Regulation of pulmonary surfactant by the adhesion GPCR GPR116/ADGRF5 requires a tethered agonist-mediated activation mechanism

  1. James P Bridges
  2. Caterina Safina
  3. Bernard Pirard
  4. Kari Brown
  5. Alyssa Filuta
  6. Ravichandran Panchanathan
  7. Rochdi Bouhelal
  8. Nicole Reymann
  9. Sejal Patel
  10. Klaus Seuwen
  11. William E Miller
  12. Marie-Gabrielle Ludwig

  • Curated by eLife

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    Evaluation Summary:

    Bridges et al., present a timely study on an adhesion G-protein coupled receptor known as GPR1116, which is an important regulator of lung function. The authors performed extensive mutagenesis and functional studies to characterize the structural determinants controlling GPR116 activity. With some additional controls, the conclusions of this study would have far-reaching implications for the development of pharmacological approaches aimed at modulating the activity of this important biological target involved in the maintenance of normal pulmonary functions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

The mechanistic details of the tethered agonist mode of activation for the adhesion GPCR ADGRF5/GPR116 have not been completely deciphered. We set out to investigate the physiological importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species-swapping approaches, we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane 7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

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  1. Version published to 10.7554/elife.69061 on eLife
  2. eLife

    Author Response

    *Reviewer #2 (Public Review):

    This manuscript describes studies on the structural determinants of activation for the adhesion GPCR (aGPCR) GPR116 both in vitro and in vivo. The authors define key residues for activation on the receptors' N-terminus (the "tethered agonist") and the extracellular loops. Thus, the studies provide novel insights into the structural determinants of GPR116 activation. However, some interpretational issues (detailed below) complicate some of the authors' conclusions. Specific comments are as follows:

    1. Results section, first paragraph, last sentence: The authors write, "These results taken together indicate that the H991A mutant is capable of proper trafficking to the membrane, is able to response to exogenous peptide, but is unable to be cleaved and activated by endogenous ligands in vivo." The last part of this sentence represents an over-interpretation, as the data shown in Figure 1 do NOT show that the non-cleavable receptor is unable to be activated by endogenous ligands in vivo. It is entirely conceivable that a non-cleavable aGPCR could still be activated by endogenous adhesive ligands if those ligands were to change the position of the tethered agonist in manner that alters receptor signaling activity.

    Thank you for highlighting this misleading wording. We rephrased the sentence to read as follows: Taken together, these results demonstrate that the H991 residue within the GAIN domain is critical for cleavage of GPR116 into NTF and CTF fragments but dispensable for trafficking of the receptor to the plasma membrane and response to exogenous peptide activation in vitro.

    1. The data shown in Fig. 1B (surface expression of non-cleavable H991A mutant) need to be quantified in some way in order to be interpretable.

    As the H991A construct does not contain a cell surface epitope tag, it is difficult to directly quantitate surface expression of this protein. The data in transiently transfected HEK293 cells (Figure 1, panels C and D) and in primary alveolar epithelial cells (Figure 2, panels C&D) clearly demonstrate that the H991A mutant is activated to comparable levels as the wild-type receptor in response to exogenous peptide stimulation. In light of these functional data, we are confident that the surface expression of H991A is comparable to that of the WT receptor in vitro and in vivo.

    1. Results section, second paragraph, penultimate sentence: The authors write, "These data demonstrate that while the non-cleavable receptor is fully activated in vitro by exogenous peptides corresponding to the tethered agonist sequence, cleavage of the receptor and unmasking of the tethered agonist sequence is critical for GPR116 activation in vivo." However, the non-cleavable GPR116 mutant actually has two key differences from WT: i) lack of full liberation of the tethered agonist sequence, and ii) lack of liberation of a free NTF, which might dissociate from the CTF and have important in vivo physiological actions on its own. Isn't it conceivable that the lack of a freely mobile NTF contributes to the similarity in lung phenotype between the non-cleavable knock-in mutant and the GPR116 knockout? Based on the data shown in Figure 2, how can the authors claim these data demonstrate that unmasking of the tethered agonist is critical for GPR116 activation? The data could equally be interpreted as showing that liberation of a free NTF is critical for the physiological effects of GPR116 in vivo.

