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  • Here we show that cpg is expressed

    2019-08-13

    Here, we show that cpg2 is expressed only in the chemical this article and encodes a protein that localizes specifically to the postsynaptic endocytic zone of excitatory synapses. We present evidence that CPG2 is a critical component of the postsynaptic endocytic pathway that mediates both constitutive and activity-regulated glutamate receptor internalization. We hypothesize that CPG2 is a key component of a specialization that is devoted to the internalization of postsynaptic proteins at synapses capable of plasticity.
    Results
    Discussion Clathrin-mediated endocytosis is a general mechanism that regulates the internalization of proteins from the cell surface in eukaryotic cells Kirchhausen 2000, Mousavi et al. 2004, and its basic machinery is conserved across species and cell types (Schmid, 1997). In cases where specialized endocytic functions are required, specific mechanisms have evolved for regulating endocytosis. In the presynaptic terminal, for example, there are novel mechanisms and proteins for meeting the demands of synaptic transmission, often consisting of terminal-specific isoforms or splice variants of general endocytic proteins Jarousse and Kelly 2001, Slepnev and De Camilli 2000. Given the importance of glutamate receptor internalization for maintaining and modifying synaptic strength Carroll et al. 2001, Malinow and Malenka 2002, neurons may have developed novel specific mechanisms for regulating the endocytosis of glutamate receptors. A postsynaptic endocytic zone has been shown to be present and stable on dendritic spine and shaft synapses (Blanpied et al., 2002). Although segregated from the PSD, it is likely an integral synaptic component (Blanpied et al., 2002), and our data strongly suggest that it is the site of glutamate receptor internalization. Since CPG2 is primarily localized to the postsynaptic endocytic zone of excitatory synapses, it is not a constitutive part of the ubiquitous endocytic machinery. While the disruption of components of the general endocytic machinery has been shown to disrupt glutamate receptor internalization Carroll et al. 1999, Luscher et al. 1999, Metzler et al. 2003, Zhou et al. 2001, CPG2 is the first known protein that is specifically involved in the internalization of postsynaptic proteins. We hypothesize that CPG2 is a component of a specialized postsynaptic endocytic mechanism dedicated to the internalization of synaptic proteins, including glutamate receptors. Knockdown of CPG2 led to an increase in postsynaptic clathrin-coated vesicles, some trafficking NMDA receptors, and a disruption of glutamate receptor internalization. This phenotype is similar to that seen in presynaptic terminals of organisms with mutations of endophilin or synaptojanin, where there is an accumulation of clathrin-coated vesicles and a disruption of the synaptic vesicle cycle, due to a defect in clathrin-coated vesicle uncoating Cremona et al. 1999, Harris et al. 2000, Kim et al. 2002, Verstreken et al. 2002, Verstreken et al. 2003. Since CPG2 contains multiple protein interaction motifs, including two spectrin-like repeats and several coiled coils, we propose a model in which CPG2 interacts with other proteins at the postsynaptic endocytic zone that are necessary for the downstream processing, such as the uncoating, of glutamate receptor-trafficking vesicles. According to this model, following CPG2 knockdown, the CPG2-associated protein network is disrupted, inhibiting vesicle clearance and causing their accumulation. Due to the slowed kinetics of this late step, the entire endocytic pathway is slowed, disrupting further glutamate receptor internalization. Future experiments should reveal the proteins with which CPG2 interacts at synapses and at what point they function to regulate the vesicle cycle. This would clarify whether CPG2 is an active regulator of the endocytic pathway or is a component of the overall endocytic machinery unique to the postsynaptic density. In CPG2 knockdown neurons, there is an increase in synaptic glutamate receptors, which is likely due to the disruption of glutamate receptor endocytosis. Although some reports have indicated that endocytosis inhibition does not affect the number of surface AMPA receptors Lledo et al. 1998, Man et al. 2000, our data are consistent with results showing that acute blockade of endocytosis causes an increase in synaptic transmission (Luscher et al., 1999). However, the increase in surface receptors is moderate (20%) despite a greater than 70% disruption in glutamate receptor internalization. This discrepancy may be due to the tight link, previously proposed, between the rates of receptor insertion into and removal from the synaptic membrane Ehlers 2000, Liang and Huganir 2001. Considering there is a decrease in glutamate receptor internalization in these neurons, there must be a compensatory decrease in glutamate receptor insertion for steady-state amount of surface receptors to remain relatively constant. Thus, CPG2 knockdown appears to slow not only the internalization, but also the insertion, of glutamate receptors, suggesting that CPG2 may be necessary for the rapid cycling of synaptic glutamate receptors.