Skip to main content
Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction

  • Anna Lauriello,

    Roles Formal analysis, Investigation, Methodology, Writing – review & editing

    Current address: Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, United States of America

    Affiliation Department of History, Carleton College, Northfield, MN, United States of America

  • Quinn McVeigh,

    Roles Formal analysis, Investigation, Methodology, Writing – review & editing

    Current address: Cancer Program, Broad Institute, Cambridge, Massachusetts, United States of America; Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, United States of America

    Affiliation Department of Biology, Carleton College, Northfield, MN, United States of America

  • Rou-Jia Sung

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft

    Affiliation Department of Biology, Carleton College, Northfield, MN, United States of America


A large family of prototoxin-like molecules endogenous to mammals, Ly6 proteins have been implicated in the regulation of cell signaling processes across multiple species. Previous work has shown that certain members of the Ly6 family are expressed in the brain and target nicotinic acetylcholine receptor and potassium channel function. Structural similarities between Ly6 proteins and alpha-neurotoxins suggest the possibility of additional ionotropic receptor targets. Here, we investigated the possibility of lypd2 as a novel regulator of AMPA receptor (AMPAR) function. In particular, we focused on potential interactions with the Q/R isoforms of the GluR2 subunit, which have profound impacts on AMPAR permeability to calcium during neuronal stimulation. We find that although lypd2 and GluR2 share overlapping expression patterns in the mouse hippocampus, there was no interaction between lypd2 and either GluR2Q or GluR2R isoform. These results underscore the importance of continuing to investigate novel targets for Ly6 interaction and regulation.


Regulation of cellular signaling processes often begins at the plasma membrane, in which regulatory proteins interact with transmembrane receptors to modulate their response following an extracellular signal. Dysregulation of these processes has significant negative consequences for cellular and organismal function, with aberrant signaling processes implicated in dozens of human disease states [1], underscoring the need for the identification and characterization of novel regulators of cell signaling. The Ly6 protein family has been shown to be a prolific source of potential regulators for cell signaling, with recent work in the last few decades implicating Ly6 proteins in the modulation of cell-cell communication across multiple systems [213]. However, the number of characterized Ly6 proteins remains low, despite the large number of candidates in the family (40+ genes in mammalian systems) [14]. Averaging 15-20kDa in size, Ly6 proteins are generally found within the extracellular space, either as a secreted protein or (in the majority of cases) by being physically tethered to the outer leaflet of the plasma membrane by a post-translational C-terminal GPI anchor modification [6, 1416]. The Ly6 proteins adopt a three-finger fold, comprised of three variable sequence loops stabilized by a “crown” of 5–6 highly conserved disulfide bonds [13, 17]. This three-finger fold is also found in α-neurotoxins commonly found in elapid snake venoms, such as α-bungarotoxin [16, 18]. These α-neurotoxins primarily exert their effects by binding to orthosteric sites on their target receptor and inhibiting the conformational changes necessary for receptor function. Given their structural similarities, early explorations of Ly6 function hypothesized that Ly6 proteins may also target ionotropic receptors in a similar fashion as α-neurotoxins [19, 20]. Indeed, the first studies done on lynx1 and lynx2 demonstrated that these Ly6 proteins also target nicotinic acetylcholine receptors (nAChRs)—α-bungarotoxin targets the α7 nAChR while lynx1 and lynx2 target both α4β2 and α7 nAChRs—by altering the kinetics of receptor desensitization and receptor stochiometry, with implications for learning and memory [1922]. Recent work has shown that Ly6h also modulates nicotinic acetylcholine receptor function via a different mechanism—by lowering levels of receptor found at the plasma membrane and thus altering the maximal response of these receptors to agonist [2325]. Outside of nicotinic acetylcholine receptors, these α-neurotoxins can also interact with other ionotropic receptors, such as L-type calcium channels and voltage-gated potassium channels [26]. Could Ly6 proteins also modulate the function of such a diverse array of receptors? Current work has identified only a few non-nicotinic acetylcholine receptor targets for Ly6 proteins, including the Shaker-type potassium channels [27], CD3ζ chains of the T cell receptor signaling complex (TCR) [28], TBK1 [29], and EGFR/PDGFR heterodimer [30]. This work demonstrates that Ly6 proteins can modulate the function of non-nicotinic acetylcholine receptors, opening up possibilities of identifying other novel targets of Ly6-mediated regulation. Moreover, the role of Ly6 proteins in regulating neuronal excitability is conserved across multiple organisms (sss (sleepless) regulates sleep in Drosophila [27, 31] and odr-2 mediates odor sensing in C. elegans [32, 33]), suggesting other Ly6 proteins may also be involved in similar processes.

