RhoGDI phosphorylation by PKC promotes its interaction with death receptor p75NTR to gate axon growth and neuron survival

How receptors juggle their interactions with multiple downstream effectors remains poorly understood. Here we show that the outcome of death receptor p75NTR signaling is determined through competition of effectors for interaction with its intracellular domain, in turn dictated by the nature of the ligand. While NGF induces release of RhoGDI through recruitment of RIP2, thus decreasing RhoA activity in favor of NFkB signaling, MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, disengaging RIP2, and enhancing RhoA activity in detriment of NF-kB. This results in stunted neurite outgrowth and apoptosis in cerebellar granule neurons. If presented simultaneously, MAG prevails over NGF. The NMR solution structure of the complex between the RhoGDI N-terminus and p75NTR juxtamembrane domain reveals previously unknown structures of these proteins and clarifies the mechanism of p75NTR activation. These results show how ligand-directed competition between RIP2 and RhoGDI for p75NTR engagement determine axon growth and neuron survival. Similar principles are likely at work in other receptors engaging multiple effectors and signaling pathways.

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I look forward to seeing a revised form of your manuscript when it is ready and please let me know if you have questions or comments regarding the revision.The manuscript by Ramanujan et al. presents a compelling study on the regulation of p75NTR signaling and its implications for neuron survival and axon growth.The paper is well-constructed, with clear demonstrations of how p75NTR's interactions with various ligands, such as NGF and MAG, and downstream effectors like RhoGDI and RIP2, lead to different neuronal outcomes.The use of NMR to elucidate the structure of the RhoGDI-p75NTR complex is particularly noteworthy and adds a valuable dimension to the understanding of receptor signaling.The novelty of the work is evident in its detailed elucidation of the mechanisms by which different ligands modulate p75NTR's engagement with downstream effectors.This contributes significantly to the field of neuroscience by providing insights into how neurons integrate multiple signals for making critical growth and survival decisions.Overall Assessment: This study represents one of the most elegant explanations of p75NTR's dual functions and its ability to switch between growth and survival signaling pathways.The research is methodologically sound, and the findings are clearly presented and wellsupported by the data.With the minor revisions suggested below, I am in favor of accepting this manuscript for publication.The study will undoubtedly be a valuable addition to the field.Minor Points: * While the paper is strong in its current form, a more explicit discussion on the implications of the findings for potential therapeutic targets would enhance its impact.This could involve a deeper analysis of how the detailed mechanism might be exploited to modulate neuron survival and axon growth in pathological conditions.* In Figure 7b, the appearance of k303A axons suggests more degeneration than other conditions.It would be helpful to clarify if this is a consistent observation or an artifact and to provide quantification for axonal integrity if available.Perhaps it simply requires a different representative image.* Expanding the summary in Figure 8 to show the relationship between Serine 34 and Serine 96 phosphorylation events would help in illustrating the interplay between these modifications more clearly.* A brief explanation of the NF-kB and Rho GTP assays in the Results section would be useful for readers who may not be familiar with these techniques.* The first mention of NIH3T3 cells in the discussion of Figure 1 should include a note that these cells do not express endogenous p75NTR, as this is a crucial piece of information for the interpretation of the data.This information is currently introduced in the discussion of Figure 2 and would be more appropriately placed earlier in the text.

