PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression Victor Anggonoa,b,c,d, Yeliz Koç-Schmitze, Jocelyn Widagdoa,b,d, Jan Kormanne, Annie Quanf, Chih-Ming Chena, Phillip J. Robinsonf, Se-Young Choia,b,g, David J. Lindena, Markus Plomanne, and Richard L. Huganira,b,1 aThe Solomon H. Snyder Department of Neuroscience and bHoward Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cClem Jones Centre for Ageing Dementia Research and dQueensland Brain Institute, The University of Queensland, Brisbane, QLD 4072, Australia; eCenter for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany; fCell Signalling Unit, Children’s Medical Research Institute, The University of Sydney, Wentworthville, NSW 2145, Australia; and gDepartment of Physiology, Dental Research Institute, Seoul National University School of Dentistry, Seoul 110-749, Republic of Korea Contributed by Richard L. Huganir, July 9, 2013 (sent for review May 17, 2013) The dynamic trafficking of AMPA receptors (AMPARs) into and out plasticity, mGluR LTD, and homeostatic plasticity (7–14). PICK1 of synapses is crucial for synaptic transmission, plasticity, learning, also contains a central box-dependent myc-interacting protein-1 and memory. The protein interacting with C-kinase 1 (PICK1) di- (Bin)/amphiphysin/reduced viability to nutrient starvation-homology rectly interacts with GluA2/3 subunits of the AMPARs. Although (Rvs) (BAR) domain, which dimerizes to form a banana-shaped the role of PICK1 in regulating AMPAR trafficking and multiple crescent and binds to phospholipids (15). Many BAR-domain forms of synaptic plasticity is known, the exact molecular mecha- proteins possess the ability to sense and/or generate curvature of nisms underlying this process remain unclear. Here, we report the plasma membrane and are believed to play key roles in en- docytosis and vesicle trafficking (16). Expression of a PICK1 a unique interaction between PICK1 and all three members of fi the protein kinase C and casein kinase II substrate in neurons BAR-domain mutant that is de cient in lipid binding impairs both (PACSIN) family and show that they form a complex with AMPARs. hippocampal (15) and cerebellar LTD (8), suggesting a role for the PICK1 BAR domain in mediating the internalization, recycling, Our results reveal that knockdown of the neuronal-specific pro- and/or intracellular retention of GluA2-containing AMPARs (1, tein, PACSIN1, leads to a significant reduction in AMPAR internal- 6). However, the molecular mechanisms by which PICK1 mediates ization following the activation of NMDA receptors in hippo- the trafficking of AMPARs are complex and remain unclear. campal neurons. The interaction between PICK1 and PACSIN1 is Protein kinase C and casein kinase II substrate in neurons regulated by PACSIN1 phosphorylation within the variable region (PACSINs), also known as syndapins, belong to the feline sar- and is required for AMPAR endocytosis. Similarly, the binding of coma-Cdc42 interacting protein 4 (Fes-CIP4) homology BAR PICK1 to the ubiquitously expressed PACSIN2 is also regulated by (F-BAR) subfamily within the BAR-domain superfamily of pro- the homologous phosphorylation sites within the PACSIN2-variable teins, which sense and/or generate lipid tubules with a larger and region. Genetic deletion of PACSIN2, which is highly expressed in shallower curvature than other BAR-domain proteins (17, 18). Purkinje cells, eliminates cerebellar long-term depression. This defi- PACSIN1 is a neuronal-specific protein that regulates activity- cit can be fully rescued by overexpressing wild-type PACSIN2, but dependent retrieval of synaptic vesicles in the presynaptic termi- not by a PACSIN2 phosphomimetic mutant, which does not bind nals (19–22) as well as the endocytosis of the NR3A subunit of PICK1 efficiently. Taken together, our data demonstrate that the NMDA receptors on the postsynaptic membrane (23). More interaction of PICK1 and PACSIN is required for the activity-depen- importantly, PACSIN1 has been implicated in various neurode- dent internalization of AMPARs and for the expression of long-term generative disorders, including Huntington and Alzheimer’s depression in the cerebellum. diseases (24, 25). PACSIN2 is ubiquitously expressed, whereas the expression of PACSIN3 is restricted to the muscle, heart, and syndapin | receptor trafficking lung (26). All three members of the PACSIN family of proteins bind to endocytic machinery such as the large GTPase dyna- min and the actin-modifier neural Wiskott-Aldrich syndrome MPA-type glutamate receptors mediate most of the fast protein (N-WASP) through their conserved src-homology 3 excitatory synaptic transmission in the brain. AMPA re- A (SH3) domains, potentially linking membrane trafficking and ceptors (AMPARs) are heterotetrameric assemblies of four – actin cytoskeletal rearrangement (27). highly homologous subunits, GluA1 4, that are highly enriched In this study, we found that PICK1 interacts with PACSINs at synapses. The regulation of AMPAR density at synapses has and forms a complex with AMPARs. This interaction is regu- emerged as a key regulator of synaptic plasticity, which is thought lated by PACSIN phosphorylation and is required for NMDA- to be one of the key cellular components underlying learning and induced AMPAR endocytosis and cerebellar LTD. Overall, our memory (1, 2). In general, an increase in the number of synaptic data provide experimental evidence supporting the functional AMPARs leads to long-term potentiation (LTP), whereas the removal of surface AMPARs by endocytosis results in long-term fi depression (LTD) (1, 3). The traf cking of AMPARs into and Author contributions: V.A. and R.L.H. designed research; V.A., Y.K.