In mammalian systems, the majority of excitatory neurotransmission is mediated by ionotropic glutamate receptors. Specifically, AMPA receptors (AMPARs) are highly expressed in the brain, particularly in the hippocampal region [34]. AMPARs are synthesized, folded, and assembled in the endoplasmic reticulum, where they must undergo stringent quality control prior to export to the Golgi and ultimately to the cell surface [3537]. Several auxiliary proteins (such as TARPs, including stargazin, and CNIHs) and small interacting regulatory proteins (such as GRIP2/ABP and PICK1) have already been identified as playing key roles in AMPAR trafficking to the cell surface [3841]. In particular, TARPs and CNIHs are both large families of AMPAR auxiliary proteins that are localized to the plasma membrane; recent structures of AMPARs in complex with these transmembrane auxiliary proteins reveal that due to their small size and proximity to the plasma membrane, many of the functional interactions with the AMPAR are with the linker regions between the ligand binding domain (LBD) and transmembrane domain (TMD) or the LBD and the N-terminal domain (NBD) [39, 40, 4248]. Given the importance of AMPARs in modulating excitatory neurotransmission in mammalian brains and the significant number of the mammalian Ly6 proteins that also play roles in regulating neuronal excitability and neuronal function, this led us to hypothesize: could Ly6 proteins also be regulators of AMPA receptor function?

As the first step for considering that question, we investigated the possibility of whether lypd2, a Ly6 protein predicted to be GPI-anchored and reported to be expressed in the hippocampus, could interact with GluR2 AMPA receptors using co-immunoprecipitation in HEK-293 cells [51]. AMPA receptors are tetrameric and can exist as heteromeric combinations of predominantly GluR1/GluR2, GluR2/GluR3, or GluR1/GluR2/GluR3/GluR2 or as homomers of either GluR1 or GluR2 [52]. Receptor composition has significant implications for receptor function and regulation, with each receptor combination showing differences in calcium permeability, receptor trafficking and assembly [53, 54]. Given this varied molecular landscape of AMPA receptor function and the lack of information regarding potential for regulation by Ly6 proteins, we chose to focus our study on homomeric GluR2R or Glu2RQ receptors, which differ in a single amino acid at position 607 located in the pore region of the receptor (Fig 1A). As a simpler system, homomeric assemblies allow us to directly assess interactions between just two proteins (the homomer and lypd2) and limits the number of possible interacting combinations that could occur with heteromeric systems. Moreover, both isoforms have been shown to have unique biochemical properties of their own. Receptors containing GluR2R remain calcium impermeable, while incorporation of GluR2Q in either heteromeric or homomeric forms retains calcium permeability; alternative splicing events during GluR2 maturation result in the Q607R conversion in the pore region to render ‘mature’ GluR2 calcium-impermeable [5557]. In addition to effects on the mature receptor, previous work has also shown that the R607Q conversion has impacts on immature receptor folding and assembly, with changes in ER export kinetics for each isoform [57]. Moreover, both isoforms are differentially regulated by auxiliary proteins, including type II TARPs [53, 5661]. In comparisons between GluR2R and GluR2Q isoforms that differ in sequence only at the 607 position, γ-5 lowered glutamate affinity and accelerated desensitization of only the GluR2R isoform, while γ-7 enhanced steady state currents in the presence of CTZ and glutamate for GluR2R and not GluR2Q [61]. As noted earlier, structures of γ-5 in complex with GluR2Q suggest the effects of this type II TARP on AMPAR function are mediated by interactions between its extracellular loops with the LBD and/or LBD-TMD linker [48]. Given this isoform-specific regulation of GluR2R vs GluR2Q by small regulatory proteins in close proximity to the linker/LBD regions of the receptor and the biochemical simplicity of working with a homomeric system, we chose to focus our efforts on whether lypd2 could interact with homomeric GluR2R or GluR2Q. As shown in Fig 1A, the relative sizes and positioning of the GluR2 tetramer relative to lynx1 (one of the few Ly6 proteins with an experimentally determined structure, shown here for accuracy in scale and structure; Fig 1B shows a predicted structure of lypd2 as modeled in AlphaFold, with significant structural similarities with lynx1) suggest that a Ly6 protein is within reach of the LBD and/or the linker regions between the LBD and the TMD (potentially the NTD as well, depending on conformational flexibility of the receptor) [50]. This physical range of functional impact is quite similar to the contact regions seen for structures of the GluR2-TARP γ-2, GluR2-TARP γ-5, and GluR2-CNIH3 complexes [42, 43, 48]. Given the internal positioning of Q607 (which is buried in the transmembrane domain and directly faces the inside of the pore), it is unlikely that any mechanism of regulatory protein interaction (either by auxiliary or Ly6 proteins) would be through direct contacts with that residue. The results of our experiments showed that lypd2 does not interact with homomeric GluR2R or GluR2Q AMPA receptors. Although we were unable to identify an interaction between these Ly6 proteins and AMPA receptors, we hope this work will prompt the field to consider investigating alternative targets of Ly6 regulation.

