GRIP1 and 2 regulate activity-dependent AMPA receptor recycling via exocyst complex interactions

Abstract

PSD-95/SAP90/DLG/ZO-1 (PDZ) domain-mediated protein-protein interactions play important roles in regulating AMPA receptor trafficking and neuronal plasticity. GRIP1 and GRIP2 are homologous multi-PDZ domain-containing proteins that bind to the Ctermini of AMPA-R GluA2 and GluA3 subunits. Previous attempts to determine the cellular roles of GRIP1 and GRIP2 in neurons have been complicated by nonspecific reagents, and by the embryonic lethality of conventional GRIP1 KO mice. To circumvent these issues we developed a conditional targeted deletion strategy to knock out GRIP1 in postnatal neurons derived from GRIP2 KO mice. Loss of GRIP1 and 2 did not affect normal AMPA-R steadystate trafficking and endocytosis, but strikingly impaired activitydependent AMPA-R recycling. This previously uncharacterized role for GRIP1 appears to be mediated by novel interactions with the cellular trafficking machinery via the exocyst protein complex. Indeed, disruption of GRIP1-exocyst binding caused a strikingly similar deficit in AMPA-R recycling. Together these findings reveal a previously unidentified role for AMPA-R-GRIP1-exocyst protein complexes in activity-dependent AMPA-R trafficking.A MPA receptors conduct the majority of fast excitatory synaptic transmission in the mammalian central nervous system. Dynamic regulation of AMPA-R trafficking in and out of synapses modulates the efficacy of synaptic transmission and is crucial for synaptic plasticity (1-3). One major molecular mechanism that governs AMPA-R trafficking is the regulated interaction of intracellular C-termini of AMPA-R subunits with PSD-95/SAP90, DLG/ ZO-1 (PDZ) domain-containing proteins (4-7). The type II PDZ motifs of GluA2 and GluA3 C-termini interact with several PDZ domain-containing proteins, including PICK1 (6, 7) and the homologous GRIP1 and GRIP2 proteins (GRIP2 is also called ABP for AMPA-R binding protein) (4,5,8,9). GRIP proteins and PICK1 interact with GluA2 at the same site, but are differentially regulated by PKC-dependent phosphorylation of GluA2 Ser880 (10-12). In addition to GluA2/3, GRIP1 and 2 binds various signaling and cytoskeletal proteins, including EphB receptor tyrosine kinase, ephrinB ligands, α-liprin scaffolding protein, proteoglycan NG2, Fras1 and 2, GRIP1-associated protein 1 (GRASP-1), kinesin-1/KIF5 microtubule motor protein and matrix metalloproteinase 5 (13-19). Thus, GRIP proteins may serve to organize macromolecular complexes, linking AMPA-Rs and other membrane proteins to the cytoskeleton and membrane trafficking and signal transduction pathways.GRIP-GluA2/3 interactions are implicated in regulating AMPA-R synaptic targeting. Overexpression of the GluA2 C terminus in neurons decreases the synaptic targeting of AMPA-Rs (4). Point mutants of GluA2 that are unable to bind GRIP proteins exhibit reduced synaptic accumulation (20). GRIP1's sixth PDZ domain interacts with the α-liprin family of proteins, and disrupting GRIP-α-liprin interactions inhibits AMPA-R surface expression and synaptic targeting (14). GRIP proteins also bind ...

Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism

“…Consistent with this hypothesis, we found that three variants (I549V, T550M, and I586L) increased interaction with GluA2, two variants (I586L and A625T) are associated with a faster recycling of GluA2, and three variants (I586L, A625T, and M794R) increased surface GluA2 in hippocampal neurons. Because loss of GRIP1/2 function was associated with reduced interaction and slower GluA2 recycling (22), our data support a gain of GRIP1 function in these variants. Although disruption of GluA2-GRIP interactions in neurons also alters synaptic function and plasticity (19,25), the effect of GRIP1 variants on these readouts remains to be elucidated.…”

“…GRIP1 was recently reported to modulate the activitydependent trafficking of a pH-sensitive pHluorin-GluA2 fusion protein (pH-GluA2) (22). We therefore examined the effects of altered GRIP1-GluA2/3 interaction of three GRIP1 variants (T550M, I586L, A625T) on pH-GluA2 internalization and recycling in live, transfected hippocampal neurons (23).…”

“…Taking advantage of the pH difference between extracellular space (pH = 7.4) and intracellular vesicles (pH < 6.0), this assay use a pH-sensitive green fluorescent protein, pHluorin fused to the N terminus of GluA2 (pH-GluA2) as a reporter to visualize the dynamic process of GluA internalization and recycling (22,23). Full details are in SI Materials and Methods.…”

“…Low-density primary hippocampal cultures were prepared from embryonic day 18 (E18) rat brains, and maintained in serum-free medium, as described previously (22). Cells were fed twice per week and used for experiments after 14 to 18 d in vitro.…”

GRIP1 is required for homeostatic regulation of AMPAR trafficking

“…The interaction between GRIP1 and GluA2/3 regulates surface expression, membrane trafficking, and synaptic targeting of AMPARs, and the regulation of this interaction is critical for neuronal development and several forms of synaptic plasticity (7,(19)(20)(21)(22)(23). Loss of GRIP1/2 slows activity-dependent AMPAR recycling (24) and blocks cerebellar long-term depression (LTD) expression (20). Though the PDZ domains of GRIP1 and GRIP2 are highly conserved, some regions between these PDZ domains are variable.…”