PREreview of "Rare variant in intracellular loop-2 of the ghrelin receptor reveals novel mechanisms of GPCR biased signaling and trafficking"

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/18630662. Balfe et al. report on the in vitro biochemical investigation of an ultra-rare physiological variant of the growth hormone secretagogue receptor (GHSR), namely the point mutation L149P at the intracellular loop 2 (ICL2) of the receptor. The authors carried out a wide variety of biochemical assays to characterise the downstream signalling pathways of L149P mutant, in comparison to the wild-type receptor, which lead them to identify specific biased signalling profiles, namely β-arrestin biased signalling, and biased intracellular trafficking to G-protein kinases 2, 3, 5 and 6. The manuscript sets out to be an important contribution to the field of biased signalling in GPCRs research, however major revisions are strongly suggested, before further evaluation. General Comments ● Several key terms are used in a seemingly random/interchangeable fashion throughout the manuscript, especially "basal activity" and "constitutive activity." Furthermore, they are used in reference to receptor and G-proteins alike, without further explanation. This makes it difficult to follow what the authors mean in each specific paragraph, often hampering a comprehensive understanding of the fine-tuned signalling properties of the L149P mutant. We suggest an overall revision of such terminology, making it consistent throughout the manuscript. ● The authors present a huge amount of graphs in the main text (>100), distributed in merely 5 figures, which massively overloads the Results section. We suggest a thorough revision of all graphs of each figure, prioritising the most striking variations/differences between wild-type and mutant receptor. Introduction Overall, the Introduction is well written and provides a clear overview of the key topics and the background knowledge required for this study. However, some aspects can still be improved: 1) Regarding the biased signaling, a scheme showing the different pathways and/or definitions would have been helpful to understand them better "..(biased ligands)......(biased receptors).....(system bias)......(location bias)... . 2) Additionally, the authors describe the preferred G-protein coupling of GHSR. Later in the manuscript, they investigate whether this coupling is altered by mutation of ICL2 (L149P). Including a schematic overview of G-protein coupling and recruitment in the Introduction would improve clarity about the current state of knowledge. So maybe put the first Part of Figure 6 (WT) already in the Introduction part. 3) While the terms "basal activity" and "constitutive activity" are used in the Methods and Results sections, the Introduction does not provide background on the high constitutive activity of GHSR, nor does it mention the A204E mutant in which this activity is abolished. Including this information in the Introduction would improve clarity and context. It could be helpful to mention this and explain the term, especially when measuring basal activity. 4) We suggest moving Figure 1a from the Results section to the Introduction. As the authors already describe the L149P mutant in the Introduction, introducing the corresponding figure at this stage would provide earlier context. 5) The manuscript would benefit from a clearer articulation of the study's motivation and overall aim. In particular, the following sentence could be rewritten to more explicitly convey the objective of the study: "In this study, we identify and characterize an ultra-rare, coding variant in intracellular loop-2 (ICL2) of the GHSR, Leu149Pro (L149P). The Leu149ICL2 site (Ballesteros-Weinstein number 34.51) is +7 amino acids downstream of the highly conserved E/DRY motif, and we have shown previously that nonnatural GHSRLeu149 mutations regulate receptor bias and trafficking 10, 14" Replace with e.g. "Previous work from our group has demonstrated that non-natural mutations at GHSR Leu149 influence receptor bias and trafficking. Building on these findings, we examine an ultra-rare coding variant in intracellular loop 2 (ICL2) of GHSR, Leu149Pro (L149P). Leu149^ICL2 (Ballesteros–Weinstein 34.51) is positioned seven residues downstream of the conserved E/DRY motif." Methods The methods are easily comprehensible. However, 1) Some parts are lacking information necessary to accurately reproduce the experiments: "The N-terminally mVenus-tagged human βarr1 (hβarr1) and βarr2 (hβarr2) plasmids were a gift from Dr. Lauren Slosky (University of Minnesota ;Minneapolis, MN)." "The mini-Gq plasmid was a gift from Dr. Nevin Lambert (Augusta University; Augusta, GA)." In some instances, even when the plasmid was a gift, the plasmid was categorized by their Addgene number. If such a number is unavailable, at least the plasmid name, the restriction sites, through which the construct was introduced, and the promotor, through which the construct is expressed, should be specified, as well as any potential antibiotic resistances in case they were used for positive selection. "The bBRET sensors mVenus-CAAX 50, (MyrPalm)-mVenus 6 , and 2xFYVE-mVenus 51 have been described previously" That is not true. Under citation number 50, there is a CAAX-mKO (Kusabira orange) control mentioned, but also not described. We strongly suggest to carefully check the provided literature citations and correct accordingly. 