Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.

The number of synaptic ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-D-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas fulllength GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.AMPA-type glutamate receptors mediate most of the fast excitatory synaptic transmission in the brain. Various forms of synaptic plasticity depend on changes in AMPA receptor (AMPAR) 2 transmission. Trafficking of AMPAR and their lateral diffusion to and from synapses are key mechanisms that govern synaptic strength (1-5). Induction of long-term depression in hippocampal cell cultures, hippocampal slice preparations, and cultured cerebellar Purkinje cells is associated with a reduction in synaptic AMPAR (6 -10). Likewise, the induction of long-term potentiation yields an increase in the number of AMPAR at synapses (11,12). AMPAR are hetero-oligomers composed of subunits GluR1 to GluR4 (GluRA to GluRD). In the hippocampus GluR1/GluR2 or GluR2/GluR3 receptors predominate (13). Although the extracellular domain and the four membrane spanning domains are largely homologous between the different subunits, the cytoplasmic carboxyl termini differ, with long termini for GluR1 and GluR4 and short termini for GluR2 and GluR3. These cytoplasmic tails are responsible for subunit-specific AMPAR trafficking to synapses (14), which is mediated through binding of PDZ (postsynaptic density 95/disc large/ zonula occludens-1) domain proteins. GluR2/GluR3 receptors cycle constitutively between internal compartments and the plasma membrane (14, 15), presumably at sites distant from the synapse (16). The interaction between GluR2 and type II PDZ domain proteins like protein interacting with C kinase 1 (PICK1), glutamate receptor interacting protein (GRIP), and AMPAR-binding protein (ABP) is necessary for this cycling (17). After int...

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High-resolution detection of specific molecules in cells is a major challenge in biology. We show that infrared scanning near-field microscopy can detect the spatial distribution of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid–type glutamate receptor clusters on hippocampal neurons. The GluR2 subunits were labeled with the die Alexa 488 and high-resolution infrared micrographs were taken at 6.25 μm. The absence of photobleaching makes this approach suitable for a long-term observation and allows to localize different infrared-absorbing molecules over the complex background of other cell components.