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Characterization of Trileucine Motif in the C-Terminus of the Equine Lutropin/Choriogonadotropin Receptor

Curr. Issues Mol. Biol. 2024, 46(11), 13179-13192; https://doi.org/10.3390/cimb46110786

by Sang-Gwon Kim¹, Munkhzaya Byambaragchaa², Sei Hyen Park¹, Ha-Rin Jeong³, Jae-Hyek Park³, Myung-Hum Park⁴, Myung-Hwa Kang⁵ and Kwan-Sik Min^1,2,3,*

Reviewer 1: Anonymous

Reviewer 2:

Konda Mani Saravanan

Reviewer 3: Anonymous

Curr. Issues Mol. Biol. 2024, 46(11), 13179-13192; https://doi.org/10.3390/cimb46110786

Submission received: 17 October 2024 / Revised: 13 November 2024 / Accepted: 16 November 2024 / Published: 18 November 2024

(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript set to test an interesting hypothesis and the results are nicely written up.

My major concern is Figure 2. I think WB is not suited for quantitation. It would be much better to just use the ELISA data. WB data are just for showing the sizes of the receptor. Panel C is not listed in the figure legend. The mature and immature bands can also be highlighted.

Minor points:

1. Line 16: lutropin is misspelled.

2. Line 171: add “with” at the end of the line.

3. 3. Line 216: no need to introduce wild-type (WT)? Same as HEK in Line 217.

4. What is the loading control in Fig. 6?

Author Response

Reviewer 1
Comments and Suggestions for Authors
This manuscript set to test an interesting hypothesis and the results are nicely written up.
My major concern is Figure 2. I think WB is not suited for quantitation. It would be much
better to just use the ELISA data. WB data are just for showing the sizes of the receptor.
Panel C is not listed in the figure legend. The mature and immature bands can also be
highlighted.
→inserted “C) Western blots of whole-cell lysates from transfection were probed with
antibody to VSVG-tag as shown” the Panel C list in the Figure 2.
Minor points:
1. Line 16: lutropin is misspelled.
→changed luteotropin/chorionic gonadotropin to lutropin.
2. Line 171: add “with” at the end of the line.
→added with rec-eCG at the Line 171.
3. Line 216: no need to introduce wild-type (WT)? Same as HEK in Line 217.
→we changed “wild-type (WT) and human embryonic Kidney” to WT and HEK” as reviewer’s
comments.
4. What is the loading control in Fig. 6?
→total ERK result was inserted in Panel B.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The tri-leucine motif is present in the C-terminus of LH/CGRs, and this study examined the function of these three leucine residues by site-directed mutagenesis. The results indicated that replacing alanine for Leu636Leu637Leu638 did not entirely diminish cAMP responsiveness, cell surface expression, or pERK1/2 activation. The authors conclude that although individual leucine residues of the trileucine motif do not impact cAMP responsiveness, the complete pattern is essential for receptor trafficking and signaling, particularly affecting PKA and pERK1/2 pathways.

I believe the writing quality is exceptional; nonetheless, the comprehensive research on characterization of Tri-Leucine Motif in the C-terminus of the Equine Lutropin/Choriogonadotropin Receptor is really intriguing. The figures and tables presented in the paper provide an avenue for future research on the receptor. Despite the authors' presentation of key literature in the introduction, the results presented are significant and contribute to the current body of knowledge. After considering a few of my comments, I endorse the manuscript for publication.

The Introduction section is poorly referenced. The authors miss relevant key citations.

More details about sequence characteristics of Choriogonadotropin Receptor should be presented in the Introduction section.

The methods section has high text similarity evidence from the plagiarism report. The text should be rewritten.

The overall methodology adopted in the paper can be presented as a flow diagram to ease the readers.

Limited discussion of the limitations of the current study. The authors should discuss advantages and disadvantages of the current study.

The conclusion could be strengthened by briefly discussing the practical challenges in elucidating the mechanisms underlying agonist-receptor complex-mediated downstream signaling.