Fig 1. Model for potential Ly6 interaction with GluR2.

A) Structures of GluR2 (3KG2) and lynx1 (2L03) shown as cartoon relative to outer leaflet of the plasma membrane [17, 49]. Each GluR2 subunit of the tetramer is shown in a different color; R607Q editing site on GluR2 is shown as spheres (in 3KG2 it has been edited to Q, as indicated), C-terminal GPI anchor on lynx1 is shown as a black curved line. B) Alphafold prediction of structure of mouse lypd2 (resides 21–100, omitting predicted N- and C-terminal signal sequences) [50]. Disulfide bonds are shown as gold sticks.

Materials and methods

DNA constructs

Rat cacng2 (stargazin; NM_053351.1) was synthesized and subcloned into pcDNA3.1+/C-(K)-DYK using CloneEZ strategy (Genscript). Rat lypd2 (NM_001130545.1) and mouse lypd2 (NM_026671.1) were synthesized and subcloned into pcDNA3.1(+)-myc-His A using HindIII/EcoRV sites (Genscript). The FLAG epitope is located immediately N-terminal to the predicted GPI attachment residue (as predicted using [62]). GFP-GluR2R plasmid was a kind gift from R. Malinow. GFP-GluR2Q plasmid was generated from GFP-GluR2R plasmid using site-directed mutagenesis using the indicated primers: R607Q_f: CCT TGG GTG CCT TTA TGC AAC AAG GAT GCG ATA TTT CGC; R607Q_r: GCG AAA TAT CGC ATC CTT GTT GCA TAA AGG CAC CCA AGG.


Mouse whole brain and hippocampal cDNA were purchased from Zyagen (Mouse C57 Brain and Hippocampus cDNA). RT-PCR was carried out for GAPDH and lypd2 using the indicated primers: mGAPDH-QF: 5’-GTTGTCTCCTGCGACTTCA-3’; mGAPDH-QR: 5’-GGTGGTCCAGGGTTTCTTA-3’; mlypd2 QF: 5’-GCATCCAACTGTGTCACCAC-3’; mlypd2 QR: 5’-GTCAGAATTGCAGCAGGACA-3’. Anticipated sizes for GAPDH and lypd2 products were 184bp and 192bp, respectively.

Cell culture

HEK-293T cells (ATCC, CRL-1573) were maintained at 37°C and 5% CO2 in culture medium consisting of 10% fetal bovine serum (Omega), 1% penicillin/streptomycin (Corning), and 1% l-glutamine (Sigma) in low-glucose DMEM with 2mM l-glutamine (Corning). Cells were grown to 60–80% confluence for transfection with X-tremeGENE HP reagent (Roche) at a 2:1 ratio of transfection reagent to DNA in Opti-MEM (Thermofisher). Transfection mixture was removed 24h after transfection and replaced with normal growth medium. Cells were allowed to recover for 24h in normal grown medium prior to immunostaining or immunoprecipitation protocols below.


HEK-293T cells were plated on glass coverslips coated with poly-D-lysine (Sigma) and cultured for 24h before transfection. Transfection was done as described above. Cells were rinsed with ice-cold PBS and fixed in 4% formaldehyde/PBS for 10min at room temperature. Cells were blocked for 1h at room temperature in 10% normal goat serum (Thermofisher) and then sequentially incubated in primary antibodies (overnight at 4C with rabbit α-GFP (Thermofisher, A11122) or mouse α-FLAG (Thermofisher, MA191878)). Labeled cells were washed three times with ice-cold PBS and incubated in secondary antibody for 1h at room temperature and DAPI (Sigma) for 1min at room temperature. Cells were mounted onto glass slides in ProLong Glass Anti-fade mountant (Thermofisher). Fluorescently conjugated secondary antibodies used were Alexa Fluor goat anti-mouse 568 and Alexa Fluor goat anti-rabbit 488 (Thermofisher). Cells were imaged on a Zeiss AxioImager at 40X magnification. Figures show representative imaged from at least three independent experiments for each set of transfected conditions.

Co-immunoprecipitation (Co-IP)

Proteins were extracted with “+SDS” lysis buffer (10mM Tris pH 7.5, 100mM NaCl, 5mM EDTA, 1% Triton X-100, 0.05% SDS, Complete protease inhibitor (Roche)). Protein concentrations were quantified via BCA Protein Assay (Thermofisher). 75 μg of total protein was reserved for input and 750μg for Co-IP. Co-IP samples were rocked for a minimum of 4 hours with 12.5 μl Protein G magnetic beads (NEB) at 4°C. Samples were washed 3X with a Co-IP +SDS wash buffer (10mM Tris pH 7.5, 100mM NaCl, 5mM EDTA, 0.05% Triton X-100, 0.05% SDS, Complete protease inhibitor (Roche)), then stored at -20C in 1X SDS Sample Buffer. “-SDS” lysis and wash buffers were the same as above except they were made without SDS; “PBS” lysis and wash buffers were phosphate buffered saline (PBS) purchased from Corning.