2) The statistical analysis part is very minimalistic: There are many graphs that are not derived using non-linear regression (e.g. AUC charts and Emax charts). How were they obtained? What do the error bars signify in those cases? The details of the statistical analyses are given in the supplementary tables, which makes it very complicated and dispersive, since the reader needs to constantly move back and forth from the main manuscript to the SI text. We suggest adding the details of the performed statistical tests in the Methods section, and the legend(s) of the relevant statistical values (e.g. t-/P-/F-values) in the respective figure captions. 3) The MβCD treatment/cholesterol depletion is not covered in the Methods: What was the MβCD concentration used in cholesterol depletion? What was the cell amount? What buffers were used and how long was the incubation with MβCD? What % cholesterol depletion was achieved and how was it measured? Was the cholesterol depletion performed on plated cells or cells in suspension? Following cholesterol depletion, what was the cell viability and how was it assessed? How was the option of cells having entered a pre-apoptotic state prior to the measurements excluded? How was the effect of MβCD on the activity of the receptor excluded? Given the promiscuous nature of MβCD, how was the chelation and therefore the effect of non-target lipids on the GHSR excluded? What was the measurement window after the cholesterol depletion? Results There are 112 graphs presented in 5 figures, making them hardly comprehensible and overseeable. Overall, this appears to prioritize quantity of experiments over depth of analysis. Combined with the wide range of topics covered, this gives the impression of several separate studies in one manuscript. Here are some improvement suggestions. The authors are however strongly suggested to check and revise each graph and figure, as we already pointed out in the general comments. 1) Break up the Figures in a way that the relevant graphs fall directly under the related paragraphs (for example, graphs 1.a and 1.b under the third paragraph of the 1st chapter of results, graphs 1.e - 1.g under the 4th paragraph of the 1st chapter of results etc). 2) See whether some data of different graphs can be presented in the same graph. Furthermore, reevaluate the relevance of detailed displays, only keep the graphs that enforce your narrative (for example, the graphs 1.k - 1.m can do without the raw data since it's not discussed either - that can be shifted to the SI. Keep only the AOC graphs, which could be combined into one for better overview). Finally, several times the manuscript states that "there was no difference in constitutive or ghrelin-stimulated responses." It is unclear why the corresponding figures are included, as they do not add new or changing information. 3) Give the graphs titles according to the experiment conducted or the result obtained, not just the experimental target (for example, 1.h portrays basal localization in early endosomes, not basal early endosomes). This should give a clearer overview of what is displayed, especially with the abundance of graphs. 4) The repetitive use of the word "vehicle" on many different occasions makes it unclear what exact vehicle this is (Is it always the same? Then why not just call it by its name? Furthermore, when the word vehicle is not used, does it mean that it was absent from the experiment?) and what it signifies - control conditions, conditions without pre-treatment, transient transfection with a different agent… If the vehicle signifies negative control, rather call it "negative control." 5) The error bars are not visible on many of the dot plots. Smaller dots would reduce this issue. 6) The statistical significance in some of the graphs is drawn between samples that cannot be directly compared, which is any 2 experiments where more than 1 parameter was changed (for example, at graph 2.y between MβCD pre-treated WT and vehicle pre-treated L149P, at graph 2.w etc). Part 1: The ultra-rare, natural variant — GHSRL149P — exhibits altered subcellular distribution and trafficking in a cholesterol-dependent manner. and Part 2: Basal and agonist-stimulated βarr1/2 recruitment to the L149P mutant is dynamin- and cholesterol-dependent. 1) Scheme 1d: Several parts are unmarked; What is cholesterol? What are the orange strings? What are the two orange circles on top of MβCD? Furthermore, the MβCD mode of