Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2

Abstract

␣-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with 2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2 and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre-and postsynaptic membrane proteins.endocytosis ͉ postsynaptic ͉ sorting signal ͉ synaptic plasticity F ast neurotransmission at excitatory synapses is mediated by heterotetrameric ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors composed of combinations of four subunits (GluR1-4). AMPA receptors interact with different factors including the transmembrane protein stargazin (1), PDZ proteins GRIP1/ABP, SAP97, and PICK1, and NSF (2). Accumulating evidence suggests that rapid changes in functional postsynaptic AMPA receptor numbers are important means of controlling synaptic efficacy (2-5). AMPA receptors undergo constitutive and regulated clathrin-and dynamin-dependent endocytosis via distinct AMPA-or NMDAinduced signaling cascades (reviewed in refs. 2-5). How exactly AMPA receptor cargo is targeted for clathrin-mediated internalization remains an open question. One possibility is that AMPA receptors are recognized by endocytic adaptor proteins such as the clathrin adaptor complex AP-2, a major endocytic protein interaction hub (6-8). NMDA-induced AMPA receptor internalization can be blocked by overexpression of a GluR2 cytoplasmic tail (CT) peptide (pep2r) or by mutating the putative AP-2 binding motif within the GluR2 CT. Infusion of hippocampal CA1 neurons with the putative AP-2-blocking peptide prevents induction of long-term depression (LTD), suggesting that the association of GluR2 with AP-2 may be an important determinant for NMDA-induced LTD (7). Whether AP-2 directly binds to GluR2 CTs and via which of its four subunits is unknown.Here we have identified the molecular determinants responsible for binding of AP-2 to the CTs of AMPA-type glutamate receptors. We demonstrate that the 2 subunit of AP-2 interacts directly and with nanomolar affinity with a basic motif found in CTs of GluR1-3 and the presynaptic vesicle protein synaptotagmin 1. Our data thus suggest that common mechanisms regulate AP-2-dependent internalization of pre-and postsynaptic membrane proteins.

Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

“…Thus, the binding of clathrin adaptor AP-2 to the cargo containing AMPA receptors is a key final step in executing AMPA receptor endocytosis during LTD. Although the m2 subunit of AP-2 was shown previously to bind directly to the membrane proximal region (MPR) of GluA2/A3 in vitro, in a manner dependent on GluA2's arginine 845 (R845) 6 , it has been unclear whether and how this process is regulated by neuronal activity. Here we showed that STG bound directly to m2 and enhanced the formation of a tripartite complex consisting of the AMPA receptor, STG and m2.…”

“…S14a). This is probably because of the different experimental conditions used; Kastning et al 6 carried out a pull-down assay using a GST-tagged MPR of GluA2 and a recombinant m2, which was prepared by in vitro transcription and translation, whereas we performed a co-immunoprecipitation assay using full-length GluA2 and m2 after solubilizing membranes. Thus, a small difference in the m2-GluA2 wt and m2-GluA2 R845A interactions may not have been detected in our assay.…”

“…As the number of surface AMPA receptors is determined by the balance between endocytosis and exocytosis (including the recycling of endocytosed receptors), we next used an antibody feeding assay 6,10 . HA-GluA2 on the cell surface of living neurons was first labelled with an anti-HA antibody and NMDA was then applied to the neurons to induce HA-GluA2 endocytosis.…”

Regulation of postsynaptic AMPA responses by synaptojanin 1

“…Both constitutive and regulated AMPA receptor endocytosis were shown to be dependent on clathrin, the clathrin adaptors, and dynamin (3,(5)(6)(7)(8)(9). Thus, clathrin-mediated endocytosis, a process known to play a fundamental role in the recycling of synaptic vesicles (10,11), also has a critical role postsynaptically, although the flux of endocytic traffic is much more intense presynaptically.…”

“…Thus, clathrin-mediated endocytosis, a process known to play a fundamental role in the recycling of synaptic vesicles (10, 11), also has a critical role postsynaptically, although the flux of endocytic traffic is much more intense presynaptically. Accordingly, a growing number of components of the clathrin-dependent endocytic machinery of the presynapse were reported, by either morphological or functional studies, to be present also at the postsynapse (8,9,12,13). One protein that is expressed at high levels in the nervous system and is concentrated in nerve terminals, where it has an important role in the clathrin-dependent recycling of synaptic vesicles, is synaptojanin 1 (SJ1) (14-17).…”