Western blotting

Samples were heated for 10 minutes at > 90°C. Proteins were run on 4–20% gels (Bio-Rad Mini-PROTEAN® TGX Stain-Free™ Protein Gels) in 1X TGS buffer (Bio-Rad) and transferred using a semi-dry transfer (Bio-Rad Trans-Blot® Turbo Transfer System) to a 0.2μm nitrocellulose membranes (Bio-Rad). Blocking was done in a 5% skim milk in 1X TBST. Membranes were blotted using primary antibodies against the mouse α-FLAG antibody (Thermofisher, MA191878), rabbit α-actin (Thermofisher, MA5-32479), or rabbit α-GFP antibody (Thermofisher, A11122). Figures show representative immunoblots from at least three independent experiments for each set of transfected conditions.


AMPA receptors are highly expressed throughout the brain, particularly in the hippocampus. We reasoned that Ly6 proteins that are also highly expressed in the brain would have a higher likelihood of interacting with AMPA receptors. Using RT-PCR analysis, we confirmed expression of lypd2 in mouse whole brain and hippocampal cDNA samples (Fig 2A). HEK 293 cells have been extensively demonstrated to be suitable for the expression of functional AMPA receptors in a heterologous system. In order to assess if they were a suitable heterologous expression system for lypd2, we performed a series of expression tests using constructs generated from mouse and rat genomes. We found that mouse and rat lypd2 expressed well and showed punctate expression at the cell surface, which is in agreement with previously established cellular localization patterns for other Ly6 proteins (Fig 2B and 2C) [23, 63]. Having established expression conditions for lypd2, all remaining experiments were conducted using the rat isoforms for these Ly6 proteins. As shown in Fig 3A, co-immunoprecipitation experiments with GluR2 homomeric AMPA receptors did not show any interaction with lypd2. These results were consistent for both GluR2R and GluR2Q isoforms.

Fig 2. Expression of rodent lypd2 in whole brain, hippocampus, and HEK-293T cells.

(A) RT-PCR showing expression of lypd2 in mouse whole brain and hippocampal cDNA (n = 3). (B) Expression of FLAG-tagged M. musculus lypd2 and R. norvegicus lypd2 in HEK-293T cells as measured by Western Blot (n = 3). Cells were transfected with indicated amounts of DNA for each construct and blotted for presence of C-terminal FLAG epitope. Empty vector (pCDNA) was used as a control. (C) Representative images of cell surface expression of lypd2 constructs visualized using immunofluorescence under non-permeabilizing conditions (n = 3). HEK-293T cells were transfected with the indicated amounts of each construct and stained for presence of C-terminal FLAG epitope. Blue: DAPI, white: FLAG.

Fig 3. Lypd2 does not interact with GluR2Q or GluR2R.

(A) Representative Western Blots of lypd2-FLAG co-immunoprecipitated with GFP-GluR2Q or GFP-GluR2R (n = 4). (B) Representative Western Blots of stargazin co-immunoprecipitated with GFP-GluR2Q (n = 4). (C) Representative Western Blots of lypd2-FLAG co-immunoprecipitated with GFP-GluR2Q prepared with different lysis and wash buffers (see Materials and Methods; n = 3). Top panels show 10% input blotted with the indicated primary antibodies; bottom panels show pulldown samples blotted with the indicated primary antibodies. HEK-293T cells were transfected with the indicated μg of DNA for each construct; cells were transfected with 0.5 μg of receptor DNA and the indicated amounts of stargazin or lypd2-FLAG DNA.

In order to determine if our buffer conditions were appropriate for detecting protein-protein interactions with GluR2, we performed a pulldown using GluR2 and stargazin. Stargazin has been previously shown to interact with and regulate the activity of GluR2, and served as a positive control for our pulldown conditions [64, 65]. As shown in Fig 3B, we were able to successfully pulldown stargazin using GluR2Q. Having established that our initial buffer conditions were suitable for pulling down GluR2-interactors, we considered the possibility that these buffer conditions were too stringent for detecting potentially weaker interactions between GluR2 and lypd2. We repeated our lypd2 GluR2Q pulldown with alternative buffers (-SDS from ours; PBS) under less stringent conditions [66]. As shown in Fig 3C, less stringent buffers continued to result in no pulldown, indicating that the lack of interaction seen is not due to our buffer conditions.


The Ly6 protein family has been implicated in the regulation of cell-cell communication pathways across multiple model systems. In several examples, the molecular mechanism underlying this regulatory function is due to alterations in the trafficking and function of ionotropic receptors such as nicotinic acetylcholine receptors and potassium channels. Here, we explored an alternative approach towards identifying potential new ionotropic receptor targets of Ly6 regulation. We focused on AMPA receptors as a possible target due to their role in mediating neuronal communication, the well-established precedence for regulation of AMPA receptor function via small regulatory proteins, and their highly dense expression in particular regions of the brain (including the hippocampus) where lypd2 is also expressed [34, 35, 37, 38, 54].

