GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction

Anna Lauriello; Quinn McVeigh; Rou-Jia Sung

doi:10.1371/journal.pone.0278278

. 2022 Nov 28;17(11):e0278278. doi: 10.1371/journal.pone.0278278

GluR2^Q and GluR2^R AMPA Subunits are not Targets of lypd2 Interaction

Anna Lauriello ^1,^¤a, Quinn McVeigh ^2,^¤b,^¤c, Rou-Jia Sung ^2,^*

Editor: Israel Silman³

PMCID: PMC9704558 PMID: 36441793

Abstract

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 GluR2^Q or GluR2^R isoform. These results underscore the importance of continuing to investigate novel targets for Ly6 interaction and regulation.

Introduction

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 [2–13]. 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, 14–16]. 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 [19–22]. 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 [23–25]. 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 [35–37]. 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 [38–41]. 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, 42–48]. 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 GluR2^R or Glu2R^Q 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 GluR2^R remain calcium impermeable, while incorporation of GluR2^Q 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 [55–57]. 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, 56–61]. In comparisons between GluR2^R and GluR2^Q isoforms that differ in sequence only at the 607 position, γ-5 lowered glutamate affinity and accelerated desensitization of only the GluR2^R isoform, while γ-7 enhanced steady state currents in the presence of CTZ and glutamate for GluR2^R and not GluR2^Q [61]. As noted earlier, structures of γ-5 in complex with GluR2^Q 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 GluR2^R vs GluR2^Q 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 GluR2^R or GluR2^Q. 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 GluR2^R or GluR2^Q 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 — 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 http://mendel.imp.ac.at/gpi/gpi_server.html [62]). GFP-GluR2^R plasmid was a kind gift from R. Malinow. GFP-GluR2^Q plasmid was generated from GFP-GluR2^R 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.

RT-PCR

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.

Immunostaining

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.

Results

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 GluR2^R and GluR2^Q isoforms.

Fig 2 — (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 — (A) Representative Western Blots of lypd2-FLAG co-immunoprecipitated with GFP-GluR2^Q or GFP-GluR2^R (n = 4). (B) Representative Western Blots of stargazin co-immunoprecipitated with GFP-GluR2^Q (n = 4). (C) Representative Western Blots of lypd2-FLAG co-immunoprecipitated with GFP-GluR2^Q 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 GluR2^Q. 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 GluR2^Q 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.

Discussion

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 GluR2^Q or GluR2^R 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.

Supporting information

S1 Raw images

(PDF)

Click here for additional data file.^{(3.9MB, pdf)}

Acknowledgments

We thank R. Malinow for the kind gift of the GFP-GluR2^R plasmid.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0278278.r001

Decision Letter 0

Israel Silman

12 Jul 2022

PONE-D-22-12127GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 InteractionPLOS ONE

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Reviewer #1: The task formulated by the authors - to detect novel receptors with which may interact the proteins of the Ly6/uPAR family - is well justified. They correctly started by emphasizing the well-known common three-finger folding of these proteins and of the three-finger alpha-neurotoxins from the snake venoms. However, they did clearly mention that there are two classes of the Ly6/uPAR proteins - secreted like SLURP-1 and membrane-bound by the GPI anchor like Lynx1. They gave the reasons why they have chosen lypd2 (because there are data on its colocalization with the AMPA receptors) but did not mention that lypd 2 is membrane-bound.

It is well-known that among the major targets of the Ly6/uPAR proteins are nicotinic acetylcholine receptors (nAChR) and the authors correctly give the references when as additional targets were found potassium channels and EGFR/PDGFR receptors.

The authors indeed demonstrated the colocalization in hippocampus of the lypd2 and of the calcium-conducting and non-conducting forms (Q/R isoforms of the GluR2 subunit) of the AMPA receptors, but found no interaction with their single chosen representative of the Ly6/uPAR family.

The authors made several mistakes in the introduction concerning snake venom neurotoxins:

line 49: major binding site of the snake venom alpha-neurotoxins is the orthosteric binding site (where agonists are bound), but not the allosteric sites (where mainly other allosteric positive or negative modulators are attached)

line 52: give references

line 53: alpha -bungarotoxin does not inhibit alpha4beta2 receptors , and instead of alpha7beta1 should be written " alpha7beta2".

line 20 and through the whole text authors use the term "ly6 proteins" , although most often in the literature are used Ly6 or Ly6/uPAR proteins.