Author Response

Reviewer 2

Comments and Suggestions for Authors

The Introduction section is poorly referenced. The authors miss relevant key citations. More details about sequence characteristics of Choriogonadotropin Receptor should be presented in the Introduction section.

→We inserted “in rat LH/CGR (rLH/CGR) and hLH/CGR [29]. The deleucine-based motifs in rLH/CGR inhibit internalization for impairment in the binding of the rLH/CGR to the endogenous nonvisual arrestins. Among them, several LH/CGRs have been shown to be located leucine motif in their C-terminal tail as shown in Figure 1.” At the introduction section.

→These studies were specifically designed to determine whether trilecuine motif affects the functional properities of the eLH/CGR.

The methods section has high text similarity evidence from the plagiarism report. The text should be rewritten.

→Material and Method section were partly rewritten by reviewer’s comment.

The overall methodology adopted in the paper can be presented as a flow diagram to ease the readers.

→We considered this points by reviewer’s comment, but we decided that we wouldn’t have to do it.

Limited discussion of the limitations of the current study. The authors should discuss advantages and disadvantages of the current study.

→ The results clearly demonstrate that each leucine residues were no affect on the properties of eLH/CGR including PKA signal transduction, loss of cell surface receptor, and phospho-ERK1/2. eLH/CGR-AAA mutant, however, specifically influences on the cAMP signaling including Rmax response. Nevertheless, we couldn’t in a clear way suggest the correlation between cAMP signaling and phospho-ERK1/2 in the presented study. Although this study did not fully explore the relationship, the highly conserved regions, such as the trileucine and palmitoylation motifs in the C-terminus of eLH/CGR, likely participate in specific signaling pathways, such as receptor internalization. Therefore the conserved regions in C-terminal of LH/CGRs should be systematically identified to which signals are pathway. We propose that the trileucine motif plays a role in downstream signaling, possibly through cooperative interactions with other motifs.

The conclusion could be strengthened by briefly discussing the practical challenges in elucidating the mechanisms underlying agonist-receptor complex-mediated downstream signaling.

→Conclusion sections are rechecked by reviewer’s comment and shorted.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments are in the separated file.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

The authors, Sang-Gwon Kim, Munkhzaya Byambaragchaa, Sei Hyen Park, Ha-Rin Jeong, Jae-Hyek Park, Myung-Hum Park and Myung-Hwa Kang and Kwan-Sik Min provide the functional roles of trileucine at the C-terminus of the equine LH/CGR using multiple cellular assays with site-directed mutagenesis in their manuscript titled Characterization of Tri-Leucine Motif in the C-terminus of the Equine Lutropin/Choriogonadotropin Receptor.

Here are major and minor comments on the authors’ manuscript.

Major points

Abstract

Could authors emphasize a little more about the research motivations/rationales for why LH/CGR is interesting for authors, why the equine species is important, and why the tri-leucine motif is important? The authors’ description for intracellular signaling is very clear.

For example, about the mutations on this site were found in horses or other species, or trafficking disturbance of this receptor causes hormonal imbalance in vivo in some species.

→We inserted “in rat LH/CGR (rLH/CGR) and hLH/CGR [29]. The deleucine-based motifs in rLH/CGR inhibit internalization for impairment in the binding of the rLH/CGR to the endogenous nonvisual arrestins.” In the Introduction Section. However, we did not suggest in the Abstract section due to a lack of space.

(2)

Section 2.3.

Are there technical or cell-specific reasons for using the different cell lines to evaluate the GPCR signaling?

→We used to CHO-K 1 cells to analysis cAMP responsiveness. However, we usually use HEK 293 cells for phosphor-ERK1/2 activation. The endogeneous expressions of beta-arrestin 1 and beta arrestin 2 in the HEK 293 cells were more expression than CHO-K1 cells.

(3-1)

L133: “CHO cells were used for cAMP analysis at 48 h post-transfection” L244-L245: “CHO-K1 cells transiently transfected with eLH/CGRs were seeded in 384-well plates (10,000 cells/well) 24 h post-transfection” If this description indicates the same experiment and the reviewer might not have overlooked other descriptions, the time point of this experiment should be same.