As noted in the introduction, we focused our efforts on the R/Q isoforms of GluR2 due to previously established work indicating isoform-specific regulation by auxiliary and/or small regulatory proteins [61]. Although our experiments indicate that we were unable to isolate stable complexes between either GluR2Q or GluR2R homomeric receptors with lypd2, they do not rule out the possibility of a Ly6-AMPAR interaction with other receptor combinations (GluR1 homomers, GluR1/GluR2, GluR2/GluR3, for example), with other GluR2 isoforms that vary in the intracellular or extracellular regions (flip/flop, R/G editing, differences in glycosylation patterns, etc), or in the presence of an additional binding partner in a trimeric complex [35, 52, 58, 67, 68]. In particular, previous work has demonstrated that native AMPARs in the brain are almost always associated with auxiliary subunits (such as γ-8 and CNIH2); therefore, if lypd2 were interacting with native AMPARs in the hippocampus it would likely be doing so in the context of a pre-existing complex of AMPAR with auxiliary subunits [52]. Future explorations of potential Ly6-AMPAR interactions could account for this complexity by including these additional auxiliary subunits in the transfection system. Moreover, additional work is also necessary to determine whether the observed overlaps in hippocampal expression for AMPARs and lypd2 (previous studies and this work, respectively) correlate with physical co-localization between lypd2 and AMPARs, a biological feature characteristic of interacting partners.

Although we were unsuccessful in identifying new targets for Ly6 regulation in this study, the importance of considering alternative strategies for studying the targets of this large and diverse protein family remains. Many of these Ly6 proteins and their associated molecular targets were identified as outcomes of phenotype-based screens; however, we posit that perhaps a biochemical approach may provide valuable information towards identifying the regulatory network of Ly6 interactors beyond the current few (such as using Ly6 proteins as bait to pull down novel prey interacting proteins that could be identified using mass spectrometry analysis) [69, 70]. The large size of this protein family suggests there could be additional Ly6 regulatory targets waiting to be identified.


We thank R. Malinow for the kind gift of the GFP-GluR2R plasmid.