------

Reviewer #2: The paper explores a possible interaction between a ly6 protein lypd2 and AMPA receptors using co-immunoprecipitation assays. It is an interesting topic certainly worth investigating. The result is negative which is in itself worth noting. The experiments are very basic, but the bigger problem is the experimental design. Overall, there is very little about where ly6-AMPAR interaction is expected. If the experiments are limited for whatever reason, it does not make much sense to look for possible interactions of lypd2 with Q or R variants of GluA2. It would make more sense to test other AMPAR subunits (A1, A3, A4, including heteromers) or complexes of AMPARs with auxiliary subunits. That would make the study more informative with the same level of experimentation.

The reason for this is predominantly extracellular location of ly6 proteins: they are either secreted or GPI-anchored to the outer leaflet of the plasma membrane whereas the Q/R editing site is in the pore of AMPARs, i.e. in the transmembrane region. It is not clear how the two could interact. Of course, the only way to rule this out is by experiments, however, if experiments are limited as it seems to be the case here, then it would make more sense to focus on variation in extracellular parts of AMPARs (provided by different AMPAR core subunits and complexes of AMPARs with auxiliary subunits) as these are more likely sites of interaction with lypd2 (ly6-related alpha-bungarotoxin also interacts with extracellular domains of acetylcholine receptors).

The authors even do pull down Stargazin and AMPAR complexes, but for some reason do not test these for the possible presence of lypd2.

The authors correctly note rich repertoire of AMPAR auxiliary subunits, however:

Line 76: please note that Stargazin is a TARP.

Lines 77-80 – it is not clear to me at all how AMPAR auxiliary proteins share cellular location and molecular architecture with ly6 proteins. Listed AMPAR auxiliary proteins are all transmembrane proteins whereas ly6 proteins are secreted or GPI-anchored to the outer leaflet of the plasma membrane. The authors state that AMPAR auxiliary subunits are heavily stabilized by disulphide bonds which is not the case. They provide a 2010 reference to support this statement, however, no structures of auxiliary subunits were available then. Meanwhile, structures of TARPs y2 (Stargazin), y8 and y5 came out as well as of CNIHs 2 and 3 which do not show extensive disulphide linking. Please, also note that AMPARs in the brain are almost always associated with auxiliary subunits (see e.g. Yu et al, Nature, 2021; https://doi.org/10.1038/s41586-021-03540-0), which means that if lypd2 was interacting with native AMPARs in hippocampus, it would have to do so with AMPARs complexed with auxiliary subunits (y8 and CNIH2).

RT-PCR is used to show expression of lypd2 in hippocampus and although AMPARs are also expressed in hippocampus, this does not strictly mean co-localization. This should be at least acknowledged in the discussion.

I don’t think the ratio of Stargazin – A2 DNA used in transfections is mentioned anywhere.

Maybe I missed it, but it is not mentioned anywhere how many times Western Blots or imaging experiments were replicated.

Figures:

The figures are quite low in resolution.

A panel or a figure to introduce the topic would be very helpful, e.g. AMPAR structure (including Q/R editing site) and a model of ly6 protein in the same membrane (UniProt entry for lypd2 provides alphafold structure which might be useful).

Raw images of gels relating to Fig. 1A are quite over-exposed. The amount of loaded ladder should be reduced to make it readable. Also, please explain what “X” stands for here.

Figure 1B – actin bands are very faint. Again, there seem to be some bands in a well marked “X”.

Figure 1C – why is over-expressed lypd2 visible as puncta? Is it aggregating?

Discussion is very limited and should be extendend (e.g. by acknowledging that the performed experiments do not entirely exclude interaction between lydp2 and AMPARs as other core subunits have not been tested nor complexes of AMPARs with auxiliary subunits).

Some typos:

Abstract: “alpha” shows up as “a”

Line 67 – delete “of”

Line 100 – was instead of were?

Line 120 – degree sign missing after 37

Line 122 – 2 mM

**********

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PLoS One. 2022 Nov 28;17(11):e0278278. doi: 10.1371/journal.pone.0278278.r002

Author response to Decision Letter 0

24 Aug 2022

We thank the reviewers for their feedback. We have addressed the comments and requests for revisions from the expert reviewers, including an additional figure and text to clarify points of confusion (see below). In doing so, we believe we have significantly improved and strengthened our manuscript. In the following, we provide an itemized list of changes we have made in addressing the provided comments.

