will compare the time course of the DOX-induced expression of GluR2 in the presence and absence of stargazin to quantitatively test if stargazin is a molecular chaperone of GluR2. We will further use GFP-tagged GluR2 in neurons to examine the effect of stargazin on receptor dynamics using time-lapse fluorescent confocal mircroscopy. Results from these experiments will determine the contribution of stargazin to the trafficking and maturation of newly assembled AMPA-Rs. The NMDA receptor (NMDA-R) is another member of the glutamate receptor family that plays critical roles in synaptic plasticity. Evidence in the field suggests that the domain arrangement and mechanism of subunit assembly may differ between NMDA-Rs and AMPA-Rs.
In Aim 3, we will therefore use the approaches we established for our studies on AMPA-Rs to NMDA-Rs. In particular, we will use EM to study the structure of heterotetrameric NMDA-Rs formed by NR1 and NR2B subunits.
Dysfunction of glutamate receptors causes a variety of neurological and psychiatric disorders and stroke. Mutations in glutamate receptor subunits have been found in patients with X-linked mental retardation, and thus altered glutamate receptor function and assembly is likely to be the direct cause of the disease. By revealing the detailed molecular basis for wild-type and mutant glutamate receptor function, trafficking and subunit assembly, this proposal intends to extend our understanding of neurological and mental disorders, which may especially lead to curing X-linked mental retardation.
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