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

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 plasma membrane cholesterol depletion is misrepresented: Was it ever proven that MβCD inserts itself inside the membrane and stays there?

2) How was AUC calculated? What’s does it tell us? How were the error bars derived?

3) “Both WT and L149P receptors displayed minimal endocytosis from non-raft (CAAX) microdomains and their magnitudes did not differ.”

There seems to be an explanation missing. How does the change in CAAX and MyrPalm localization with the WT or L149P receptors translate to endocytosis?

4) “...we stimulated bBRET sensor-expressing cells with a saturating concentration of ghrelin (1μM) and measured the change in CAAX, MyrPalm, and 2xFYVE localization with the WT or L149P receptors over two-hours.”

The corresponding graphs are named misleadingly. This doesn’t sound like hGhrelin kinetics, but rather like changes in receptor localization upon agonist activation.

5) “Conversely, both the rate and magnitude of ghrelin-induced L149P endosomal translocation was markedly increased relative to WT (Figure 1m), consistent more with a Class B-like trafficking profile.”

The highlighted part is an interpretation that a) belongs in the discussion, b) demands an example, and c) merits a citation.

6) “Notably, WT receptor endocytosis from lipid rafts was reduced to a level similar to the L149P mutant under cholesterol-replete (vehicle-treated) conditions.”

How were those cholesterol-replete conditions achieved? Where is the protocol for that? Why don’t these results reflect the basal state? Or is the word “replete” misused in a way that it simply signifies the native cholesterol state without added MβCD, and not the repletion state that follows depletion?

7) “These results together support that reduced basal βarr2 recruitment (see Figure 2i) and reduced agonist βarr1/2 efficacy (see Figures 2c,g and 2j,n) at the L149P mutant are likely related to its PM expression bias and/or distinct endosomal trafficking profile (see Figure 1).”

This is a big conclusion to make and therefore belongs in the discussion supported by further explanation.

8) “...we did observe a mild reduction in basal βarr1 recruitment to the MβCD-pretreated L149P relative to the vehicle-pretreated WT receptor (Figure 2w).”

This is not what the graph shows; the bar diagram shows four columns for the four samples with the same intensity. Is the observed “mild reduction” indeed biochemically/biologically relevant?

9) “In contrast to βarr1, MβCD pretreatment decreased basal βarr2 recruitment to the WT receptor but did not affect L149P (Figure 2z), suggesting that reduced L149P-βarr2 recruitment is membrane cholesterol-dependent.”

This makes no sense. If the basal βarr2 recruitment of L149P was not affected by the MβCD pretreatment, why would it be cholesterol-dependent?

10) “...although the effects were larger for the L149P mutant (Figures 2aa-bb) and similar in magnitude to βarr1 (see Figures 2x-y).”

Would be easier to see if the numbers were provided, this way it’s hard to visualize the comparative relative difference (or similarity) between the two. I suggest adding the Emax and or EC50 values directly in the graph or in the text, instead of in the supplementary table, to avoid the confusion.

11) “Notably, MβCD pretreatment reduced ghrelin Emax at the WT receptor to a level statistically similar to the vehicle-pretreated L149P, suggesting that the moderate reduction in βarr2 recruitment efficacy to L149P at baseline (~35%) is cholesterol-dependent.”

Reference the relevant graph - is it 2.bb? If so, add the statistical relevance between the samples, although it is not an ideal comparison, since more than one parameter was changed between the two.

12) “Our results also suggest that the βarr1/2 recruitment characteristics of L149P are, at least partially, related to its differential PM expression bias, endosomal trafficking, and association with membrane cholesterol (see Figure 1).

This sentence is almost an exact copy of the sentence in the point seven (see above) - but it fits here better. The comment from point seven still holds though.

Part 3: L149P-mediated inhibition of G protein signaling is Gα subtype-specific and cholesterol-independent.

and

Part 4: βarr recruitment to the L149P mutant requires GRK2/3-mediated phosphorylation but does not require G protein-dependent PKC or GRK2/3 activation.

and

Part 5: The L149P mutation switches the GRK recruitment profile of the GHSR.

1) Multiple sensors are introduced in these sections (“BRET-based mini-Gq Sensor” (Part 3), “BRET-based receptor-GRK6 YFP recruitment assay” (Part 4) and “BRET-based receptor-GRK5/6YFP recruitment assay” (Part 5)). These experiments are relatively small and often lead to speculative statements, with brief attention given before moving on to the next assay. We suggest to extend the explanation on the relevance of these assays.

2) “Constitutive activity” is introduced in the results section and represents a recurring theme throughout these last three parts of the Results section. However, it is neither introduced in the Introduction nor consistently described in the Methods section, where it is only mentioned once (for Figures 3–5), although being addressed in Figures 1 and 2.

The authors should clarify what they mean with “constitutive activity”.

3) As already noted in the General Comments, the terms “constitutive”, “basal”, “activity”, “efficacy” and “dissociation” are used inconsistently and confusing, especially in regard to the receptor and the different G-proteins. For example:

“The GHSR exhibits remarkably high constitutive Gα_q activity (>50% ghrelin E_max ³³); thus, to provide a direct measure of constitutive activity, we included an empty vector (EV) condition to normalize basal receptor responses (i.e., derive Δ net BRET relative to EV). Constitutive Gα_q dissociation from L149P was reduced by >70% relative to the WT receptor (Figure 3b).”