Response to Reviewers

Reviewer 1:

“The task formulated by the authors - to detect novel receptors with which may interact the proteins of the Ly6/uPAR family - is well justified. They correctly started by emphasizing the well-known common three-finger folding of these proteins and of the three-finger alpha-neurotoxins from the snake venoms. However, they did clearly mention that there are two classes of the Ly6/uPAR proteins - secreted like SLURP-1 and membrane-bound by the GPI anchor like Lynx1. They gave the reasons why they have chosen lypd2 (because there are data on its colocalization with the AMPA receptors) but did not mention that lypd 2 is membrane-bound.”

We thank the reviewer for this careful attention to detail and have noted on line 126 that lypd2 is predicted to be a GPI-anchored protein.

“The authors made several mistakes in the introduction concerning snake venom neurotoxins: line 49: major binding site of the snake venom alpha-neurotoxins is the orthosteric binding site (where agonists are bound), but not the allosteric sites (where mainly other allosteric positive or negative modulators are attached),”

We thank the reviewer for noting this error and have clarified on line 55 that alpha-neurotoxins primarily exert their effects through the orthosteric site, not the allosteric site.

“line 52: give references. line 53: alpha -bungarotoxin does not inhibit alpha4beta2 receptors, and instead of alpha7beta1 should be written "alpha7beta2.”

We have included additional references on line 58 on the earliest work studying lynx1 modulation of nicotinic acetylcholine receptors (references 19 and 20). We have corrected and clarified the effects of bungarotoxin on inhibition of homopentameric alpha7 receptors while lynx1 and lynx2 have modulatory effects on alpha7 and alpha4beta2 receptors on lines 59-61, and included an additional reference (reference 22).

“line 20 and through the whole text authors use the term "ly6 proteins" , although most often in the literature are used Ly6 or Ly6/uPAR proteins.”

We have gone through the manuscript and replaced “ly6 proteins” or “ly6” with “Ly6 proteins” and “Ly6.”

Reviewer 2:

“Overall, there is very little about where ly6-AMPAR interaction is expected. If the experiments are limited for whatever reason, it does not make much sense to look for possible interactions of lypd2 with Q or R variants of GluA2. If the experiments are limited for whatever reason, it does not make much sense to look for possible interactions of lypd2 with Q or R variants of GluA2. It would make more sense to test other AMPAR subunits (A1, A3, A4, including heteromers) or complexes of AMPARs with auxiliary subunits. That would make the study more informative with the same level of experimentation. The reason for this is predominantly extracellular location of ly6 proteins: they are either secreted or GPI-anchored to the outer leaflet of the plasma membrane whereas the Q/R editing site is in the pore of AMPARs, i.e. in the transmembrane region. It is not clear how the two could interact. Of course, the only way to rule this out is by experiments, however, if experiments are limited as it seems to be the case here, then it would make more sense to focus on variation in extracellular parts of AMPARs (provided by different AMPAR core subunits and complexes of AMPARs with auxiliary subunits) as these are more likely sites of interaction with lypd2 (ly6-related alpha-bungarotoxin also interacts with extracellular domains of acetylcholine receptors).”

We thank the reviewer for this feedback. We agree that it does not make sense to look for interactions of lypd2 with the amino acids at position 607, representing the Q/R editing site on GluR2, and it was not our original intention to identify or propose direct interactions between lypd2 and the Q/R amino acid site. To help clarify this confusion, we have:

1) Included Figure 1 (lines 117-123) at the reviewer’s suggestion below to highlight where we expect Ly6-GluR2 receptor interactions to occur at the structural level. In this model, based on the scale of each molecule relative to each other, it is likely that a Ly6 protein could make contact with the ligand binding domain and/or the linker between the ligand binding domain and the transmembrane domain.

2) Striven to clarify our logic with regards to why we chose to focus on the Q and R variants of the GlRA2 homomer in our study in lines 131-166. Our interest in these isoforms was on the basis of literature indicating isoform-specific differences in interactions and regulations with small regulatory proteins such as the type-II TARPs �-5 and �-7.

3) We have also expanded the discussion (lines 322-328) to acknowledge that there are other AMPAR variations in terms of subunit composition and editing events that we did not explore that could also be possibilities for interaction.

