PONE-D-20-00450

Melanopsin Carboxy-terminus Phosphorylation Plasticity and Bulk Negative Charge, not Strict Site Specificity, Achieves Phototransduction Deactivation

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This manuscript investigates the possible correlation between the manifold potential C-terminal phosphorylation sites in mouse melanopsin and the desensitization of its light-activated state. To this purpose the authors embark upon an impressive collection of experimental approaches, including site-directed mutagenesis, second messenger monitoring, MS-based peptide mapping and in-silico modeling. Involvement of phosphorylation of C-terminal sites in desensitization of GPCRs is well documented, and there is variable evidence this is also the case for melanopsins. This manuscript focuses on identification of (groups of) responsible sites. The authors generate novel insight, but the overall outcome is a bit disappointing. Very specific conclusions cannot be drawn, and the discussion section contains redundant elements, and lacks appropriate inclusion of literature data.

Major comments:

1. As far as I could find, two gene lineages of melanopsins are known, OPN4x and OPN4m. Mammals only contain the m-type, but also may generate splice variants. For mouse melanopsin a short and a long splice variant were reported (Pires (2009) J Neurosci) and this manuscripts studies the long one. This should be clearly mentioned in the text and the method section. In the discussion it could be speculated whether the results obtained for the long variant would also hold for the short version, since that might also contribute to the diversity in the ipRGC population.

2. There is considerable overlap with the approach in ref. 14 (Mure (2016) Neuron). That paper also investigates the contribution of phosphorylation of potential sites to desensitization of mouse melanopsin-L, and includes in-vivo analysis of transgenic mice. It is essential to the quality of the discussion section and in fact the entire paper that results in ref. 14 are compared with those reported in this manuscript, to be concluded by an integrative discussion.

3. A fair number of host cells have been used for the heterologous expression of melanopsins (see e.g. compilation in Shirzad (2016) Prog Retin Eye Res). HEK cells generate low expression levels but seem appropriate for analyzing signaling pathways. However, considering the large population of potential phosphorylation sites, and the alleged involvement of a fair number of kinases, it is questionable whether HEK cells form a proper representative for ipRGCs, since the kinase population and kinase and arrestin levels will vary between cell types. Hence, both the activation level, the kinetics of de-activation and the effect of mutations will differ in HEK cells compared to ipRGCs and probably even between ipRGC classes. Direct translational interpretations therefore are out of proportion. This element is briefly touched in the discussion, but should be further embraced, possibly also in the light of the in-vivo data in ref. 14.

4. Fluorescent monitoring of the Ca2+ level in cells is a good qualitative indicator for activation and desensitization of melanopsins. However, would it also allow the subtle quantitative comparison between WT and mutants, as executed in this manuscript ? For instance, the 487 nm excitation for the Ca2+ sensor will also excite melanopsin and metamelanopsin, thereby altering the photo-equilibrium between these states during the desensitization process, probably affecting the desensitization kinetics. The latter will then also depend on the expression level of functional melanopsin. Has it e.g. been assessed whether activation and deactivation kinetics are similar for different expression levels of WT melanopsin ? If the absorbance bands of melanopsin and metamelanopsin, and the functional expression levels somewhat differ between WT and mutants, this will affect the activation and deactivation kinetics, and impair quantitative comparison between all these species. These elements also need to be properly discussed.

5. Actually, the mass spectroscopic analysis is the most complex, but also most elegant experimental approach, since it exploits the WT protein and does not depend on mutational perturbation. Unfortunately, the shortest time interval undertaken is 1 min, in which the deactivation process has largely been completed. Has a shorter time interval not been considered ?

Minor comments:

6. Top sentence in the legend of Fig. 3 is incomplete ("At least eight proximal C-terminus phosphorylation sites in the are needed to deactivate melanopsin").

7. Re lines 245-247: Possibly the phosphomimetic mutant more rapidly, but reversibly, recruits arrestins, thereby slowing down activation ?

8. Tables 5 and 6 are necessary, but too distractive in the main text. I suggest to transfer these to the supporting information.

9. line 455: though -> through ?

10. line 525: "Small aliquots (5 μL) of purified melanopsin was injected ...". I presume this was after proteolytic treatment ?

