Excitatory synapses are the contact sites through which neurons communicate with each other. These synapses are asymmetric structures that are formed by pre- and postsynaptic terminals containing distinct sets of proteins. Incoming action potentials are converted into chemical signals (neurotransmitters) at presynaptic terminals, which subsequently pass through the synaptic cleft and are reconverted into electrical signals at postsynaptic sites (Lisman et al., 2007). These synaptic contacts are not static but are able to undergo structural changes and thereby modify neuronal network computation (Nishiyama and Yasuda, 2015). At postsynaptic sites, interacting proteins are densely packed into a sub-membrane structure called the postsynaptic density (PSD) (Sheng and Hoogenraad, 2007). Scaffold proteins of the PSD-95 family membrane-associated guanylate kinases (MAGUKs) are highly abundant components of the PSD and function as central regulators of postsynaptic organisation (Zhu et al., 2016a). PSD-95 family MAGUKs contain three PDZ domains that are known to directly interact with N-methyl-D-aspartate (NMDA) receptor C-termini (Kornau et al., 1995), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor C-termini (Leonard et al., 1998), and AMPA receptor auxiliary subunit C-termini (Dakoji et al., 2003). The PDZ domains are followed by an SH3 – guanylate kinase (GK) domain tandem (Funke et al., 2005). The MAGUK SH3 domain lost its function to bind proline-rich peptides; instead it forms an intramolecular interaction with the GK domain (McGee et al., 2001). Similarly, the PSD-95 GK domain is atypical in that it is unable to phosphorylate GMP but has evolved as a protein interaction domain (Johnston et al., 2011). Binding of known interactors to the GK domain typically involves residues of the canonical GMP-binding region (Reese et al., 2007; Zhu et al., 2011; Zhu et al., 2016b). This modular array of protein interaction domains allows PSD-95 MAGUKs to function as bidirectional organisers of synaptic function. First, neurotransmitter receptors can be incorporated or removed from postsynaptic membranes, depending on molecular interactions with these sub-membrane scaffold proteins. Second, together with other scaffold proteins at postsynaptic sites, they align downstream effectors and cytoskeletal proteins. Accordingly, PSD-95 family MAGUKs are essential for the establishment of long-term potentiation (LTP) by regulating the content of AMPA receptors at dendritic spines (Ehrlich and Malinow, 2004; Opazo et al., 2012; Sheng et al., 2018). In line with this is the observation that acute knockdown of PSD-95 MAGUKs leads to a decrease in postsynaptic AMPA and NMDA receptor-mediated synaptic transmission as well as a reduction in PSD size (Chen et al., 2015). Taken together, exploring protein complex formation directed by PSD-95 MAGUK family members is of central importance for understanding regulation of synaptic transmission. We have previously shown that the synaptic MAGUK protein PSD-95 oligomerises upon binding of monomeric CRIPT-derived PDZ3 ligands (ligands that specifically bind to the third PDZ domain) (Rademacher et al., 2013) and speculated that ligand – PDZ3 domain binding induces conformational changes in the C-terminal domains that lead to complex formation. Our initial observations of PDZ ligand-induced effects in PSD-95 MAGUK proteins have recently been supported by other studies (Zeng et al., 2016; Zeng et al., 2018).
In this study, we use a bimolecular fluorescence complementation (BiFC) assay to show that PSD-95 oligomerisation can be triggered by Neuroligin-1 (NLGN1) and that this is dependent on the C-terminal SH3-GK domain tandem. Moreover, we identify new interaction partners of PSD-95 C-terminal domains by quantitative mass spectrometry. We provide evidence that the heterotrimeric G protein subunit Gnb5 is a novel GK domain interactor and that its ability to bind to PSD-95 is likewise promoted by binding of a ligand to the PSD-95 PDZ3 domain.