“The authors even do pull down Stargazin and AMPAR complexes, but for some reason do not test these for the possible presence of lypd2.”

Given that AMPAR receptors have been shown to participate in dimeric complexes (detected as an interaction between the receptor and a single auxiliary protein of interest using a transfected cell culture system absent other auxiliary proteins), our goal in this study was to delineate the possibility of an interaction between Ly6 proteins and AMPARs using a similar approach. Consequently, the purpose of the stargazin pulldown was to serve as a positive control for our pulldown conditions, not for the purposes of detecting a trimeric complex. We thank the reviewer for this feedback, however, and we highlight in our expanded discussion in lines 327-336 that it is certainly possible that there could be trimeric complexes between AMPAR, Ly6 proteins, and these auxiliary proteins and indicate that this could be a source of future exploration.

“The authors correctly note rich repertoire of AMPAR auxiliary subunits, however: Line 76: please note that Stargazin is a TARP.”

We thank the reviewer for this careful attention to detail and have noted on line 97 that stargazin is a TARP.

“Lines 77-80 – it is not clear to me at all how AMPAR auxiliary proteins share cellular location and molecular architecture with ly6 proteins. Listed AMPAR auxiliary proteins are all transmembrane proteins whereas ly6 proteins are secreted or GPI-anchored to the outer leaflet of the plasma membrane. The authors state that AMPAR auxiliary subunits are heavily stabilized by disulphide bonds which is not the case. They provide a 2010 reference to support this statement, however, no structures of auxiliary subunits were available then. Meanwhile, structures of TARPs y2 (Stargazin), y8 and y5 came out as well as of CNIHs 2 and 3 which do not show extensive disulphide linking.”

We thank the reviewer for this feedback, and have updated both our text in the manuscript and our references accordingly. The new structural information available on AMPAR-auxiliary protein structures is very exciting, and indeed shifts our perspective on the similarities between these auxiliary proteins and Ly6s—as we have modified in the text, it is evident that the similarities are less in molecular structure and more in the size and placement of these small regulatory proteins. Both Ly6s, TARPS, and CNIHs are located in close proximity to the plasma membrane, which limits their contact points to largely the ligand binding domain and/or the linker region between the ligand binding domain and the transmembrane domain of the AMPAR. We have clarified our language in the introduction to reflect this in lines 99-111 and 161-163.

“Please, also note that AMPARs in the brain are almost always associated with auxiliary subunits (see e.g. Yu et al, Nature, 2021; https://doi.org/10.1038/s41586-021-03540-0), which means that if lypd2 was interacting with native AMPARs in hippocampus, it would have to do so with AMPARs complexed with auxiliary subunits (y8 and CNIH2). RT-PCR is used to show expression of lypd2 in hippocampus and although AMPARs are also expressed in hippocampus, this does not strictly mean co-localization. This should be at least acknowledged in the discussion.”

We thank the reviewer for this thoughtful feedback. We have expanded the discussion in lines 322-336 to acknowledge the questions left unexplored in our current study and to consider these possibilities as avenues for future exploration.

“I don’t think the ratio of Stargazin – A2 DNA used in transfections is mentioned anywhere.”

We have updated the figure legend on line 303-304 for Figure 3 with the ratio of transfected DNAs.  

“Maybe I missed it, but it is not mentioned anywhere how many times Western Blots or imaging experiments were replicated.”

All figures for Westerns and imaging experiments are representative images from at least three replicates; we have noted this in lines 225-226 and 247-248 in the methods.

“The figures are quite low in resolution.”

The figures were created using digital image files saved from our GE ImageQuant Western Blot Imager. We note the reviewer’s feedback, but unfortunately are only able to work with the image resolution from the saved images. We will examine the settings on our instrument to see if there is the possibility of improving the image resolution for future work.

“A panel or a figure to introduce the topic would be very helpful, e.g. AMPAR structure (including Q/R editing site) and a model of ly6 protein in the same membrane (UniProt entry for lypd2 provides alphafold structure which might be useful).”

We thank the reviewer for their feedback and have created Figure 1 to show a juxtaposition of structural data for GluR2 and lynx1 as well as the Alphafold model of lypd2. Since the structure of lynx1 has been experimentally determined, we chose to use lynx1 in panel A to maintain accuracy in scale and structural detail; however, as shown in panel B it is likely that lypd2 shares significant structural homology to lynx1. We have introduced and explained this model in the text as well, in lines 117-123 and 155-163.  