In this sentence the authors talk about directly measuring GHSR constitutive activity, but then conclude in the same sentence about constitutive Gα_q dissociation. This is, language-wise, confusing, even more since the manuscript is lacking clear definitions of the used terminology. This terminology problem is particularly evident in this section of the manuscript.

Thus, the authors should clarify all the above-mentioned terms, and especially the distinction between constitutive and basal activity, and use the latter two accordingly throughout the text.

4) The manuscript introduces inhibitors and compounds that are not mentioned afterwards in the Discussion, namely inhibitor Go6983 and compound 101. We suggest revising this, mentioning these compounds in the Discussion section.

5) Figure 4 contains the highest number of graphs in one figure, and is thus the most overwhelming figure of the manuscript. Figures 4.a, 4.h, 4.o, 4.v and 4.cc repeat in turn the same explanatory drawing of the respective assay - the same holds in Figures 3 and 5. As long as the experimental setup is identical, a single comparative presentation would improve clarity and already eliminate some pictures from the overcrowded figures.

6) Additionally, supplementary figures are mentioned, which add to the already extensive set of main figures. This further increases the amount of data the reader needs to integrate. For example:

“...at the WT receptor was reduced moderately by PTX (Supplementary Figure 2a), while L149P-βarr1 recruitment was unaffected (Supplementary Figures 2b-c). For βarr2, ghrelin potency and efficacy were not affected by PTX at either receptor (Supplementary Figure 2d-f).”

“… the effect did not reach significance for ΔGRK5/6 and statistical comparisons were precluded for ΔGRK2/3 due to high background and poor curve fit.”

We strongly suggest that the authors shift consistently all graphs showing negligible and/or non significant differences between samples to the SI file, instead of keeping some of the graphs in the mani text, and others in the SI. This would give a clear graph selection rationale.

7) As already noted for parts 1 and 2, some of the statements appear interpretative and may be better suited for the Discussion section, especially the concluding paragraphs of each part, where the findings are summed up and conclusions across multiple experiments are drawn. For example:

“Thus, G-alpha i/o signaling does not likely account for G-beta/gamma-independent beta-arrestin or GRK2 recruitment to L149P”.

“We speculate that the increase in WT-βarr2 recruitment in ΔGRK5/6 cells is due to a compensatory upregulation of GRK2/3, as observed elsewhere26.”

“Collectively, the results…”, “...the results in Figure 5 support that…”, “...selective GRK5/6-dependent βarr recruitment to L149P might be due to enhanced GRK6 ‘precoupling’.”

Discussion

1) The Discussion would benefit from clearer links to the corresponding results and figures. Several statements are presented without explicit literature citations or figures/results references, making it difficult to assess which experimental data support them. This conveys the impression of a philosophical debate rather than a proper scientific discussion. For guidance, please take example from the 4th paragraph of the discussion - that is written well. Furthermore, Figure 6 nicely summarizes the key findings of the study. Nevertheless, the link between the experiments and the proposed interpretation could be made more explicit. Structuring the discussion around this figure could help clarify these connections for the reader.

2) There is a possible helix-disrupting effect of the L149P mutation on ICL2 suggested; however, these conclusions are not clearly linked to specific results or figures.

3) There are conclusions drawn that aren’t supported neither by the presented results nor literature citations. Examples:

“These results suggest that distinct ICL3 or C-tail phosphorylation patterns (‘barcodes’) could account for distinct trafficking of the L149P mutant.”

This reference to ICL3 as a connection for L149P–GRK6 precoupling is not sufficiently justified, as the manuscript previously discusses only ICL2. Clarifying the role of ICL3 in this context would improve coherence.

“In contrast, the L149P mutant displayed robust agonist-directed endosomal translocation in a Class B-like manner.“

“...it is possible that the WT-GHSR internalizes, in part, via caveolin-dependent pathways while L149P preferentially utilizes clathrin-mediated endocytosis.”

“These results support well-established ideas suggesting that (i) ICL2 conformations critically regulate transducer coupling selectivity, (ii) G protein- and βarr-coupled GPCRs can adopt distinct conformational states, and (iii) βarr recruitment is dependent on receptor subcellular distribution and microdomain partitioning [location bias].”

“Thus, enhanced GRK6 pre-coupling could ‘prime’ L149P conformations for βarr coupling, desensitization, and Class B-like receptor trafficking.”

4) There is a repeating concept of conformational states occurring. However, this study does not focus on the conformational landscape of the receptor but rather its localization, downstream signalling and internalization. Furthermore, regarding, for example, changes in downstream signalling, the mutation may as well cause dysregulation due to changes in sequence specificity, ICL2 specific conformational changes that don’t translate to the rest of the receptor etc. Therefore, from this research alone, one cannot draw conclusions about receptor conformations. If conformational states are to be mentioned regardless, appropriate literature needs to be cited, and the tentative nature of the conclusions needs to be highlighted. Examples:

“...a natural missense mutation in Leu149ICL2 induces conformational states unfavorable to cholesterol-enriched PM microdomains and G protein activation,...”

“These results support well-established ideas suggesting that (i) ICL2 conformations critically regulate transducer coupling selectivity, (ii) G protein- and βarr-coupled GPCRs can adopt distinct conformational states…”

5) “In a physiological context, we speculate that the L149P mutation might reduce energy intake, body weight, and growth hormone secretion...”

As we already noted for the Introduction, there exist other, less rare GHSR mutants that exhibit reduced or inhibited constitutive activity, where the physiological context is better known. A reference to those would reinforce the above statement.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.