→changed “48 h” at the Figure 3 legend.

(3-2)

L169-L170: “Cells were seeded into 96-well plates (1 x 104 cells) at 24 h post-transfection” Why is post-transfection duration different from the one for the cAMP assay? Readers would assume that the transfected receptor expression levels might be different.

→cAMP analysis was conducted at 48 hrs post-transfection. This protocol does not include the washing step. However, the cells were seeded before 24 h at the cell surface loss experiment, and next day it was conducted. Thus all experiments was fit at 48 hrs post-transfection.

(4) Figure 2.

Related to (3), what is the timepoint of this experiment after transfection for HEK293 cells? There is no description unless the reviewer might have overlooked. The reviewer thinks that considering the purpose of the authors’ project assay timings regarding (3) and (4) should be the same.

→We indicated that the timepoint at the 2.6 Agonist-induced cell surface loss of equine LH/CGR s of Materials and Method Section. At 0 time, the cell surface expression was set to 100% in untreated cells.

(5)

Figure 2. What is the authors’ explanation/interpretation for the different results in ELISA and WB experiments to detect the same cell surface protein? L288-L290: “A) Enzyme-linked immunosorbent assay (ELISA) and B) Western blotting were used to assess surface expression of eLH/CGRs. According to this description, both assays seem to assess surface expression of the same protein. The reviewer could not fully understand the explanation in the discussion section. Or was the lysate for the western blotting whole-cell lysate?

→In the discussion section, we inserted that “as shown in western blot results. However, ELISA’s results are taken to be more accurate than those of the western blot.”

→In western blot analysis, whole-cell lysates were used.

(6) Table 1 and Table 2.

Are statistical tests and interpretations based on them not applicable here?

→Statistical results are inserted in Table 1 and Table 2.

(7) L266: “cAMP responsiveness” Is Rmax reduction by single amino acid substitutions not included in “cAMP responsiveness”?

→At the presented results, we do not find the differences of EC₅₀ level spite of Rmax reduction at the two mutants of eLH/CGR-LAL and -LLA. Thus, we suggest that the differences resulted in the leucine motif of amino acids.

(8) Figure 6.

(8-1) WT reduction is appropriately 20% at 15 min in Figure 6C, but it is approximately 55% in Figure 6D at 15 min, which is not a negligible difference because the purpose of the experiments is same. Are there any reliable control/housekeeping proteins?

→inserted “total ERK’s results” by reviewer’s comment.

(8-2) Same proteins are measured, but locational relationships pERK1/2 and ladders are obviously different in Figures 6A and 6B, which gives an unreliable impression of WB experiments to readers.

→We conducted many times for this experiment. We also found that WT phosphor-ERK1/2 bands were differences between Figure 6A and 6B’ results at the 10 min post treatment.

(8-3)

At 0 min, there seems to be almost no pERK1/2 protein on the blot in a nontreated condition. How about total ERK1/2?

→inserted “total ERK’s results” by reviewer’s comment.

(8-4) Please indicate pERK1/2 and total ERK with molecular weights for ladders.

→inserted “molecular weight” by reviewer’s comment.

(9)

L428: “funding acquisition, M.-K.S.” Does the contribution only to funding acquisition and read the manuscript meet the authorship criterion in the CIMB journal?

→deleted “funding acquisition” by reviewer’s comment.

Minor points

(1)There is “tri-leucine” and “trileucine”. There is usage of “pERK1/2” and “phospho-ERK1/2”.

→changed “tri-leucine and pERK1/2” to trileucine and phosphor-ERK1/2” in the all sentence

(2) L108: “equine LH/CGR-wt” Although it is already abbreviated as “the full-length eLH/CGR-wild type (eLH/CGR-wt)” at L102, “equine LH/CGR-wt” is used many times in the following manuscript.

→changed “tri-leucine and pERK1/2” to trileucine and phosphor-ERK1/2”

(3) Figure 1C. Does “Δ” mean deletions?

→deleted “Δ” at the Figure 1.

(4) L217: “human embryonic kidney (HEK) 293 cells” It is already abbreviated at L84 for the first time and other parts.