-S., J.W., J.K., A.Q., out of synapses is highly dynamic and is regulated by various C.-M.C., S.-Y.C., and D.J.L. performed research; M.P. contributed new reagents/analytic AMPAR-interacting proteins that bind to the cytoplasmic tail of tools; V.A., J.W., P.J.R., S.-Y.C., D.J.L., M.P., and R.L.H. analyzed data; and V.A. and R.L.H. the receptors (1). wrote the paper. Protein interacting with C-kinase 1 (PICK1) interacts directly Conflict of interest statement: R.L.H. provides antibodies to Millipore Corporation for sale with the C termini of the GluA2 and GluA3 subunits of AMPARs and is entitled to a share of royalties received by Johns Hopkins University on sales of products described in this article. This arrangement is managed under a licensing agree- through its postsynaptic density-95/discs-large/zona occludens-1 ment between Millipore Corporation and Johns Hopkins University, and the terms are (PDZ) domain (4, 5). PICK1 has been shown to regulate the managed by Johns Hopkins University in accordance with its conflict-of-interest policies. surface expression, trafficking, and synaptic targeting of AMPARs R.L.H. is a paid consultant to Millipore Corporation. (6), and genetic deletion of PICK1 has revealed its important role 1To whom correspondence should be addressed. E-mail: [email protected]. in several forms of synaptic plasticity, such as hippocampal and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ cerebellar LTD, hippocampal LTP, Ca -permeable AMPAR 1073/pnas.1312467110/-/DCSupplemental. 13976–13981 | PNAS | August 20, 2013 | vol. 110 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1312467110 Downloaded by guest on September 28, 2021 duality of presynaptic trafficking machinery in regulating post- determine if the interaction between PICK1 and PACSINs occurs synaptic AMPAR trafficking and synaptic plasticity. in vivo, we performed immunoprecipitation assays with total mouse brain lysates using specific antibodies against PACSIN1, 2, and 3. Results Western-blotting analysis revealed that PICK1 coimmunoprecipi- PACSIN Interacts with PICK1 and Forms a Complex with AMPARs. It is tated with PACSIN1−3, but not with anti-matrilin 3 antibody, which well established that PICK1 plays an important role in multiple was used as a negative control (Fig. 1D). forms of synaptic plasticity by modulating the subunit composi- To determine whether PACSIN associates with AMPARs, we tion, trafficking, and surface expression of AMPARs (1). How- transfected HEK 293T cells with constructs encoding HA- ever, little is known about the detailed molecular mechanisms by GluA2, myc-PICK1 and GFP-PACSIN1. Myc-PICK1 coimmu- which PICK1 regulates AMPAR trafficking. To investigate this noprecipitated with HA-GluA2, but GFP-PACSIN1 only coim- we performed a yeast two-hybrid screen and discovered a unique munoprecipitated with HA-GluA2 when myc-PICK1 was present interaction between PICK1 and PACSIN3. To validate this in- in the cells (Fig. 2A). These data suggest that PACSIN1 forms teraction, we first transfected HEK 293T cells with full-length a complex with GluA2 in vitro via PICK1 binding. Immunopre- constructs encoding GFP, GFP-PACSIN1−3, and myc-PICK1, cipitation from total mouse brain extracts using specific anti- either individually or in combination. GFP-PACSIN1−3, but not bodies against PACSIN1, 2, and 3 revealed that GluA2/3 GFP, coimmunoprecipitated with myc-PICK1 when coexpressed subunits of AMPARs are associated with all three PACSIN in these cells and demonstrated that these coimmunoprecipita- proteins in vivo (Fig. 2B). Together, these results suggest that tions were abolished in the absence of myc-PICK1, confirming PACSINs may play a role in the regulation of AMPAR traf- the specificity of the interaction between PICK1 and all three ficking in neurons. PACSIN proteins in vitro (Fig. 1A). This interaction was further confirmed by the ability of recombinant GST-PACSIN1−3, but PACSIN1 Loss-of-Function Impairs Activity-Dependent AMPAR not GST alone, to pull down PICK1 when incubated with total Internalization. As all three PACSIN proteins bind endocytic lysates from HEK 293T cells that overexpressed Strep-tagged proteins and have been implicated in endocytosis (26), we hy- PICK1 (Fig. 1B). Immunofluorescence labeling of cultured rat pothesized that PACSIN might regulate AMPAR endocytosis in hippocampal neurons with specific antibodies against PICK1 and neurons.