Referee #2:
This study focuses on the receptor p75NTR, a receptor that mediates signaling by several structurally unrelated ligands, and couples to signaling by several alternative pathways, via sometimes competitive binding of a variety of signaling adapter proteins.The study reveals specific mechanisms that dictate which signaling outcome prevails when the receptor encounters two ligands, MAG and NGF simultaneously.The investigators find that NGF promotes recruitment of RIP2 to p75NTR, displacing RhoGDI and thereby decreasing RhoA activity while increasing NF-kappaB activation, while MAG induces PKC-mediated phosphorylation of Ser34 of RhoGDI, promoting association of RhoGDI with p75NTR and increasing RhoA activation.
The study provides structural data yielding insight into the mechanisms of these interactions.Further, the study reveals how association of these signaling proteins with the juxtamembrane domain of p75NTR imparts a more rigid structure on this otherwise unstructured domain, and providing a possible mechanism that allows coupling of structural changes in the receptors extracellular domain to structural changes in the intracellular domain Overall, the paper is technically impressive and rigorous, well-written and interesting.Major issues: I found only one serious short-coming in the presentation.The authors correctly note that the mechanism by which MAG promotes the association with p75NTR is unclear.However, what they fail to indicate is that part of this lack of clarity reflects the fact that MAG does not bind to p75NTR.Two MAG-binding proteins, NogoR and LRP1 have been reported to associate with p75NTR in a MAG-dependent way, thereby acting as coreceptors.The authors refer to MAG as a p75NTR ligand, but this is misleading.MAG is a ligand that activates p75NTR, but it is not a ligand that binds p75NTR.I know the authors are aware of this fact, and I suspect that they elected to avoid discussion of this complexity because none of their studies bear directly on whether either of these co-receptors is involved.However, I think failing to discuss this will ultimately be confusing to the readers.I found a number of small textual errors that need to be corrected for clarity, as follows: Page 6. "while Ser96 is dispensable for RhoGDI binding" should read "while Ser96 phosphorylation is dispensable for RhoGDI binding" PAGE 6: "we generated phosphor-specific antibodies against phosphor-Ser96" should read "we generated phospho-specific antibodies against phospho-Ser96".An auto-correct issue.Page 8: "Mutant S34A RhoGDI-59 did not co-immunoprecipitated" should read "Mutant S34A RhoGDI-59 did not coimmunoprecipitate" Page 13: "effects axon growth and neuron survival" should read "effects on axon growth and neuron survival" Page 14: "Even within the same cell type, can p75NTR induce" should read "Even within the same cell type, p75NTR can induce" PAGE 15: "mechanism by each PKC stimulates" should read "mechanism by which PKC stimulates" 1

Data added to the revised version
We have added one piece of data to the revised version of our manuscript.The reviewers may recall that in the diagram presented in Figure 8 at the end of the paper, we mentioned the possibility that RhoGDI may interact in "trans" with p75 NTR dimers, such that NTD and CTD of the same RhoGDI molecule bind to JXT and DD, respectively, of different p75 NTR molecules in the receptor dimer.We have now been able to demonstrate this in a co-immunoprecipitation experiment using complementation between two different p75 NTR molecules carrying mutations in either JXT or DD, respectively.These data are now shown in Figure 6A along with explanatory graphics presented in Figures EV1E and F. The corresponding paragraph in the Results section also explains this in more detail.
We have also updated the solution structure of the RhoGDI NTD/p75 NTR JXT complex (Figure 5) after refinement of the model (improved parameters in Table EV1).

Reviewer #1
While the paper is strong in its current form, a more explicit discussion on the implications of the findings for potential therapeutic targets would enhance its impact.This could involve a deeper analysis of how the detailed mechanism might be exploited to modulate neuron survival and axon growth in pathological conditions.
A discussion of this point has been included (p. 18-19) In Figure 7b, the appearance of k303A axons suggests more degeneration than other conditions.It would be helpful to clarify if this is a consistent observation or an artifact and to provide quantification for axonal integrity if available.Perhaps it simply requires a different representative image.
Axonal integrity was not compromised in this condition as verified by counterstaining for Tuj1 ).This is now indicated in the figure legend.
Expanding the summary in Figure 8 to show the relationship between Serine 34 and Serine 96 phosphorylation events would help in illustrating the interplay between these modifications more clearly.
The figure could not be expanded but was modified so as to denote the relationship between the two Ser residues.
A brief explanation of the NF-kB and Rho GTP assays in the Results section would be useful for readers who may not be familiar with these techniques.
This was now included in the Results and Methods sections.
The first mention of NIH3T3 cells in the discussion of Figure 1 should include a note that these cells do not express endogenous p75NTR, as this is a crucial piece of information for the interpretation of the data.This information is currently introduced in the discussion of Figure 2 and would be more appropriately placed earlier in the text.
This was now included in the Results section.

