The STEP61 Interactome Reveals Subunit-Specific AMPA Receptor Binding and Synaptic Regulation
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The STEP61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation Sehoon Wona, Salvatore Incontrob, Yan Lic, Roger A. Nicollb,d,1, and Katherine W. Rochea,1 aReceptor Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; bDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; cProtein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; and dDepartment of Physiology, University of California, San Francisco, CA 94158 Contributed by Roger A. Nicoll, February 27, 2019 (sent for review January 16, 2019; reviewed by David S. Bredt and Lin Mei) Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific of proteins, therefore destabilizing receptor surface and synaptic protein phosphatase that regulates a variety of synaptic proteins, expression (23). These phosphorylation events are inversely related, including NMDA receptors (NAMDRs). To better understand STEP’s and the relevant kinases and phosphatases work in concert to de- effect on other receptors, we used mass spectrometry to identify fine synaptic NMDAR expression. the STEP61 interactome. We identified a number of known inter- Striatal-enriched protein tyrosine phosphatase [STEP; also actors, but also ones including the GluA2 subunit of AMPA recep- known as protein tyrosine phosphatase nonreceptor type 5 (PTPN5)] tors (AMPARs). We show that STEP61 binds to the C termini of is expressed in the cortex, hippocampus, and striatum of brain GluA2 and GluA3 as well as endogenous AMPARs in hippocampus. (24, 25). The STEP family of proteins contains several splice The synaptic expression of GluA2 and GluA3 is increased in STEP-KO variants. All STEP splice variants (STEP61, STEP46, STEP38, and mouse brain, and STEP knockdown in hippocampal slices increases STEP20) are highly expressed in striatum. In contrast, the full- AMPAR-mediated synaptic currents. Interestingly, STEP61 overexpres- length gene product, STEP61, is the only variant expressed in sion reduces the synaptic expression and synaptic currents of both cortex and hippocampus (26–28). The brain-specific tyrosine AMPARs and NMDARs. Furthermore, STEP61 regulation of synaptic phosphatase STEP has been implicated in neuronal diseases (29) AMPARs is mediated by lysosomal degradation. Thus, we report a and neuropsychiatric disorders caused by its dysregulation (30– comprehensive list of STEP binding partners, including AMPARs, 61 34). Previous studies have shown that STEP binds to protein NEUROSCIENCE and reveal a central role for STEP61 in differentially organizing syn- kinases such as Fyn, ERK1/2, and Pyk2, and these are regulated aptic AMPARs and NMDARs. by STEP dephosphorylating their critical functional tyrosine residues (35–38). Importantly, STEP dephosphorylates GluN2B STEP | LC/MS/MS | AMPA receptor | dephosphorylation | lysosome Y1472, allowing the AP-2 clathrin-associated adaptor protein complex to bind to GluN2B, thereby leading to internalization of lutamate receptors are crucial for many aspects of synaptic GluN2B-containing NMDAR complexes (39–41). We recently Gfunction in the mammalian central nervous system, such as reported that STEP61 binds to PSD-95 and that PSD-95 triggers mediating the majority of excitatory neuronal transmission and STEP61’s ubiquitination and degradation. Thus, STEP negatively inducing many forms of synaptic plasticity (1–5). Ionotropic gluta- regulates NMDARs by enhancing AP-2 binding and internali- mate receptors are classified into three large families: NMDA zation, but STEP is down-regulated by the NMDAR scaffolding receptors (NMDARs), kainate receptors, and AMPA receptors (AMPARs). NMDARs fulfill essential roles in neuronal devel- Significance opment, synaptic plasticity, and learning and memory (6–8) and – assemble as heterotetramers (GluN1, GluN2A D, GluN3A/B), Striatal-enriched protein tyrosine phosphatase (STEP) regulates most often consisting of two GluN1 subunits and two GluN2 sub- the trafficking and function of a variety of synaptic proteins, units. Functional AMPARs are homo- or heterotetramers com- ’ – including receptors and protein kinases. In addition, STEP s posed of combinations of four subunits (GluA1 4) (9). AMPARs expression and activity are altered in many neuropsychiatric account for much of the moment-to-moment rapid synaptic com- disorders. Here, we isolate the STEP61 interactome, which in- munication. In the hippocampus, canonical long-term potentiation cludes known interactors such as NMDA receptor (NMDAR) is induced by NMDAR activation and expressed by an increase – subunits, PSD-95, and Fyn kinase. In addition, we identified the in