    We thank the reviewer for this comment and, in retrospect, agree that we may have overstated the interpretation of our results for the H991A transgenic mouse. While it is possible that the free NTF may be responsible for the physiological effects of GPR116 in vivo, in light of recently published data by Mitgau et al. (BioRxiv https://doi.org/10.1101/2021.09.13.460127), we believe this not to be the case for the following reasons. First, the H991A and WT receptors are activated to an identical level by exogenous peptide stimulation in a transformed cell line (HEK293) and in primary alveolar type 2 epithelial cells (Figures 1 and 2), irrespective of if the NTF is free floating in solution in the context of the WT receptor. These data would argue against a role of the free NTF in receptor activity. Second, in a recent publication by Mitgau et al., the authors clearly demonstrate that activation of GPR126, an adhesion GPCR that is also cleaved at the GPS and activated by exogenous peptides corresponding to the tethered agonist, by antibodies that bind and crosslink the NTF is completely dependent on cleavage at the GPS. They further demonstrate that antibody-mediated activation does not lead to liberation of the NTF from the CTF. Rather, they postulate that proper GPS processing, as occurs for the WT receptor, leads to a favorable protein confirmation of the tethered agonist, which is indispensable for GPR126 activity. Given these results, we postulate that cleavage at the GPS of WT GPR116 results in a conformation that is critical for the tethered agonist sequence to reach and bind the ECLs, resulting in activation of the receptor, similar to that observed with GPR126. We have edited our interpretation of these data in the revised manuscript.

    1. Figure 3: If the authors' hypothesis is that the tethered agonist must be liberated in order to allow activation of GPR116, then why do ANY of the Flag-tagged mutant constructs exhibit constitutive signaling activity? Doesn't the N-terminal Flag tag prevent the tethered agonist from being exposed? How can these data be reconciled with the authors' model?

    It is unlikely that the 27 amino acid N-terminal FLAG epitope tag envelopes the tethered agonistic peptide to the same extent as the tertiary structure of the carboxy terminus of the NTF (based on published structures for other aGPCRs). Additionally, we provided data demonstrating that an untagged version of the CTF protein is activated to a similar extent at FLAG-tagged CTF in response to activating peptides (Supplemental Figure 2A). Based on our data from mutagenesis experiments and modeling of GPR116 with the agonist, we do not believe the tethered agonist dives deeply within the binding pocket but rather interacts with critical amino acids at the surface of ECL2 to induce conformational changes to the receptor and downstream activation.

    1. The data shown in Fig. 3D are lacking statistical comparisons, so it is not possible to tell whether any of the differences between the mutants are statistically significant.

    Statistical analyses for data in this panel have been added

    1. The data shown in Fig. 4D (surface expression of the ECL mutants) need to be quantified in some way.

    We have added additional data to this figure (Fig4 F-G-H) using the V5-tagged mFL construct as control. As the tag is C-terminal, we quantified by flow cytometry the total expression using an anti-V5 antibody, to complement to immunocytochemistry data showing membrane expression.

    1. In interpreting the results of the ECL mutations on GPR116 signaling activity, it is unclear why the authors so explicitly propose that these data demonstrate that the tethered agonist must be interacting with ECL2. Isn't it possible that ECL2 mutants with impaired receptor signaling activity simply lock the receptor in an inactive state? In this way, the effects of the ECL2 mutations could be explained without invoking a physical interaction between the putative tethered agonist and ECL2.

    Yes, this interpretation is also possible. We have rephrased the Results and Discussion sections accordingly to reflect this possibility.

  3. eLife

    Evaluation Summary:

    Bridges et al., present a timely study on an adhesion G-protein coupled receptor known as GPR1116, which is an important regulator of lung function. The authors performed extensive mutagenesis and functional studies to characterize the structural determinants controlling GPR116 activity. With some additional controls, the conclusions of this study would have far-reaching implications for the development of pharmacological approaches aimed at modulating the activity of this important biological target involved in the maintenance of normal pulmonary functions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Adhesion GPCRs are a relatively understudied GPCR family. One of the mechanisms they are activated is by a tethered agonist peptide that interacts with the transmembrane domain to activate the receptor. The authors first show that a knock-in mouse expressing a non-cleavable GPR116 mutant to prevent the release of the tethered agonist peptide phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for GPR116 function in vivo. They then use mutagenesis and activity assays to find residues in the tethered agonist which are most important for receptor activation, as well as mutating loops in the extracellular face of the receptor to find residues important in mediating the response to the tethered agonist. The authors also highlight residues important for differential mouse vs. human GPR116 activation and propose that ECL2 is very important for GPR116 activation.
    The work is overall solid. However, there are some concerns with the lack of expression data shown, with the lack of citing and mentioning previous work about the peptide-receptor interactions, with the model and some structural interpretations that needs to be carefully made.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript describes studies on the structural determinants of activation for the adhesion GPCR (aGPCR) GPR116 both in vitro and in vivo. The authors define key residues for activation on the receptors' N-terminus (the "tethered agonist") and the extracellular loops. Thus, the studies provide novel insights into the structural determinants of GPR116 activation. However, some interpretational issues (detailed below) complicate some of the authors' conclusions. Specific comments are as follows:

    1. Results section, first paragraph, last sentence: The authors write, "These results taken together indicate that the H991A mutant is capable of proper trafficking to the membrane, is able to response to exogenous peptide, but is unable to be cleaved and activated by endogenous ligands in vivo." The last part of this sentence represents an over-interpretation, as the data shown in Figure 1 do NOT show that the non-cleavable receptor is unable to be activated by endogenous ligands in vivo. It is entirely conceivable that a non-cleavable aGPCR could still be activated by endogenous adhesive ligands if those ligands were to change the position of the tethered agonist in manner that alters receptor signaling activity.

