Neuroscience Letters 444 (2008) 27–30 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Biochemical evidence for the differential association of metabotropic glutamate receptors within synaptic complexes Ayodeji A. Asuni, Kate Lidwell, Vincent O’Connor ∗ School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK article info Article history: Received 8 June 2007 Received in revised form 2 August 2008 Accepted 5 August 2008 Keywords: Metabotropic glutamate receptors Scaffolds Synaptic webs Homer PICK-1 Synaptosomes abstract The distribution of metabotropic glutamate (mGlu) receptors within the synapse is an important deter- minant of function. mGlu have been grouped together into three main sub-classes: Group I mGlu (1 and 5) are predominantly situated on the post-synaptic membrane, whereas Group III (4, 6, 7 and 8) are largely pre-synaptic. Group II mGlu (2 and 3) are distributed peripheral to the active zone, on both sides of the synaptic cleft. Methods based on a distinct pH-dependent extractability of the pre- and post-synaptic marker proteins can provide insight into the molecular organization of synaptic junctions [G.R. Phillips, J.K. Huang, Y. Wang, H. Tanaka, L. Shapiro, W. Zhang, W. Shan, K. Arndt, M. Frank, R.E. Gordon, M.A. Gawinowicz, Y. Zhao and D.R. Colman, The presynaptic particle web: ultrastructure, com- position, dissolution and reconstitution, Neuron 32 (2001) 63–77]. We have applied such procedures to rat brain cortical synaptosomes to explore the biochemical evidence for the accepted localisations of metabotropic glutamate receptors. As shown previously a number of post-synaptic marker proteins remained detergent-insoluble at both pH 6 and pH 8. There was an increased extraction of a number of pre-synaptic plasma membrane and cytomatrix proteins consistent with dissolution of the pre- synaptic aspect of synaptic junctions at elevated pH. We similarly observed modest extraction of Group I mGlu at either pH consistent with their post-synaptic organization. However, we observed increased extractability of Group II mGlu at pH 8. The extractability of Group III mGlu was slightly increased at pH 8 but these receptors were largely refractory to extraction. We have also applied the approach to scaffolding proteins implicated in mGlu localisation to define the biochemical correlates of mGlu scaffolding. © 2008 Elsevier Ireland Ltd. All rights reserved. Metabotropic glutamate receptors play an important role in synap- tic transmission, and the function of the different sub-types are dependent on distinct cellular expression and by their further compartmentalized sub-cellular organization [9]. The eight cloned members of this G protein-coupled receptor family have been divided into three distinct groups according to sequence homol- ogy and signalling characteristics [3]. Group I mGlu (1 and 5) are localised primarily to post-synaptic sites but have a peri-synaptic distribution rather than being immediately opposite release sites. Group II mGlu (2 and 3) are found both pre- and post-synaptically but are distributed to extra-synaptic sites some distance from release sites. Group III mGlu (4, 6, 7 and 8) are found predom- inantly pre-synaptically and close to release sites within active zones [9]. Rather than receptors acting alone within the membrane, a wealth of recent evidence describes the presence of a scaffold of ∗ Corresponding author. Tel.: +44 2380597651; fax: +44 2380594459. E-mail address: voconno@soton.ac.uk (V. O’Connor). interacting proteins, linking receptors with cytoskeletal elements, signalling molecules and downstream events [12]. In the case of mGlu family, scaffolding proteins of the Homer and Tamalin family have been identified as receptor-interacting proteins (RIPs) Group I receptors. PICK-1 was shown to interact with mGlu7a, playing a role in its pre-synaptic targeting [7] and there is an ever growing list of further receptor-interacting proteins that contribute to the modulation of mGlu function [8]. In order to further elucidate the importance of the synaptic scaffolding complex, we have exploited an established method to provide information on the solubility of different synaptic proteins and provide biochemical correlates for the anatomically defined localisations of these receptors [16]. Cortices from five adult female Wistar rats (∼5 g total wet weight) were homogenised in 10 volumes buffer A (0.32 M sucrose, 1 mM NaHCO 3 , 1 mM MgCl 2 , 0.5 mM CaCl 2 , 0.1 mM phenylmethyl- sulfonylfluoride (PMSF) and centrifuged at 710 × g for 10 min at 4 ◦ C. The supernatant was retained and the pellet was resuspended in an equal volume of buffer A and centrifuged again at 710 × g for 10min at 4 ◦ C. The supernatants were pooled and centrifuged at 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.009