Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation

Abstract

Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains.P ostsynaptic densities (PSDs) at glutamatergic synapses organize and hold NMDA receptors (NMDARs), AMPA receptors (AMPARs), and other signaling molecules in place, apposed to sites of neurotransmitter release. Just below the PSD plasma membrane lies a latticework of vertical and parallel filaments that provides a structural scaffold to stabilize synaptic signaling molecules within PSDs (1, 2). Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are members of a family of membrane-associated guanylate kinases (MAGUKs) (3). PSD95 is the most abundant scaffold protein in adult synapses, with ∼300 PSD95 molecules (2.3% of the mass of the PSD) in the average PSD, and is part of the lattice forming the core of the PSD (4). SAP97 is also a component of the PSD lattice. Estimates of its PSD copy numbers range from 90 SAP97 molecules per average PSD [0.9% of the mass of the PSD (4)] to lower values (5). As MAGUKs, PSD95 and SAP97 share a series of highly homologous protein-interacting domains but diverge at their N-terminal domains, which affects their trafficking into and out of the PSD, as well as interactions with AMPARs and NMDARs (3, 6, 7). The SAP97β-isoform, like almost all SAP97 molecules, contains an N-terminal L27 domain that interacts with other L27 domaincontaining proteins, particularly with a differ...

De‐palmitoylation by N‐(tert‐Butyl) hydroxylamine inhibits AMPAR‐mediated synaptic transmission via affecting receptor distribution in postsynaptic densities

“…Considering that AMPARs interact with PSD95 through stargazin, we examined the association between stargazing and PSD95 after NtBuHA treatment. Co‐immunoprecipitation assay revealed that NtBuHA significantly decreased the stargazing‐PSD95 association (NtBuHA: 0.74 ± 0.02 vs Control: 1.00 ± 0.07, n = 4, unpaired Student's t test, P < 0.05, Figure B‐C).Our results demonstrated that de‐palmitoylation selectively disturbed the association of PSD95 with AMPARs, not NMDARs, which was consistent with previous report …”

“…PSDs are a family of homologous scaffold protein distributed mainly in synapses. It has been reported that PSD95, the key postsynaptic scaffold protein in PSD family, associated with AMPARs or NMDARs only in its palmitoylated conformation . Thus, we hypothesized that de‐palmitoylation of PSD95 by NtBuHA may abolish the association of iGluRs with PSD95.…”

“…S‐palmitoylation refers to the formation of a reversible thioester linkage of a palmitoyl lipid as sticky “tag” to the thiol of cysteine residue, which is tuned by the palmitoyl acyl transferase‐catalyzed palmitoylation and a reverse process, palmitoyl‐protein thioesterase‐catalyzed de‐palmitoylation . Both AMPARs and their binding partners are especially key targets for palmitoylation . For instance, AMPARs subunits, such as GluA1 and GluA2, have a cysteine residue close to the pore (C585 in GluA1, C610 in GluA2), which can be palmitoylated, following by a decrease in their insertion into the plasma membrane and an increase in their accumulation in the Golgi apparatus .…”

“…In contrast, palmitoylation of N‐terminal polybasic domain of protein kinase A‐anchoring protein 79/150 (AKAP79/150), a key AMPAR regulatory scaffold protein, facilitates surface delivery of AMPAR . Postsynaptic density protein 95 (PSD95), the key postsynaptic scaffold protein for GluRs, is associated with AMPARs or NMDARs only in its palmitoylated conformation . As for AMPARs, NMDAR function is also affected by palmitoylation, although the regulation of NMDARs by palmitoylation is complex .…”

Transcellular Nanoalignment of Synaptic Function

“…Mechanisms producing nanoclustering of MAGUKs are unclear, though may involve their palmitoylation (Fukata et al, 2013) which is required for receptor binding (Jeyifous et al, 2016) and executed by activity-regulated palmitoyl transferases (Fukata et al, 2004; Fukata et al, 2013; Noritake et al, 2009). However, the overall PSD-95 pattern of a synapse likely involves extensive interactions as part of the high degree of PSD organization in the “vertical” dimension perpendicular to the membrane.…”