Evolutionary History of Calcium-Sensing Receptors Unveils Hyper/Hypocalcemia-Causing Mutations

  1. Aylin Bircan
  2. Nurdan Kuru
  3. Onur Dereli
  4. Berkay Selçuk
  5. Ogün Adebali

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Abstract

Calcium-sensing receptor evolution highlights hyper/hypocalcemia-causing mutations The Calcium Sensing Receptor (CaSR) is a key player in regulating calcium levels and has been linked to disorders like hypercalcemia and hypocalcemia. Despite advancements in understanding CaSR’s structure and functions, there are still gaps in our understanding of its specific residues and their differences from receptors within the same class. In this study, we used phylogeny-based techniques to identify functionally equivalent orthologs of CaSR, predict residue significance, and compute specificity-determining position (SDP) scores to understand its evolutionary basis. The analysis revealed exceptional conservation of the CaSR subfamily, with high SDP scores being critical in receptor activation and pathogenicity. To further enhance the findings, gradient-boosting trees were applied to differentiate between gain- and loss-of-function mutations responsible for hypocalcemia and hypercalcemia. Lastly, we investigated the importance of these mutations in the context of receptor activation dynamics. In summary, through comprehensive exploration of the evolutionary history of the CaSR subfamily, coupled with innovative phylogenetic methodologies, we identified activating and inactivating residues, providing valuable insights into the regulation of calcium homeostasis and its connections to associated disorders.

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    Referee #3

    Evidence, reproducibility and clarity

    Major comments:

    1. I don't understand the meaning of the sentence beginning on line 61. There are published structures of taste receptors and many papers have looked at activation mechanisms.

    2. The results are extremely difficult to follow. There is far too much information in here about methods and without sub-headings is impossible to follow. I'd suggest moving a lot of the methods information to the Materials and Methods and then adding more sub-headings.

    3. In Figure 1, the conclusion seems to be that the authors identified GPRC6A and taste receptors as most closely related. However, they stated in the Introduction that these were known to be most closely related, so this just confirms what is known. The authors should acknowledge this and shorten this section significantly.

    4. On line 349 the authors state that 'any substitution on the receptor disrupts the function of the receptor'. This is not true. There are several known benign mutations that have no effect on CaSR function.

    5. I found the section based around Table 2 very difficult to follow. Initially I presumed these were variants that have not been functionally characterized that the authors would predict, then test in vitro. However, this is not the case as several have been functionally assessed (e.g. I857X, T186N, T699N, R701G, T808P). The authors should add another column to state which have been functionally assessed and what this showed. This is important as their predictions are clearly wrong for some residues (e.g. T699N has been functionally assessed and shown to be LOF). This makes it difficult to understand what the point of the tool is. The authors should expand out their analysis to look at many more residues that are known to cause disease to really assess how useful the tool is (e.g. those reported in multiple families or those that have been functionally assessed). They should also test on residues with both GOF/LOF mutations.

    6. It was unclear why the authors focussed on one cryo-EM model. There are multiple models that have been published that implicate different residues in receptor activation. The authors should look at these models too.

    7. The authors state that mutations in the TM domain result in GOF. There are many examples of known inactivating mutations in the TMD and several switch residues (with LOF/GOF). This statement needs revising.

    8. The discussion largely re-states the results and doesn't place the research within the context of the current literature. This needs extensive re-writes.

    Minor comments:

    1. Abstract - The first sentence doesn't seem to fit with the rest of the abstract. I suggest removing.

    2. The authors should define 'clade' on its first usage as it is not a common word.

    3. The authors italicise some sentences for unknown reasons. This needs removing.

    4. Figure 2 and Figure 7 need revising as they are too small and/or illegible.

    Significance

    Mutations in the CaSR cause diseases of calcium homeostasis. Specifically inactivating mutations cause disorders of hypercalcemia, while activating mutations result in hypocalcemia. Bircan et al explore the evolutionary conservation of CaSR and try to use these findings to predict whether residues would be associated with hyper/hypocalcemia. This could be useful to researchers focussed on CaSR, particularly clinical geneticists or practising clinicians that may identify genetic variants in the receptor and require tools to predict pathogenicity. However, the manuscript does not fulfil these aims in its current form.

    It is very difficult to follow what the authors have done and what the purpose of the research is. The results section needs more sub-headings as at the moment it is too long, has too many methodological details and is very difficult to follow. The discussion needs completely re-writing as it doesn't really discuss the findings in the context of the current literature. I have tried to outline the areas that need improving the most.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Bircan et al. employ phylogeny-based methods and machine learning to determine positions in the Calcium Sensing Receptor (CaSR) that are specific for this receptor compared to those residues that are important for CaSR and related subfamilies. Using machine learning, the authors predict whether selected mutations in CaSR will lead to loss- or gain-of-function and compare this with experimental results from literature.

    Minor comments:

    • line 13/14: 'there are still gaps in our understanding of its specific residues' - possibly change to 'there are still gaps in our understanding of the specific function of its residues'?

    • line 17/18: 'The analysis revealed exceptional conservation of the CaSR subfamily, with high SDP scores being critical in receptor activation and pathogenicity' - are the SDP scores critical or some aspect of the receptor, i.e. the residues with high SDP scores?