the density of synaptic AMPARs (10 12). In fact, mechanisms AMPA receptor (AMPAR) GluA2 subunit as a binding protein. underlying dynamic changes in synaptic AMPARs have been We show that STEP61 binds to the C termini of GluA2 and studied extensively, including lateral diffusion, exocytosis, and – GluA3, but not GluA1. Furthermore, we show that STEP61 de- endocytosis followed by degradation (13 15). phosphorylates GluA2, thereby regulating synaptic GluA2- One key mechanism regulating synaptic expression of both containing AMPARs via endocytosis and lysosomal degrada- AMPARs and NMDARs is receptor phosphorylation (16). Many tion. STEP61 preferentially regulates synaptic AMPARs and studies have demonstrated that activity-dependent subunit- extrasynaptic NMDARs, revealing a role for STEP61 in defining specific phosphorylation of NMDARs and AMPARs has pro- activity-dependent glutamate receptor localization. found effects on synaptic localization and channel function (17–19). The GluN2 subunits of NMDARs have long cytosolic tails, which Author contributions: S.W., S.I., R.A.N., and K.W.R. designed research; S.W., S.I., and Y.L. contain many phosphorylated S/T or Y residues. In particular, the performed research; S.W., S.I., Y.L., R.A.N., and K.W.R. analyzed data; and S.W., S.I., Y.L., GluN2B subunit has several well-characterized phosphorylation R.A.N., and K.W.R. wrote the paper. sites that have a profound effect on NMDAR trafficking (20, 21). Reviewers: D.S.B., Johnson & Johnson; and L.M., Case Western Reserve University. Two key phosphorylation events occur at the extreme C terminus of The authors declare no conflict of interest. GluN2B and control surface and synaptic expression: (i)GluN2B Published under the PNAS license. Y1472 is phosphorylated by the Src family tyrosine kinase Fyn 1To whom correspondence may be addressed. Email: [email protected] or rochek@ within an endocytic motif (22) and blocks internalization by inhib- ninds.nih.gov. iting binding to clathrin adaptor proteins, and (ii) GluN2B S1480 is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. phosphorylated by casein kinase 2 within the PDZ domain in an 1073/pnas.1900878116/-/DCSupplemental. activity-dependent manner and blocks binding to the PSD-95 family www.pnas.org/cgi/doi/10.1073/pnas.1900878116 PNAS Latest Articles | 1of10 Downloaded by guest on September 26, 2021 protein PSD-95. There are many studies on the mechanisms by as well as AMPAR-mediated synaptic currents. Our findings show that which STEP regulates NMDAR trafficking; however, AMPAR STEP61 differentially organizes synaptic AMPARs and NMDARs. regulation by STEP is poorly understood, and STEP is not thought to directly bind to AMPARs (27, 42). Results In this study, we use mass spectrometry (MS) to identify Identification of the STEP61 Interactome. To identify STEP61 bind- STEP61 binding proteins. We prepared lysates from cortex and ing proteins, we performed coimmunoprecipitation (co-IP) as- hippocampus of adult mouse brain to exclude binding partners says with STEP antibody from crude synaptosomal (P2) fractions specific to the shorter striatum-specific STEP variants. We identi- from cortex and hippocampus of adult mouse brain. We analyzed fied the STEP61 interactome, which included many of the expected STEP61 binding proteins using liquid chromatography tandem binding partners, such as GluN2B, GluN1, AP-2, PSD-95, and Fyn. MS (LC/MS/MS) (Fig. 1 A and B). We specifically used cortex and Interestingly, we also identified the AMPAR subunit GluA2. Ad- hippocampus because we recently showed that STEP61 is the only ditional analyses reveal that STEP61 directly binds to the C termini STEP splice variant expressed in cortex and hippocampus (27, 28). of GluA2 and GluA3, but not GluA1. We also found that STEP61 In contrast, all STEP splice variants are expressed in striatum. binding to GluA2 is necessary for tyrosine dephosphorylation of We analyzed co-IPs from STEP-KO brain as a control for non- GluA2. We investigated the effect of STEP61 expression on en- specific binding to STEP antibody or other nonspecific con- dogenous AMPARs and observed that the synaptic expression of tamination. From WT mouse brain samples, we identified 315 GluA2 and GluA3 is significantly increased in STEP-knockout candidate proteins from three independent experiments (SI (STEP-KO) brain, whereas only extrasynaptic NMDA receptors Appendix, Fig. S1 and Table S1), which we categorize in Fig. 1B: are increased by STEP deletion (28). STEP-KO lysates increased (i) 30% cytoskeletal-associated proteins and motor proteins (e.g., tyrosine phosphorylation of GluA2, consistent with AMPAR bind- α-actinin, DBN1, myosin-10, MAP2, Arp2/3 complex); (ii)20% ing