  1. 1. Bernard G, Shevell MI. Channelopathies: A Review. Pediatr Neurol. 2008 Feb;38(2):73–85. pmid:18206787
  2. 2. Hijazi A, Masson W, Auge B, Waltzer L, Haenlin M, Roch F. boudin is required for septate junction organisation in Drosophila and codes for a diffusible protein of the Ly6 superfamily. Development. 2009 Jul 1;136(13):2199–209. pmid:19502482
  3. 3. Nilton A, Oshima K, Zare F, Byri S, Nannmark U, Nyberg KG, et al. Crooked, Coiled and Crimpled are three Ly6-like proteins required for proper localization of septate junction components. Development. 2010 Jul 15;137(14):2427–37. pmid:20570942
  4. 4. Herberg S, Gert KR, Schleiffer A, Pauli A. The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization. Science. 2018;361:1029–33. pmid:30190407
  5. 5. Kim NC, Marqués G. The Ly6 neurotoxin-like molecule target of wit regulates spontaneous neurotransmitter release at the developing neuromuscular junction in Drosophila. Dev Neurobiol. 2012 Dec;72(12):1541–58.
  6. 6. Miwa JM, Anderson KR, Hoffman KM. Lynx Prototoxins: Roles of Endogenous Mammalian Neurotoxin-Like Proteins in Modulating Nicotinic Acetylcholine Receptor Function to Influence Complex Biological Processes. Front Pharmacol. 2019 Apr 30;10:343. pmid:31114495
  7. 7. Eshel R, Zanin A, Kapon D, Sagi-Assif O, Brakenhoff R, van Dongen G, et al. Human Ly-6 antigen E48 (Ly-6D) regulates important interaction parameters between endothelial cells and head-and-neck squamous carcinoma cells. Int J Cancer. 2002 Apr 20;98(6):803–10. pmid:11948455
  8. 8. Bamezai A, Rock KL. Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells. Proc Natl Acad Sci. 1995 May 9;92(10):4294–8. pmid:7753800
  9. 9. Okumura R, Kurakawa T, Nakano T, Kayama H, Kinoshita M, Motooka D, et al. Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia. Nature. 2016 Apr;532(7597):117–21. pmid:27027293
  10. 10. Mar KB, Rinkenberger NR, Boys IN, Eitson JL, McDougal MB, Richardson RB, et al. LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step. Nat Commun. 2018 Dec;9(1):3603. pmid:30190477
  11. 11. Ji D, Liu P, Wang F, Zhang S, Li H. Identification and expression of a novel member of Ly-6 superfamily in zebrafish Denio rerio. Dev Genes Evol. 2012 Apr;222(2):119–24. pmid:22415300
  12. 12. Liu X, Cao X, Wang S, Ji G, Zhang S, Li H. Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio. Biosci Rep. 2017 Feb 28;37(1):BSR20160265. pmid:27980020
  13. 13. Lyukmanova EN, Shulepko MA, Shenkarev ZO, Bychkov ML, Paramonov AS, Chugunov AO, et al. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors. Sci Rep. 2016 Aug;6(1):30698. pmid:27485575
  14. 14. Lee PY, Wang JX, Parisini E, Dascher CC, Nigrovic PA. Ly6 family proteins in neutrophil biology. J Leukoc Biol. 2013 Oct;94(4):585–94. pmid:23543767
  15. 15. Loughner CL, Bruford EA, McAndrews MS, Delp EE, Swamynathan S, Swamynathan SK. Organization, evolution and functions of the human and mouse Ly6/uPAR family genes. Hum Genomics. 2016 Dec;10(1):10. pmid:27098205
  16. 16. Tsetlin VI. Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators. Trends Pharmacol Sci. 2015 Feb;36(2):109–23. pmid:25528970
  17. 17. Lyukmanova EN, Shenkarev ZO, Shulepko MA, Mineev KS, D’Hoedt D, Kasheverov IE, et al. NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1. J Biol Chem. 2011 Mar 25;286(12):10618–27. pmid:21252236
  18. 18. Kryukova EV, Egorova NS, Kudryavtsev DS, Lebedev DS, Spirova EN, Zhmak MN, et al. From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs. Front Pharmacol. 2019 Jul 3;10:748. pmid:31333465
  19. 19. Miwa JM, Iban̆ez-Tallon I, Crabtree GW, Sánchez R, S̆ali A, Role LW, et al. lynx1, an Endogenous Toxin-like Modulator of Nicotinic Acetylcholine Receptors in the Mammalian CNS. Neuron. 1999 May;23(1):105–14. pmid:10402197
  20. 20. Ibañez-Tallon I, Miwa JM, Wang HL, Adams NC, Crabtree GW, Sine SM, et al. Novel Modulation of Neuronal Nicotinic Acetylcholine Receptors by Association with the Endogenous Prototoxin lynx1. Neuron. 2002 Mar;33(6):893–903. pmid:11906696
  21. 21. Miwa JM, Stevens TR, King SL, Caldarone BJ, Ibanez-Tallon I, Xiao C, et al. The Prototoxin lynx1 Acts on Nicotinic Acetylcholine Receptors to Balance Neuronal Activity and Survival In Vivo. Neuron. 2006 Sep;51(5):587–600. pmid:16950157
  22. 22. Nichols WA, Henderson BJ, Yu C, Parker RL, Richards CI, Lester HA, et al. Lynx1 Shifts a4b2 Nicotinic Receptor Subunit Stoichiometry by Affecting Assembly in the Endoplasmic Reticulum. 2014;289(45):10.
