The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

Recent studies on G‐protein‐coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The γ‐amino‐ n ‐butyric acid type B (GABAB) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane α‐helices, loops and the C‐terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G‐protein signalling. However, the HD of GB1 improves coupling efficacy. Conversely, although GB1 ECD is sufficient to bind GABAB ligands, the ECD of GB2 increases the agonist affinity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild‐type functioning of the GABAB receptor and highlight further the importance of the dimerization process in GPCR activation.

The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA_B receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.

Both subunits in the metabotropic glutamate receptor 1 dimer move relative to each other before one adopts an active conformation.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is assembled from one NR1 subunit, expressed in eight splice variants, and four NR2 subunits (NR2A-D). The combination of subunits and splice variants determines the pharmacological and physiological properties of the receptor. In the present study we investigated the relationship between NR1 splice variants and NR2 subunits in rat brain using a series of antibodies selective for the four NR1 cassettes, which vary in the NR1 splice variants, and for NR2A and NR2B. Sodium deoxycholate at pH 9.0 solubilized about 35% of the receptor, which was intact based on co-immunoprecipitation of NR1 and NR2 subunits and chemical cross-linking of the solubilized receptor. The cross-linked product contained three high molecular weight components, Mr = 603,000, 700,000, and 750,000, which were immunolabeled with antibodies to NR1 and to NR2 subunits. Immunoprecipitation analyses using antibodies selective for NR2A and NR2B showed no preferential assembly between NR2 subunits and NR1 splice variants. There was little co-immunoprecipitation of NR2A and NR2B, suggesting that most NMDA receptor complexes contain only one of these subunits. However, receptor complexes can contain at least two different NR1 splice variants. In developing conditions for the solubilization of intact NMDA receptor complexes, we observed a differential solubilization of NR1 and NR2 subunits. NR2 was nearly insoluble in Triton X-100 in both microsomal and synaptic membrane fractions, while NR1 was readily soluble in the microsomal fraction but insoluble in the synaptic membrane fraction. These results suggest that the NR1 subunit is modified when it is incorporated into the synaptic membrane, possibly by strengthening its interaction with NR2 or another synaptic protein.