    • lines 42-44: 'L-amino acid binding site at the interdomain cleft of LB1-LB2 and multiple Ca2+ amino acid binding sites on the VFT domain' - Should this be 'Ca2+ binding sites' instead?

    • lines 45-47 'While Ca2+ is the composite agonist for the CaSR, L-amino acids promote receptor activation along with Ca2+, but they are not able to activate the receptor alone'. - Unclear

    • lines 139, 146 'a ML tree' - should be 'an ML tree'

    • line 153 'γ-aminobutyric acid-B receptorsreceptors' - remove 'receptors'

    • line 162-164 'Comparison analysis of branch lengths (Patil, 2021) among common species between CaSR, GPRC6A and taste receptors shows that the CaSR subfamily is significantly more conserved than its closest subfamilies' - could you please give a very short explanation here for the non-specialists?

    • Fig 2A is unfortunately mostly unreadable. I would suggest replacing panel A with (an) alternative panel(s) clearly showing the stated results and moving the tree into the supplementary and/or making it available in a format that can be studied more closely.

    • Fig. 4A, right side. Both the x-axis and the bar colour are labelled 'SDP scores', but they don't agree with each other. Please clarify what is what.

    • Fig. 5 the numbers associated with the colour scales are unfortunately not readable

    • lines 394/5: 'Because CaSR is a highly conserved subfamily, any substitution on the receptor disrupts the function of the receptor and causes either GoF or LoF mutations.' - do you mean that no mutation in CaSR may be neutral?

    • Fig 7 is mentioned earlier than Fig 6.

    • line 516/7 and 532/3: ' we repeated the train-validation-test splitting procedure fifty times' - repetitive

    • Fig 6: what are the features in the bottom panel of 6B?

    Significance

    General assessment: The study uses computational methods to assess the importance of residues in the CaSR for function. The results are compared with the literature, as far as data are available. The study could be made more accessible to non-experts by putting results in context, more explanations in the figure legends and by making sure that the results mentioned in the text can easily be followed by looking at the figures. Another option could be to change subtitles in the results section to summarise the main findings of the section.

    Advance: This study uses phylogeny-based methods to advance our understanding of the role of residues in a GPCR and adds to our pool of techniques available for addressing such questions.

    Audience: The described research should be of interest for researchers working on CaSR, those interested in the evolution of GPCRs, and those studying the impact of point mutations in GPCRs on function and/or human health. I do not have sufficient expertise to evaluate the phylogeny-based methods used in this manuscript. At present the manuscript seems more likely to be of interest to a specialised audience, which could very likely be changed by making the manuscript more accessible to GPCR researchers that don't have a background in phylogeny-based methods.

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    Referee #1

    Evidence, reproducibility and clarity

    This study aims at identifying key positions in the CaSR responsible for its specific properties. For that aim, they aligned all class C GPCR sequences and classified them according to their sequence identity and degree of conservation among orthologs. The final aim was to be able to predict the functional consequences of point mutations, as based on the degree of conservation of each residue within the orthologs, and within homologs. They include some considerations to predict whether the mutations of some of the conserved residues may lead to gain or loss of function. I definitively believe much can be learned from the sequence evolution of a protein, as highly conserved residues mean something. As such, a study like this one is of interest. However, I am far from convinced on their final approach to predict LoF and GoF mutations. Indeed, as far as I understood, they did consider the residue position, and its conservation either in the orthologs only, or also in some homologous sequences. However, they did not consider the type of mutation. In my opinion, a mutation at a given position may well be either LoF or GoF depending on the new residues. One can easily understand that a mutation into Gly or Trp may have very different effect. They also considered that mutations in the 7TM core domain are more prone to generate GoF. This is a statistical view of what could be going on, but by no way this can be included as a criteria to decide on the consequence of the mutation, as both LoF and GoF mutations can be found in this domain. Lastly, there exist a very long list of mutation of the CaSR with known functional consequences. These must be used to validate the authors' approach. In my opinion, validating theyr approach would mean making a long list of what their analysis can prediction with a large number of positions of the CaSR, not considering our actual knowledge along these lines, and then compare they prediction with what is already known. Eventually, for a few predictions for which there is no data supporting either their LoF or GoF effect, these should simply be tested to give the readers an expectation on the viability of their approach.

    Significance

    I must clearly state that I not a specialist of the bioinformatic approaches used in this study, and as such cannot judge all these aspects of the work presented in this story. However, I am also far from convinced with the analysis and the conclusions, that, in my opinion, are not in line with my views on this topic. One key aspect is that the authors only considered mutations at specific positions in the CaSR, with the aim to predict their loss of gain of function effect. However, in my opinion, such a functional consequence not only depends on the residue mutated, but also into which residue it is converted. I cannot see that a mutation into Ala, Gly or TRP could have the same effect. As such, I cannot recommend acceptance of this paper.

  5. Version published to 10.1101/2023.06.11.544489v2 on bioRxiv
  6. Version published to 10.1101/2023.06.11.544489v1 on bioRxiv