  23. 23. Puddifoot CA, Wu M, Sung RJ, Joiner WJ. Ly6h Regulates Trafficking of Alpha7 Nicotinic Acetylcholine Receptors and Nicotine-Induced Potentiation of Glutamatergic Signaling. J Neurosci. 2015 Feb 25;35(8):3420–30. pmid:25716842
  24. 24. Wu M, Liu CZ, Barrall EA, Rissman RA, Joiner WJ. Unbalanced regulation of α7 nAChRs by Ly6h and NACHO contributes to neurotoxicity in Alzheimer’s disease. J Neurosci. 2021 Aug 26;41(41):8461–8474. pmid:34446574
  25. 25. Moriwaki Y, Kubo N, Watanabe M, Asano S, Shinoda T, Sugino T, et al. Endogenous neurotoxin-like protein Ly6H inhibits alpha7 nicotinic acetylcholine receptor currents at the plasma membrane. Sci Rep. 2020 Dec;10(1):11996. pmid:32686737
  26. 26. Kini RM, Doley R. Structure, function and evolution of three-finger toxins: Mini proteins with multiple targets. Toxicon. 2010 Nov;56(6):855–67. pmid:20670641
  27. 27. Wu MN, Joiner WJ, Dean T, Yue Z, Smith CJ, Chen D, et al. SLEEPLESS, a Ly-6/neurotoxin family member, regulates the levels, localization and activity of Shaker. Nat Neurosci. 2010 Jan;13(1):69–75. pmid:20010822
  28. 28. Kosugi A, Saitoh S ichiroh, Noda S, Miyake K, Yamashita Y, Kimoto M, et al. Physical and Functional Association between Thymic Shared Antigen-1/Stem Cell Antigen-2 and the T Cell Receptor Complex. J Biol Chem. 1998 May 15;273(20):12301–6. pmid:9575182
  29. 29. Liu Z, Jiang C, Lei Z, Dong S, Kuang L, Huang C, et al. Phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein PINLYP regulates type I interferon innate immunity. Proc Natl Acad Sci. 2022 Jan 5;119(1):e2111115119. pmid:34969857
  30. 30. Bychkov ML, Shulepko MA, Shlepova OV, Kulbatskii DS, Chulina IA, Paramonov AS, et al. SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer. Front Cell Dev Biol. 2021 Sep 14;9:739391. pmid:34595181
  31. 31. Wu M, Robinson JE, Joiner WJ. SLEEPLESS Is a Bifunctional Regulator of Excitability and Cholinergic Synaptic Transmission. Curr Biol. 2014 Mar;24(6):621–9. pmid:24613312
  32. 32. Chou JH, Bargmann CI, Sengupta P. The Caenorhabditis elegans odr-2 Gene Encodes a Novel Ly-6-Related Protein Required for Olfaction.:14.
  33. 33. Hong M, Ryu L, Ow MC, Kim J, Je AR, Chinta S, et al. Early Pheromone Experience Modifies a Synaptic Activity to Influence Adult Pheromone Responses of C. elegans. Curr Biol. 2017 Oct;27(20):3168–3177.e3. pmid:28988862
  34. 34. Shen K, Zeppillo T, Limon A. Regional transcriptome analysis of AMPA and GABAA receptor subunit expression generates E/I signatures of the human brain. Sci Rep. 2020 Dec;10(1):11352. pmid:32647210
  35. 35. Bredt DS, Nicoll RA. AMPA Receptor Trafficking at Excitatory Synapses. Neuron. 2003 Oct;40(2):361–79. pmid:14556714
  36. 36. Jacobi E, von Engelhardt J. AMPA receptor complex constituents: Control of receptor assembly, membrane trafficking and subcellular localization. Mol Cell Neurosci. 2018 Sep;91:67–75. pmid:29842943
  37. 37. Sheng M, Lee SH. AMPA Receptor Trafficking and the Control of Synaptic Transmission. Cell. 2001 Jun;105(7):825–8. pmid:11439178
  38. 38. Bissen D, Foss F, Acker-Palmer A. AMPA receptors and their minions: auxiliary proteins in AMPA receptor trafficking. Cell Mol Life Sci. 2019 Jun;76(11):2133–69. pmid:30937469
  39. 39. Chen S, Gouaux E. Structure and mechanism of AMPA receptor—auxiliary protein complexes. Curr Opin Struct Biol. 2019 Feb;54:104–11. pmid:30825796
  40. 40. Kato AS, Gill MB, Yu H, Nisenbaum ES, Bredt DS. TARPs differentially decorate AMPA receptors to specify neuropharmacology. Trends Neurosci. 2010 May;33(5):241–8. pmid:20219255
  41. 41. Schwenk J, Harmel N, Brechet A, Zolles G, Berkefeld H, Müller CS, et al. High-Resolution Proteomics Unravel Architecture and Molecular Diversity of Native AMPA Receptor Complexes. Neuron. 2012 May;74(4):621–33. pmid:22632720
  42. 42. Zhao Y, Chen S, Yoshioka C, Baconguis I, Gouaux E. Architecture of fully occupied GluA2 AMPA receptor–TARP complex elucidated by cryo-EM. Nature. 2016 Aug 4;536(7614):108–11. pmid:27368053
  43. 43. Nakagawa T. Structures of the AMPA receptor in complex with its auxiliary subunit cornichon. Science. 2019 Dec 6;366(6470):1259–63. pmid:31806817
  44. 44. Herguedas B, Watson JF, Ho H, Cais O, García-Nafría J, Greger IH. Architecture of the heteromeric GluA1/2 AMPA receptor in complex with the auxiliary subunit TARP γ8. Science. 2019 Apr 26;364(6438):eaav9011. pmid:30872532
  45. 45. Cais O, Herguedas B, Krol K, Cull-Candy SG, Farrant M, Greger IH. Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating. Cell Rep. 2014 Oct;9(2):728–40. pmid:25373908
