Endocytosis and synaptic removal of NR3A-containing NMDA receptors by PACSIN1/syndapin1

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Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1-and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.

Section: Discussionsupporting

confidence: 56%

Identification of neuronal substrates implicates Pak5 in synaptic vesicle trafficking

Strochlic

Concilio

Viaud

et al. 2012

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“…The biology of PACSIN1 overlaps heavily with known pathways affected in Huntington disease, namely the recycling of NMDA receptors (19) and the formation of neuronal spines (21). PACSIN1 is known to directly interact with human huntingtin within the polyproline region (18), and we have additionally shown that PACSIN1 interacts with the N17 domain.…”

Section: Measuring the Conformation Of The Amino Terminus Within Thementioning

confidence: 72%

“…This interaction is enhanced in the presence of expanded polyglutamine (18). PACSIN1 is a predominantly cytoplasmic neuronal protein that has functions in NMDA receptor recycling (18,19), actin/microtubule reorganization (20), and neuronal spine formation (21). Both N17 and PACSIN1 are substrates of casein kinase 2 (CK2) (12,17).…”

mentioning

confidence: 99%

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Caron

Desmond

Xia

et al. 2013

134

150

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, but the mechanism of this pathogenic length is poorly understood. Using Förster resonance energy transfer, we describe an intramolecular proximity between the N17 domain and the downstream polyproline region that flanks the polyglutamine tract of huntingtin. Our data support the hypothesis that the polyglutamine tract can act as a flexible domain, allowing the flanking domains to come into close spatial proximity. This flexibility is impaired with expanded polyglutamine tracts, and we can detect changes in huntingtin conformation at the pathogenic threshold for HD. Altering the structure of N17, either via phosphomimicry or with small molecules, also affects the proximity between the flanking domains. The structural capacity of N17 to fold back toward distal regions within huntingtin requires an interacting protein, protein kinase C and casein kinase 2 substrate in neurons 1 (PACSIN1). This protein has the ability to bind both N17 and the polyproline region, stabilizing the interaction between these two domains. We also developed an antibody-based FRET assay that can detect conformational changes within endogenous huntingtin in wild-type versus HD fibroblasts. Therefore, we hypothesize that wild-type length polyglutamine tracts within huntingtin can form a flexible domain that is essential for proper functional intramolecular proximity, conformations, and dynamics.polyglutamine diseases | conformational switching | FLIM-FRET | neurodegeneration | fluorescence lifetime imaging microscopy H untington disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene (1). The pathogenic threshold of CAG expansion is ∼37 repeats, with increased repeats leading to an earlier age-onset of HD (2-4). This CAG mutation results in an expanded polyglutamine tract in the amino terminus of the gene's protein product, huntingtin (1). To date, no phenotypes at the level of huntingtin molecular biology or animal models can be attributed to polyglutamine lengths near the human pathogenic disease threshold (5).Polyglutamine or glutamine-rich domains are also found in transcription factors such as the Sp-family and cAMP response element-binding protein (CREB) and are defined as proteinprotein interaction motifs used to scaffold and regulate transcription by RNA polymerase II (6, 7). In the transducin-like enhancer of split (TLE) corepressor proteins, the glutamine-rich domains are thought to allow dimerization (8). However, the role of polyglutamine in proteins such as huntingtin may be distinct from that of a protein-protein interaction or dimerization domain.The polyglutamine tract of huntingtin is flanked on...

“…PACSIN1 is a neuronal-specific protein that regulates activitydependent retrieval of synaptic vesicles in the presynaptic terminals (19)(20)(21)(22) as well as the endocytosis of the NR3A subunit of NMDA receptors on the postsynaptic membrane (23). More importantly, PACSIN1 has been implicated in various neurodegenerative disorders, including Huntington and Alzheimer's diseases (24,25).…”

Section: Syndapin | Receptor Traffickingmentioning

confidence: 99%

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression

Anggono

Koç-Schmitz

Widagdo

et al. 2013

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The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.syndapin | receptor trafficking