“Raw images of gels relating to Fig. 1A are quite over-exposed. The amount of loaded ladder should be reduced to make it readable. Also, please explain what “X” stands for here.”

“X” refers lanes containing other samples not relevant to this publication and cropped out of the final figures in the manuscript; we have appended the labeling associated with our raw image files to reflect this annotation. We have also included an additional exposure of the gel for the whole brain sample in which the ladder was not overexposed to demonstrate the correct size of the expected PCR products.

 “Figure 1B – actin bands are very faint. Again, there seem to be some bands in a well marked “X”.”

We note the reviewer’s critique here, and acknowledge that the actin bands are very faint. However, we do not have the ability to reexpose that specific blot as the physical blot itself was discarded after those initial exposures. The lanes marked X have been previously described in the legend for Fig 1A, and apply to all subsequent figures.

 “Figure 1C – why is over-expressed lypd2 visible as puncta? Is it aggregating?”

GPI anchored Ly6 proteins have been previously observed to form puncta, possibly due to localization to microdomains in the plasma membrane. It is thought that these puncta represent functional organization of the Ly6 proteins at the plasma membrane (in contrast to non-functional aggregates), although additional details remain unknown. We have included additional references in the text (line 261). 

“Discussion is very limited and should be extendend (e.g. by acknowledging that the performed experiments do not entirely exclude interaction between lydp2 and AMPARs as other core subunits have not been tested nor complexes of AMPARs with auxiliary subunits).”

We thank the reviewer for this feedback and have expanded the discussion in lines 323-337 to highlight the these potential avenues of future exploration with regards to other AMPARs containing other core subunits as well as the possibility of trimeric complexes between AMPARs, auxiliary subunits, and lypd2.

“Some typos: Abstract: “alpha” shows up as “a” Line 67 – delete “of” Line 100 – was instead of were? Line 120 – degree sign missing after 37 Line 122 – 2 mM”

We thank the reviewer for such a close reading of the text, and these typos have been addressed in the revised manuscript text.

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PLoS One. doi: 10.1371/journal.pone.0278278.r003

Decision Letter 1

Israel Silman

12 Sep 2022

PONE-D-22-12127R1GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 InteractionPLOS ONE

Dear Dr. Sung,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. As I wrote under Comments to the Author, Reviewer 2 suggests an extensive set of experiments that I do not think that you need to execute in the present manuscript. The reviewer does, however, raise several, more minor, issues that you are requested to address as fully as possible in your revised version. Please submit your revised manuscript by Oct 27 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Israel Silman

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

As you will see, Reviewer 2 has provided an extensive review which suggests that you perform many additional experiments. Although the experiments suggested are constructive, and worth executing, I do not think that you need to address them in the present manuscript. The reviewer does, however, make several more minor criticisms that you should try to address as fully as possible in your revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: I am glad that my critical comments were taken into account and the required changes were introduced into the revised version.

Reviewer #2: I thank the authors for going through all my points and in particular for including a new figure (Fig. 1) as I know this can take time.

I do think the study addresses an interesting question and provides a result (lypd2 does not interact with GluA2 homomers), but my main concerns still remain and that is the logic behind the experiments and comparisons the authors draw between Ly6 and AMPAR auxiliary subunits.

Yes, they are both (Ly6 and auxiliary proteins) associated with membrane, but all AMPAR auxiliary proteins are transmembrane proteins and interact fairly tightly with AMPAR transmembrane regions. Hence, their interactions with AMPARs can be expected to be sensitive to Q/R editing site in the pore. Ly6 proteins are anchored to the membrane, but are not transmembrane proteins. Their interaction points are most likely to be with the extracellular domains of AMPARs as shown in Fig. 1.

So why not test then other AMPARs subunits and/or variants which differ in the extracellular region and not just in the pore region?

Yes, Q/R editing site is very important for kinetics, trafficking, assembly etc. but majority of these come from the fact that this residue is in the pore and directly determines calcium permeability and pore properties.

Currently the logic is: protein A and protein B perhaps interact across this interface. To test this interaction, we will use mutation in A away from the interaction interface as it is known to be important for A.

Why not find examples of compounds/peptides that interact with extracellular domains of AMPARs and differ between Q and R variants (I am not aware of any, but I might be wrong)? Or perhaps there is a mutation in the pore region of AChRs that affects binding of alpha-bungarotoxin? If so, this would introduce well the rest of the paper.