11. line 534: "concentrated eluates were size separated....". These are eluates form the affinity purification column ?

12. Figure 2A: Why is the fluorescent intensity in this and subsequent figures normalized ? I presume, that many mutants will reach higher maximal intensity. This also is useful information and the corresponding deactivation rates are presented anyway in the corresponding column figure. At least, if I understand this properly from the methods section, the deactivation rates are calculated from the absolute data ?

13. Figure 3A: This figure a.o. shows the phosphonull + P12 mutant, containing P-I + P-II phosphorylatable sites. However in Fig. 4A phosphonull + P12 apparently stands for P-II + P-III sites, in Fig. 4B phosphonull + P12 apparently stands for P-II + P-IV sites, and in Fig. 4C phosphonull + P12 apparently stands for P-II + P-III sites, as well as for P-II + P-IV sites. Please explain or correct.

14. Figure 3B: This figure lacks the activation signal. Were the first 20 seconds or so cut off ? Maybe include these on a smaller time-scale. Also, many curves are hardly distinguishable. Either use off-sets or absolute intensities.

15. Figure 4C: The deactivation rate for the phosphonull mutant + P-II + P-III sites is very much larger compared to WT. However, this is absolutely not obvious from Fig. 4A. The main differences in Fig. 4A seem to be a somewhat slower activation rate (but only if the absolute maximal intensities are similar) and maybe a somewhat lower final level (although probably not significant different). Please explain.

16. Figure 5: Diamond shaped residues in the "foreign" C-terminals are not easily detected. Better diamond-shape the other residues and encircle the potential phosphorylation sites.

17. Figure 6: How was the curve of the phosphonull + P12 mutant (P-ID + P-IID) normalized ? Here it certainly is much more informative to show absolute intensities.

18. Figure 7: The fragmentation spectra are not very clear to me. Maybe include one-page size spectra in the supporting information. What is the meaning of the bold lines in the spectra ? Do these arise from the corresponding peptides ?

19. Figure S4: I could not find a reference to this figure in the text. If you want to use it still, I suggest to only display the relevant C-terminal sequences.

Reviewer #2: This work from Valdez-Lopez and colleagues systematically interrogates the location, mechanism, and role of phosphorylation of the C-terminus of melanopsin in deactivation. As expected from this group, the experiments are well-designed and test both necessity and sufficiency of phosphorylation at given locations. The authors report some very interesting findings and will be of wide interest to the field. I have only a couple, relatively minor, critiques.

1. The last line in the abstract states, “This degree of functional versatility could help explain the diverse ipRGC light responses as well as non-image and image forming behaviors, even though all six subtypes of ipRGCs express the same melanopsin gene Opn4.”

This seems like an overly sweeping claim. For example, much of the diversity between subtypes is now thought to result from different downstream targets of the melanopsin phototransduction cascades (Jiang et al., 2018 and Sonoda et al., 2018). It also glosses over the fact that there are two splice isoforms of Opn4 that are differentially expressed by ipRGC subtypes. Finally, the phrasing of “as well as non-image and image forming behaviors” makes the meaning of this phrase unclear, are the authors stating that the functional versatility of the melanopsin protein could explain the diversity of visual behaviors driven by ipRGCs? Again, this is too broad a claim because the ipRGC role in behavior is also determined by a host of other factors, not least the differential projection patterns of ipRGC subtypes, as well as their intrinsic physiological properties.

In short, this sentence should be removed or modified.

2. Some of the text in the figures is almost microscopic, including graph labels in, for example, Figure 4 (but also elsewhere in general) and including labeling of protein structure in, for example, Figure 5 (expecially in A-D). If the text in Figure 5 on the protein structures is not important, it should be removed, otherwise, please enlarge it.

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Reviewer #1: No

Reviewer #2: No

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Melanopsin Carboxy-terminus Phosphorylation Plasticity and Bulk Negative Charge, not Strict Site Specificity, Achieves Phototransduction Deactivation

PONE-D-20-00450R1

Dear Dr. Robinson,

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Additional Editor Comments:

Thank you for submitting a revised version. I am happy that all the reviewers' concerns and comments have been addressed in this excellent paper.