Biochemical evidence for the differential association of metabotropic glutamate receptors within synaptic complexes

The distribution of metabotropic glutamate (mGlu) receptors within the synapse is an important determinant 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, composition, 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.