Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1

Xiaohua Wang; James Lundblad; Stephen M. Smith

doi:10.1371/journal.pone.0266993

Peer Review History

Original SubmissionMarch 30, 2022
25 May 2022 Decision Letter - Xiangming Zha, Editor PONE-D-22-09433Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a mouse model of Familial Hypocalciuric Hypercalcemia Type 1PLOS ONE Dear Dr. Smith, 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. Please submit your revised manuscript by Jul 09 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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If consent was waived for your study, please include this information in your statement as well. 6. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors studied a model of familial hypocalciuric hypercalcemia type 1. The model consists of transfecting and co-transfecting the wild type (WT) and mutated (mCaSR) in human embryonic kidney (HEK) cells. In the abstract, the authors claim that mCaSR homodimer is localized to the cytoplasm and lack function, the functional WT CaSR homodimer is localized to the membrane, and the heterodimer is localized to the membrane but has decreased sensitivity to extracellular Ca2+ ions. The data supports the paper’s conclusions as far as the results in the HEK cell line. The data in Figure 5 with different transfection percentage of wild type and mutated CaSR would be stronger if the author demonstrated that the protein levels of CaSR and mCaSR correspond to the transfected DNA levels, as it is often not the case (for example, mutants are often targeted for degradation more than wild type proteins). The more precise title to the paper would be “Reduced affinity of Ca2+-sensing wild type-mutant heterodimers and reduced mutant homodimer trafficking combine to impair Ca2+ sensing by Ca2+-sensing receptor with the exon 5-deleted mutation”, as the current title claims results in the mouse model though data were obtained in a cell line, and the existence of the same mechanism in the mouse model still needs to be shown. The wording in the abstract and the conclusions also have to be adjusted according to what was actually shown in the paper. Minor points: Line 33: a space between “expressing” and “mutant” is missing. Line 78: “in”? Line 87: “to” is missing. Line 109: “Human”. Line 113: full stop is missing. Line 117: extra “in”. Line 207: Is F-G panels are another example similar to D-E? Reviewer #2: The authors successfully showed that exon 5-deleted mutant CaSR, which causes NSHPT in mice when homozygously expressed, showed no functional cell surface expression and lost its ability to sense [Ca2+]o change in HEK 293 cells. They also showed that mutant CaSR trafficking to the cell surface could be rescued by heterodimerization with WT CaSR. The result looks promising, and I hope they will pursue their findings in animal studies to combat CaSR mutation-related diseases. Minor correction: 1. Page 11, line 205-207: Clarify figure 1F,G. 2. Figure 1: D-E and F-G should explain clearly in the figure legend. ****** 6. 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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Revision 1
16 Jun 2022 Author Response I write to resubmit our manuscript [PONE-D-22-09433] after responding to the reviewers and modifying in accordance with the journal requirements. We thank the reviewers for their careful reading of our manuscript and believe that the document has been improved after their helpful reviews. Please find a point by point below. In addition, we have altered the title, acknowledgements, and figures in accordance with the suggestions. Please do not change the Funding Statement. The unadjusted blot images are submitted as a supplementary information file. Patients and animals were not utilized in this study which was approved by the OHSU IACUC and OHSU safety committee. Detailed response to Reviewers: Reviewer #1: The authors studied a model of familial hypocalciuric hypercalcemia type 1. The model consists of transfecting and co-transfecting the wild type (WT) and mutated (mCaSR) in human embryonic kidney (HEK) cells. In the abstract, the authors claim that mCaSR homodimer is localized to the cytoplasm and lack function, the functional WT CaSR homodimer is localized to the membrane, and the heterodimer is localized to the membrane but has decreased sensitivity to extracellular Ca2+ ions. The data supports the paper’s conclusions as far as the results in the HEK cell line. The data in Figure 5 with different transfection percentage of wild type and mutated CaSR would be stronger if the author demonstrated that the protein levels of CaSR and mCaSR correspond to the transfected DNA levels, as it is often not the case (for example, mutants are often targeted for degradation more than wild type proteins). The reliable direct measurement of protein levels in single cells is difficult if one is trying to assay only that reaching the plasma membrane. Measurements of protein in populations of cells may result in considerable variance due to inclusion of transfected and untransfected cells. We attempted to measure the protein levels at the membrane by tagging the WT or mCaSR with EGFP and employing the fluorescence as a measure of the amount of protein reaching the membrane (Fig 5 C). We were able to do this as using 25% of the WT DNA resulted in ~25% of the EGFP at the membrane. A similar level of fluorescence (~25%) was detected at the membrane when half of the DNA was mCaSR-EGFP and half was CaSR-myc, consistent with the expectation shown in the model (Fig 5D, 3rd row). The small amounts of fluorescence measured when DNA for mCaSR-EGFP alone was used was due to scattered light from protein confined to the cytoplasm (see Fig 1I). The more precise title to the paper would be “Reduced affinity of Ca2+-sensing wild type-mutant heterodimers and reduced mutant homodimer trafficking combine to impair Ca2+ sensing by Ca2+-sensing receptor with the exon 5-deleted mutation”, as the current title claims results in the mouse model though data were obtained in a cell line, and the existence of the same mechanism in the mouse model still needs to be shown. The wording in the abstract and the conclusions also have to be adjusted according to what was actually shown in the paper. We agree that the title was misleading and have changed it to: Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1. Minor points: Line 33: a space between “expressing” and “mutant” is missing. Corrected. Line 78: “in”? Corrected. Line 87: “to” is missing. Corrected. Line 109: “Human”. Corrected. Line 113: full stop is missing. Corrected. Line 117: extra “in”. Corrected. Line 207: Is F-G panels are another example similar to D-E? The F-G panels are examples similar to D-E but provide examples of the variability across a culture. I have modified the legend to make this clear. Reviewer #2: The authors successfully showed that exon 5-deleted mutant CaSR, which causes NSHPT in mice when homozygously expressed, showed no functional cell surface expression and lost its ability to sense [Ca2+]o change in HEK 293 cells. They also showed that mutant CaSR trafficking to the cell surface could be rescued by heterodimerization with WT CaSR. The result looks promising, and I hope they will pursue their findings in animal studies to combat CaSR mutation-related diseases. We are pleased that you found our results promising. Minor correction: 1. Page 11, line 205-207: Clarify figure 1F,G. The F-G panels are examples similar to D-E but provide examples of the variability across a culture. I have modified the text to include these panels and the legend to make this clear. 2. Figure 1: D-E and F-G should explain clearly in the figure legend. Please see response to question 1. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0266993.r002
1 Jul 2022 Decision Letter - Xiangming Zha, Editor Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1 PONE-D-22-09433R1 Dear Dr. Smith, 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. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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. Kind regards, Xiangming Zha, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0266993.r003
Formally Accepted
11 Jul 2022 Acceptance Letter - Xiangming Zha, Editor PONE-D-22-09433R1 Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1 Dear Dr. Smith: 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Xiangming Zha Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0266993.r004

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