INTERACTIONS BETWEEN TRANSMEMBRANE AMPA RECEPTOR REGULATORY PROTEIN (TARP) ISOFORM GAMMA 8 AND AMPA RECEPTORS IN THE MOUSE BRAIN

Peter Donoghue1, Victoria Hann1, Helen Payne1, Paul Chazot1, Michael Spedding2, Christopher Thompson1
1Durham University, Durham, United Kingdom, 2Institut de Recherches Servier, Suresnes, France

AMPA receptors are known to mediate fast excitatory neurotransmission in the CNS and have been shown to be present both synaptically and extra-synaptically, and are believed to be an important component in a range of neurological phenomena such as LTP. However, AMPA receptors play a role in a variety of neurological mood disorders not traditionally associated with glutamatergic neurotransmission (Bleakman et al., 2007). Due to this greater implication between the relationship of AMPA receptors and a variety of neurological conditions, a diverse range of potential targets for therapeutic agents possessing modulatory effects on AMPA dynamics are being considered. One group of potential drug targets are a family of proteins shown to have importance in the cell surface trafficking, synaptic targeting and functional heterogeneity of AMPARs known as transmembrane AMPA receptor trafficking proteins (TARPs), of which there are 5 described isoforms, γ2, γ3, γ4, γ5, γ8 (Milstein et al., 2007).

Using a novel antibody generated to a CT-sequence of the TARP γ8 isoform, and commercial antibodies to each of the GluR subunits, we have investigated by immunohistochemistry (as described in Payne et al., 2006), the relationship between TARP γ8 distribution compared with that of the GluRs. The anti- TARP γ8 isoform antibody was shown to selectively label recombinant γ8, but not γ2, γ3 or γ4 expressed in HEK 293 cells by both immunoblotting and immunocytochemistry.

Most notably GluR1 and GluR2 appear to have a pattern of expression mirroring that of TARP γ8, with the peak levels of GluR 1 and 2 expression being in the same brain structures as the regions of highest TARP γ8 expression, including the hippocampal formation (CA1 and 2), thalamus, cerebellum and laminae IV and V of the cerebral cortex.

Expanding on the distribution data, we have also utilised immunopurification techniques (Payne et al., 2006) to show that TARP γ8 not only co-localises with the AMPAR subunits in specific regions of the brain, but that they also interact as part of a protein complex. Using the anti-TARP γ8 CT antibody column, GluR1 and GluR2 were co-purified from both mixed cortical/hippocampal and cerebellum preparations. Current work is focussed on identifying further TARP γ8 protein partners using a proteomics approach.

Bleakman et al (2007) CNS Neurol Disord Drug Targets 6(2):117-26
Milstein et al (2007) Neuron. 55(6):905-918.
Payne et al. (2006) J Neurosci. 26(33):8600-8608

This work was funded by BBSRC (VH) and BBSRC/Servier CASE award (PD).