  46. 46. Riva I, Eibl C, Volkmer R, Carbone AL, Plested AJ. Control of AMPA receptor activity by the extracellular loops of auxiliary proteins. eLife. 2017 Aug 30;6:e28680. pmid:28871958
  47. 47. Ben-Yaacov A, Gillor M, Haham T, Parsai A, Qneibi M, Stern-Bach Y. Molecular Mechanism of AMPA Receptor Modulation by TARP/Stargazin. Neuron. 2017 Mar;93(5):1126–1137.e4. pmid:28238551
  48. 48. Klykov O, Gangwar SP, Yelshanskaya MV, Yen L, Sobolevsky AI. Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L. Mol Cell. 2021 Dec;81(23):4771–4783.e7.
  49. 49. Sobolevsky AI, Rosconi MP, Gouaux E. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature. 2009 Dec;462(7274):745–56. pmid:19946266
  50. 50. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021 Aug 26;596(7873):583–9. pmid:34265844
  51. 51. Sjöstedt E, Zhong W, Fagerberg L, Karlsson M, Mitsios N, Adori C, et al. An atlas of the protein-coding genes in the human, pig, and mouse brain. 2020;18.
  52. 52. Yu J, Rao P, Clark S, Mitra J, Ha T, Gouaux E. Hippocampal AMPA receptor assemblies and mechanism of allosteric inhibition. Nature. 2021 Jun 17;594(7863):448–53. pmid:33981040
  53. 53. Isaac JTR, Ashby MC, McBain CJ. The Role of the GluR2 Subunit in AMPA Receptor Function and Synaptic Plasticity. Neuron. 2007 Jun;54(6):859–71. pmid:17582328
  54. 54. Song I, Huganir RL. Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci. 2002 Nov;25(11):578–88. pmid:12392933
  55. 55. Wright A, Vissel B. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain. Front Mol Neurosci [Internet]. 2012 [cited 2022 Apr 20];5. Available from: pmid:22514516
  56. 56. Greger IH, Khatri L, Kong X, Ziff EB. AMPA Receptor Tetramerization Is Mediated by Q/R Editing. Neuron. 2003 Nov;40(4):763–74. pmid:14622580
  57. 57. Greger IH, Khatri L, Ziff EB. RNA Editing at Arg607 Controls AMPA Receptor Exit from the Endoplasmic Reticulum. Neuron. 2002 May;34(5):759–72. pmid:12062022
  58. 58. Koike M, Tsukada S, Tsuzuki K, Kijima H, Ozawa S. Regulation of Kinetic Properties of GluR2 AMPA Receptor Channels by Alternative Splicing. J Neurosci. 2000 Mar 15;20(6):2166–74. pmid:10704491
  59. 59. Migues PV, Hardt O, Wu DC, Gamache K, Sacktor TC, Wang YT, et al. PKMζ maintains memories by regulating GluR2-dependent AMPA receptor trafficking. Nat Neurosci. 2010 May;13(5):630–4.
  60. 60. Mahajan SS, Ziff EB. Novel toxicity of the unedited GluR2 AMPA receptor subunit dependent on surface trafficking and increased Ca2+-permeability. Mol Cell Neurosci. 2007 Jul;35(3):470–81. pmid:17544687
  61. 61. Kato AS, Siuda ER, Nisenbaum ES, Bredt DS. AMPA Receptor Subunit-Specific Regulation by a Distinct Family of Type II TARPs. Neuron. 2008 Sep;59(6):986–96. pmid:18817736
  62. 62. Eisenhaber B, Bork P, Yuan Y, Löffler G, Eisenhaber F. Automated annotation of GPI anchor sites: case study C. elegans. Trends Biochem Sci. 2000 Jul;25(7):340–1. pmid:10871885
  63. 63. Mallya M, Campbell RD, Aguado B. Transcriptional Analysis of a Novel Cluster of LY-6 Family Members in the Human and Mouse Major Histocompatibility Complex: Five Genes with Many Splice Forms. Genomics. 2002 Jul;80(1):113–23. pmid:12079290
  64. 64. Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, et al. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature. 2000 Dec;408(6815):936–43. pmid:11140673
  65. 65. Vandenberghe W, Nicoll RA, Bredt DS. Stargazin is an AMPA receptor auxiliary subunit. Proc Natl Acad Sci. 2005 Jan 11;102(2):485–90. pmid:15630087
  66. 66. George SR, O’Dowd BF, editors. G protein-coupled receptor-protein interactions. Hoboken, N.J: Wiley-Liss; 2005. 329 p. (Receptor biochemistry and methodology).
  67. 67. Wen W, Lin CY, Niu L. R/G editing in GluA2Rflop modulates the functional difference between GluA1 flip and flop variants in GluA1/2R heteromeric channels. Sci Rep. 2017 Dec;7(1):13654. pmid:29057893
  68. 68. Kato AS, Gill MB, Ho MT, Yu H, Tu Y, Siuda ER, et al. Hippocampal AMPA Receptor Gating Controlled by Both TARP and Cornichon Proteins. Neuron. 2010 Dec;68(6):1082–96. pmid:21172611
  69. 69. Gottschalk A, Almedom RB, Schedletzky T, Anderson SD, Yates JR, Schafer WR. Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J. 2005 Jul 20;24(14):2566–78. pmid:15990870
  70. 70. Savas JN, Stein BD, Wu CC, Yates JR. Mass spectrometry accelerates membrane protein analysis. Trends Biochem Sci. 2011 May;S0968000411000582. pmid:21616670