"Given that AMPAR receptors have been shown to participate in dimeric complexes (detected as an interaction between the receptor and a single auxiliary protein of interest using a transfected cell culture system absent other auxiliary proteins), our goal in this study was to delineate the possibility of an interaction between Ly6 proteins and AMPARs using a similar approach. Consequently, the purpose of the stargazin pulldown was to serve as a positive control for our pulldown conditions, not for the purposes of detecting a trimeric complex."

AMPARs have also been expressed as a trimeric complex in a heterologous system (A1 + y8 + CNIH-2 in HEK cells in Kato et al., Neuron, 2010; https://doi.org/10.1016%2Fj.neuron.2010.11.026; PMID: 21172611), so still not sure it makes sense to use it only as a pulldown control and not in an experiment which would substantially strengthen the paper (in my opinion).

Minor comments:

1) Thank you for making it clear that n is 3 or more for all experiments, but it would be good to specify the exact number of replicates (n = x) for each experiment. Some quantification of at least some of the experiments would also strengthen the paper.

2) Figure 1 – nice figure! Orienting the two peptides in the same way would help comparison and it would also make it clearer how similar the fold is.

3) Line 104-105: "AMPA receptors are tetrameric and can exist as heteromeric combinations of GluR1 with either GluR2 or GluR3 or as homomers of either GluR1 or GluR2."

To me this reads like AMPARs can form A1/A3 heteromers. At least in vivo, much more dominant heterotetramer combinations are A1/A2/A1/A2, A3/A2/A3/A2 and A1/A2/A3/A2 (see e.g. Yu et al, Nature, 2021; https://doi.org/10.1038/s41586-021-03540-0).

4) "Several auxiliary proteins, including TARPs (such as stargazin), GRIP2/ABP, PICK1, and CNIHs and stargazin, have already been identified as playing key roles in AMPAR trafficking to the cell surface (38–40)."

GRIP2 and PICK1 do interact with AMPARs, but are not considered to be their auxiliary subunits in the way TARPs (and others) are (Schwenk et al., Neuron, 2012).

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2022 Nov 28;17(11):e0278278. doi: 10.1371/journal.pone.0278278.r004

Author response to Decision Letter 1

25 Oct 2022

Reviewer #1:

I am glad that my critical comments were taken into account and the required changes were introduced into the revised version.

Reviewer #2:

I thank the authors for going through all my points and in particular for including a new figure (Fig. 1) as I know this can take time.

We thank both reviewers for their attention to our manuscript, and we feel the manuscript has been significantly improved on the basis of their feedback.

Reviewer #2

“Yes, they are both (Ly6 and auxiliary proteins) associated with membrane, but all AMPAR auxiliary proteins are transmembrane proteins and interact fairly tightly with AMPAR transmembrane regions. Hence, their interactions with AMPARs can be expected to be sensitive to Q/R editing site in the pore. Ly6 proteins are anchored to the membrane, but are not transmembrane proteins. Their interaction points are most likely to be with the extracellular domains of AMPARs as shown in Fig. 1.”

We respectfully disagree with the expectation that the interaction between the transmembrane domains of AMPARs and auxiliary proteins are the molecular basis for their regulation of AMPAR function. The interaction between transmembrane domains may be important for complex formation and stability, but prior work mapping the functional interface between TARPs and AMPARs have shown that 1) the extracellular loops of TARPs mediate their functional effects on AMPARs and 2) the regions important for mediating these effects on AMPARs include the linker region between the N-terminal domain (NTD) and ligand binding domain (LBD) for �-2 and the linker region between the LBD and the transmembrane domain (TMD) for �-8 1,2,3. Ly6 proteins, despite not having a transmembrane domain, are positioned well to interact with any of these linker regions to potentially mediate functional impacts in a similar mechanism as the extracellular loops of TARPs.

1Ben-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.

2Cais 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.

3Herguedas 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).

We have added additional language in the introduction (lines 81-87 and 124-128) to 1) clarify that auxiliary protein mediated regulation of AMPARs are likely to be mediated through contacts between the TMD-LBD or LBD-NBD linkers, not direct contact with the Q/R site and 2) include the additional citations above.

“So why not test then other AMPARs subunits and/or variants which differ in the extracellular region and not just in the pore region?”