Reviewer #2
6th Dec 2023 1st Authors' Response to Reviewers 2 I found only one serious short-coming in the presentation.The authors correctly note that the mechanism by which MAG promotes the association with p75NTR is unclear.However, what they fail to indicate is that part of this lack of clarity reflects the fact that MAG does not bind to p75NTR.Two MAG-binding proteins, NogoR and LRP1 have been reported to associate with p75NTR in a MAG-dependent way, thereby acting as coreceptors.The authors refer to MAG as a p75NTR ligand, but this is misleading.MAG is a ligand that activates p75NTR, but it is not a ligand that binds p75NTR.I know the authors are aware of this fact, and I suspect that they elected to avoid discussion of this complexity because none of their studies bear directly on whether either of these co-receptors is involved.However, I think failing to discuss this will ultimately be confusing to the readers.
We have modified the Introduction section to clarify this point.But we would like to note that, as far as we are aware, no evidence has been presented that MAG does not interact with p75 NTR in the complex with co-receptors NgR and Lingo-1, only that it does not in their absence (Yamashita et al, 2002).(Incidentally, this paper does not actually show this either, but refers to "unpublished preliminary experiments".We will welcome any additional information the reviewer may have that would help to clarify this point.)In the GDNF/GFRa1/RET and TGFβ-TBRII-TBRI complexes, the ligands (GDNF and TGFβ) are unable to bind their signaling receptors (RET and TBRI/ALK5, respectively) in the absence of the binding co-receptors (GFRα1 and TBRII, respectively), but crosslinking and x-ray crystallography studies have conclusively established that both ligands do make direct contacts with the signaling receptors.However, this binding is too weak to be detected with the isolated signaling receptors (Ibáñez, 2013;Derynck & Budi, 2019).Likewise, MAG may interact directly with p75 NTR through contacts that are too weak to be detected in the absence of the NgR and Lingo-1 co-receptors.In the absence of similar evidence, it cannot be concluded at present that MAG does not make direct contacts with p75 NTR .
We have also made all the corrections indicated below (and thank this reviewer for his/her thorough reading of our manuscript): Page 6. "while Ser96 is dispensable for RhoGDI binding" should read "while Ser96 phosphorylation is dispensable for RhoGDI binding" PAGE 6: "we generated phosphor-specific antibodies against phosphor-Ser96" should read "we generated phospho-specific antibodies against phospho-Ser96".An auto-correct issue.
Page 8: "Mutant S34A RhoGDI-59 did not co-immunoprecipitated" should read "Mutant S34A RhoGDI-59 did not co-immunoprecipitate" Page 13: "effects axon growth and neuron survival" should read "effects on axon growth and neuron survival" Page 14: "Even within the same cell type, can p75NTR induce" should read "Even within the same cell type, p75NTR can induce" PAGE 15: "mechanism by each PKC stimulates" should read "mechanism by which PKC stimulates" 19th Dec 2023 1st Revision -Editorial Decision Manuscript number: EMBOR-2023-58305V2 Title: RhoGDI phosphorylation by PKC regulates its interaction with death receptor p75NTR to gate axon... Author(s): Ajeena Ramanujan, Zhen Li, Yanchen Ma, Zhi Lin, and Carlos Ibanez Dear Prof. Ibanez Thank you for your patience while we have editorially reviewed your revised manuscript.I am now writing with an 'accept in principle' decision, which means that I will be happy to accept your manuscript for publication once a few minor issues/corrections have been addressed, as follows.
-Page 13, top: the reference to Figure 6A is incorrect when you describe the effect of NGF treatment on p75[NTR]-RhoGDI interaction.I believe this should refer to Figure 6B.From there on all references to Figure 6 panels need to be checked.I think the addition of panel A has messed up the callouts.
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(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines.For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (