Tuesday, January 26, 2010

Assembly and Stoichiometry of the AMPA Receptor and Transmembrane AMPA Receptor Regulatory Protein Complex (TARP):

Today in lab meeting I presented this recent paper from the Tomita lab regarding TARP binding to AMPA Receptors (AMPAR).  Tomita,  as a post-doc in the Bredt lab, was one of the first people to investigate TARP binding and function in depth.  TARPs, as the name suggests, are auxiliary subunits of AMPAR, and are known to modulate AMPAR diffusion and conductances.  They contain a PDZ-binding domain that can bind PSD-95, and thus indirectly link AMPAR to PSD-95.  Single particle tracking studies have shown this binding can regulate AMPAR mobility in the synpase (Bats and Choquet, 2007).  As for the conductances, recordings from AMPAR in oocytes show that AMPAR desensitization is slowed and reduced in the presence of TARPs (Priel et. al. 2005).

In this paper, the authors used fairly simply immunoblotting techniques to investigate how AMPAR form tetramers, how many TARPs can bind to AMPAR, and how TARP binding modulates AMPAR currents. Their basic method was to create AMPAR variants with different weights, so that when they formed tetramers from the different size monomers, the tetramers ran at different speeds.  Around half of the amino acids in AMPAR are in the extracellular n-terminal domain (NTD), making the weight difference significant.

Their first interesting result used AMPAR lacking their NTD, but weighted differently using GFP.  They found that when they mixed AMPARs lacking their NTD, they could form tetramers containing 1, 2, 3, or 4 of the GFP subunits, showing that the tetramers were not formed by dimer-of-dimers, but from monomers.  In contrast, when they mixed full-length AMPAR with AMPAR lacking their NTD, their gel yielded only three bands, showing that the full length AMPAR formed dimers before being dimerized again into tetramers.  From this they concluded that the NTD of AMPAR is important for the initial formation of dimers.

They next turned to finding how many TARPs can interact with one AMPAR.  By transfecting differing amounts of TARP cRNA, running their gels, and staining for GluR1, they were able to find five different size bands, relating binding of 0-4 TARPs.  When they used the minimal transfection situation, which allowed only one TARP to bind, they found that the binding of one TARP was enough to affect the channel conductance.

The above result, however, was in oocytes, and not actual neurons.  To investigate the TARP binding in neurons, they used cerebellar granule cells in a stargazer mutant line, which lacks the TARP stargazin.  By blotting for GluR2/3 in heterozygous mice, they found only two bands of protein, suggesting that AMPARs form associations with TARPs at a fixed stoichiometry.  This fixed stoichiometry suggests that the TARP levels are either minimal or saturated.  When they stained for stargazin, they found no unbound stargazin, and thus concluded that only one TARP binds to each AMPAR.

The most interesting aspect of this paper, to me, is how they were able to investigate what I consider a biophysical property - dimerization and stoichiometry - using simple biophysical techniques (and probably common ones in biophysics at that).  The finding that the n-terminus is important for the initial dimerization does not seem new to me, as it has previously been shown that the n-terminus alone can form dimers (Leuschner and Hoch, 1999), and I have read reviews that the LIVBP portion of the NTD is the initial site of dimerization (Greger and Ziff, 2007).  However, the finding that one TARP alone is enough to modulate AMPAR function is intriguing.  I am not sure that I believe their finding that one and only one TARP binds to AMPAR in vivo, given how indirect the evidence is, and that they mention disagreement with another paper in their discussion.

Bats, C., Groc, L., & Choquet, D. (2007) The Interaction between Stargazin and PSD-95 Regulates AMPA Receptor Surface Trafficking. Neuron 53, 719-734.

Greger, I. H., Ziff, E. B., & Penn, A. C. (2007) Molecular determinants of AMPA receptor subunit assembly. Trends in Neurosciences 30, 407-416.

Leuschner, W. D. & Hoch, W. (1999) Subtype-specific assembly of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits is mediated by their n-terminal domains. J Biol Chem 274, 16907-16916.

Priel, A., Kolleker, A., Ayalon, G., Gillor, M., Osten, P., & Stern-Bach, Y. (2005) Stargazin Reduces Desensitization and Slows Deactivation of the AMPA-Type Glutamate Receptors. J Neurosci 25, 2682-2686.

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