Our experiments do not rule out the possibility of other domains on the AMPAR as playing a role in the interaction. We have included language in the discussion (lines 292-293) to clarify this; we describe the logic of our reasoning in a response to a separate reviewer comment further below.

“Yes, Q/R editing site is very important for kinetics, trafficking, assembly etc. but majority of these come from the fact that this residue is in the pore and directly determines calcium permeability and pore properties.”

The biochemical mechanism behind the role of the Q/R editing site in determining calcium permeability and pore properties is quite clear and has been previously established; however, this only encapsulates direct effects post-assembly once the full pore has been established within a correctly folded and assembled AMPAR tetramer. It has also been shown that the editing at this site plays a critical role in the steps of AMPAR maturation prior to complete assembly. Greger et al4 shows changes in export kinetics from the ER between Q and R forms, indicating that the change in amino acid at this position has potential impacts on AMPAR function at a stage in its biogenesis where pore formation may not have occurred (i.e during protein folding and/or assembly). Moreover, Cais et al2 observed Q/R isoform-specific differences in association between �-2 and AMPARs lacking an NTD (decreased co-immunoprecipitation with GluR2R vs GluR2Q), suggesting that the effects of the Q/R editing site extend beyond direct impacts to the pore.

4Greger IH, Khatri L, Ziff EB. RNA Editing at Arg607 Controls AMPA Receptor Exit from the Endoplasmic Reticulum. Neuron. 2002 May;34(5):759–72.

Consequently, the role of the Q/R site in AMPAR function extends beyond its direct function in the pore to include indirect effects on AMPAR biogenesis as well. We have added language to 1) distinguish the effects of the Q/R editing on immature vs mature AMPAR (lines 117-120) and 2) highlight that any mechanism of isoform specific regulation is unlikely to occur through direct contact with the Q/R site itself (line 154).

“Currently the logic is: protein A and protein B perhaps interact across this interface. To test this interaction, we will use mutation in A away from the interaction interface as it is known to be important for A.”

We would like to clarify that our logic is the following (using similar language as the reviewer):

1) Protein A (AMPAR) and protein B (Ly6) could interact at the cell surface. As shown in Fig 1, the interaction interface is likely to be extracellular due to predicted anchoring of Ly6 proteins to the extracellular face of the membrane.

2) Prior work on other family members for protein B suggest roles in regulation of ionotropic receptor function. Prior work on Protein A (an ionotropic receptor) demonstrates multiple examples of regulation via extracellular interactions with small regulatory proteins.

3) Specifically, Protein A has different isoforms and combinations that each have unique biochemical properties and biological functions. One type of regulation is the effects of small regulatory proteins on properties of homomeric Protein AQ and Protein AR.

4) As there has been no prior work investigating the possibility of interaction and/or regulation between Protein A and Protein B, we choose to begin our work by examining the simplest molecular version of Protein A that still has relevance for function: homomeric Protein AQ and Protein AR.

Our hypothesis: If protein B is involved in regulation of function of protein AQ and protein AR, then protein B may interact differently with protein AQ and protein AR.

As noted in our previous revision, we found Reviewer #2’s comments very helpful in clarifying our hypothesis and we included substantial changes to our text to highlight that our hypothesis does not rule out the possibility of protein B interacting or regulating other forms of protein A. Given that there was no information available on regulation of protein A by protein B, we began with this hypothesis with these two isoforms as a simple starting point in our investigations (working with a homomeric receptor with only one amino acid change) that would still contribute knowledge to the field (working with isoforms that have been previously studied and have significant functional implications). Consequently, our choice to study the Q/R isoforms was not to “use the mutation to test the interaction interface,” but rather as a simpler system for evaluating potential effects on AMPAR function. While we understand there can be a variety of approaches towards investigating a completely uncharacterized system (here, Ly6 protein interaction with AMPAR), we hope this clarifies our approach to the reviewer. We have included additional language in lines 108-118 to clarify this in the text.

“Why not find examples of compounds/peptides that interact with extracellular domains of AMPARs and differ between Q and R variants (I am not aware of any, but I might be wrong)?”

To our knowledge, we are also unable to identify the reagents the reviewer is suggesting, and so would be unable to do this experiment.

“Or perhaps there is a mutation in the pore region of AChRs that affects binding of alpha-bungarotoxin? If so, this would introduce well the rest of the paper.”

