An investigation into NMDA receptor subtype synaptic targeting and lateral mobility mediated by the PSD-95 MAGUK family of scaffold proteins

Information in our brains is processed by a network of nerve cells. An important sub-component of a neurone is the synapse which is where communication between adjacent neurones occurs. Neurotransmitter receptor proteins are found in the membrane at the synapse. They are pivotal in receiving the message from one neurone and then inducing a response in the recipient nerve cell. NMDA receptors are a type of excitatory, glutamate neurotransmitter receptor. They play an integral role in memory formation and in neurological diseases such as schizophrenia, stroke, epilepsy and chronic pain. There are several types of closely related NMDA receptors. These differ with respect to their pharmacological properties; where they are localized in the neuronal membrane, i.e whether at synapses or close to the synapse in what are termed extra-synaptic sites and, in the different signalling pathways and responses they activate inside the neuronal cell. In collaboration with Dr Daniel Choquet's research group in France, we have recently discovered that NMDA receptor subtypes differ in their respective mobilities in neuronal membranes. One type (NR1/NR2A receptors) stays within the synapse whereas NR1/NR2B receptors are more mobile and can readily move between synaptic and extra-synaptic sites. NMDA receptors are anchored at synapses by their association with a family of scaffolding proteins, the PSD-95 proteins. Because of the observed differences in their respective lateral mobilities, it is suggested that NMDA receptor subtypes interact differently with the PSD-95 proteins. This differential interaction is crucial for the correct functioning of the neurones since different intracellular signalling pathways appear to be activated by synaptic versus extra-synaptic receptors; in fact these functional responses are diametrically opposed leading to either programmed cell death as in 'apoptosis' or in the promotion of neuronal cell survival. This research proposal aims to investigate the factors that determine whether an NMDA receptor subtype is directed to synapses or to extra-synaptic sites by studying the interaction of NMDA receptor subtypes and the scaffolding family of proteins, the PSD-95 proteins. Understanding these processes may contribute towards the development of NMDA receptor subtype targeted therapies.

NMDA receptors are major mediators of fast excitatory neurotransmission. They play a pivotal role in synaptic plasticity, in development of the nervous system and in neurological disorders. NMDA receptors are formed by the assembly of NR1 and NR2 subunits and are associated at synapses with the post-synaptic density-95 (PSD-95) family of scaffolding proteins. It is becoming clear that the two major NMDA receptor subtypes in adult brain, NR1/NR2A and NR1/NR2B, may couple to different members of this family, PSD-95 and SAP102. This selective association may determine whether the receptors are synaptic or extra-synaptic and which downstream signalling pathways are activated. Relatedly, in collaboration with Dr Choquet, we discovered that NR2A subunit-containing receptors were relatively immobile compared to NR2B subunit-containing receptors. It is hypothesized that this differential mobility is mediated via coupling of NMDA receptor subtypes to the different scaffold proteins. The scaffolding molecules associate with assembled NMDA receptors via a 4 amino acid motif localized at the distal NR2 C-termini. Since each NR2 subunit has this motif, it is unclear why NR1/NR2B NMDA receptors should be more mobile and have a preference for association with SAP102 rather than PSD-95 and vice versa. We have preliminary data which shows that additional but different sequences contribute to NR2A/PSD-95 and NR2B/PSD-95 association suggesting that that protein-protein affinity differences may control association of subtypes with selective scaffold proteins within the same family. In this proposal, we wish to test this hypothesis. The aim is to study NR2/PSD-95 and NR2/SAP102 interactions by mapping of the respective binding domains; the determination of the affinities of protein-protein association and in single particle tracking experiments to ascertain if indeed it is the scaffold that controls lateral mobility and ultimately downstream signalling leading to neuronal responses.