To our knowledge there is not a mutation in the pore region of nAChRs that affects binding of bungarotoxin. We did find a work studying the influence of a pore mutation T288N on GABAA receptor trafficking5; however, this was absent data related to any potential impacts on auxiliary and/or small regulatory protein function.

5Hernandez, C.C., Zhang, Y., Hu, N. et al. GABA A Receptor Coupling Junction and Pore GABRB3 Mutations are Linked to Early-Onset Epileptic Encephalopathy. Sci Rep 7, 15903 (2017). https://doi.org/10.1038/s41598-017-16010-3.

"AMPARs have also been expressed as a trimeric complex in a heterologous system (A1 + y8 + CNIH-2 in HEK cells in Kato et al., Neuron, 2010; https://doi.org/10.1016%2Fj.neuron.2010.11.026; PMID: 21172611), so still not sure it makes sense to use it only as a pulldown control and not in an experiment which would substantially strengthen the paper (in my opinion).”

We acknowledge and appreciate the reviewer’s opinion on this experiment, and have included the new citation in line 294. As we noted in our previously revised text, our experiments do not exclude the possibility of a trimeric complex and that is a source for future work.

“1) Thank you for making it clear that n is 3 or more for all experiments, but it would be good to specify the exact number of replicates (n = x) for each experiment. Some quantification of at least some of the experiments would also strengthen the paper.”

We have updated the specific n for each experiment in the figure legends. With regards to quantification—we are unsure as to which experiments the reviewer is referring to. For example, while we recognize the value in quantifying Western Blot intensities for certain experiments (for example, quantifying differences in surface expression for surface biotinylation or labeling experiments), given that our results did not show an interaction we are unsure what the benefit of quantifying those bands (or in our case, the lack of bands) would be.

“2) Figure 1 – nice figure! Orienting the two peptides in the same way would help comparison and it would also make it clearer how similar the fold is.”

Thank you. The convention for orienting structures of Ly6 proteins in the literature is generally with the disulfide bonds at the “top” of the image and the three loops facing “down;” we have maintained this orientation in panel B to maintain consistency with depictions in the field. To help the reader orient, we intentionally kept the color and representation of the disulfide bonds (gold sticks) consistent between Lynx1 in panel A and the model of lypd2 in panel B to facilitate comparison between the two proteins.

“3) Line 104-105: "AMPA receptors are tetrameric and can exist as heteromeric combinations of GluR1 with either GluR2 or GluR3 or as homomers of either GluR1 or GluR2."

We thank the reviewer for their close reading of the text, and have adjusted the language in lines 104-105 to be clearer.

“4) "Several auxiliary proteins, including TARPs (such as stargazin), GRIP2/ABP, PICK1, and CNIHs and stargazin, have already been identified as playing key roles in AMPAR trafficking to the cell surface (38–40)."

GRIP2 and PICK1 do interact with AMPARs, but are not considered to be their auxiliary subunits in the way TARPs (and others) are (Schwenk et al., Neuron, 2012).”

We have adjusted the language in lines 76-78 to reflect this and included two additional citations (both the reviewer’s suggestion and the one below).

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.

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PLoS One. doi: 10.1371/journal.pone.0278278.r005

Decision Letter 2

Israel Silman

14 Nov 2022

GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction

PONE-D-22-12127R2

Dear Dr. Sung,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Israel Silman

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: I thank the authors for addressing all of my comments. I think the manuscript reads much better now.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0278278.r006

Acceptance letter

Israel Silman

16 Nov 2022

PONE-D-22-12127R2

GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction

Dear Dr. Sung:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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PERMALINK

GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction

Anna Lauriello

Quinn McVeigh

Rou-Jia Sung

Roles

Abstract

Introduction

Fig 1. Model for potential Ly6 interaction with GluR2.

Materials and methods

DNA constructs

RT-PCR

Cell culture

Immunostaining

Co-immunoprecipitation (Co-IP)

Western blotting

Results

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

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

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Israel Silman

Roles

Author response to Decision Letter 0

Decision Letter 1

Israel Silman

Roles

Author response to Decision Letter 1

Decision Letter 2

Israel Silman

Roles

Acceptance letter

Israel Silman

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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GluR2^Q and GluR2^R AMPA Subunits are not Targets of lypd2 Interaction

Fig 3. Lypd2 does not interact with GluR2^Q or GluR2^R.