    2. The data shown in Fig. 1B (surface expression of non-cleavable H991A mutant) need to be quantified in some way in order to be interpretable.

    3. Results section, second paragraph, penultimate sentence: The authors write, "These data demonstrate that while the non-cleavable receptor is fully activated in vitro by exogenous peptides corresponding to the tethered agonist sequence, cleavage of the receptor and unmasking of the tethered agonist sequence is critical for GPR116 activation in vivo." However, the non-cleavable GPR116 mutant actually has two key differences from WT: i) lack of full liberation of the tethered agonist sequence, and ii) lack of liberation of a free NTF, which might dissociate from the CTF and have important in vivo physiological actions on its own. Isn't it conceivable that the lack of a freely mobile NTF contributes to the similarity in lung phenotype between the non-cleavable knock-in mutant and the GPR116 knockout? Based on the data shown in Figure 2, how can the authors claim these data demonstrate that unmasking of the tethered agonist is critical for GPR116 activation? The data could equally be interpreted as showing that liberation of a free NTF is critical for the physiological effects of GPR116 in vivo.

    4. Figure 3: If the authors' hypothesis is that the tethered agonist must be liberated in order to allow activation of GPR116, then why do ANY of the Flag-tagged mutant constructs exhibit constitutive signaling activity? Doesn't the N-terminal Flag tag prevent the tethered agonist from being exposed? How can these data be reconciled with the authors' model?

    5. The data shown in Fig. 3D are lacking statistical comparisons, so it is not possible to tell whether any of the differences between the mutants are statistically significant.

    6. The data shown in Fig. 4D (surface expression of the ECL mutants) need to be quantified in some way.

    7. In interpreting the results of the ECL mutations on GPR116 signaling activity, it is unclear why the authors so explicitly propose that these data demonstrate that the tethered agonist must be interacting with ECL2. Isn't it possible that ECL2 mutants with impaired receptor signaling activity simply lock the receptor in an inactive state? In this way, the effects of the ECL2 mutations could be explained without invoking a physical interaction between the putative tethered agonist and ECL2.

  6. eLife

    Reviewer #3 (Public Review):

    The data reported in this study highlight the physiological relevance of an autoproteolysis event which exposes a receptor's tethered ligand leading to its activation through a network of residues stabilizing the receptor's active conformation. With an emphasis on the orphan adhesion G Protein Coupled Receptor (aGPCR) member GPR116, the authors describe that a proteolytically-deficient mutant of the receptor recapitulates many of the phenotypic elements that are characteristic of lung defects observed in a GPR116-deficient mouse line previously developed and analyzed by the same group, thus alluding to the critical role played by an aGPCR canonical intramolecular cleavage in maintaining physiological receptor function. While the authors bring a quintessential support for this hypothesis by analyzing lung cells phenotypes in this CRISPR-generated genetically modified mouse mutant, these observations come amidst a divided field. Indeed, attempts to verify if aGPCR functions rely on a strict requirement for proteolytic cleavage have led to conflicting data and have highlighted the need for deeper range analytical strategies. Adding to this controversial hypothesis, some aGPCR members lack the ability for autoproteolytic cleavage, thus further interrogating the functional relevance of this event as a pan-aGPCR activation mechanism. In order to further support their hypothesis and due to limited knowledge on the nature of a potential endogenous ligand for GPR116, the authors have undertaken an elaborate structure-function analysis of the relationship taking place between the cryptic peptide sequence and GPR116 receptor domains. Their experimental strategy focused on the peptide's ability to activate receptor functions by exploiting a cross-species characteristic underlining the human receptor's differential ability to transduce ligand-mediated intracellular signaling cascades compared to its mouse homologue. This mutational scanning strategy conducted on the tethered ligand peptide as well as on critical receptor domains identified in cross-species comparison led the authors to delineate the activation determinants that accompany the ligand-receptor complex in achieving a conformational state capable of engaging intracellular effectors leading to the modulation of intracellular calcium levels. While the lack of constitutive activity detected in the full-length receptor underlines an absolute requirement for extrinsic factors to induce physiological activating conditions (such as a yet unknown endogenous ligand, mechanical force or other) which consequently may point to a different pattern of ligand/receptor activation determinants, the description of residues constituting the intramolecular network of activation is not expected to deviate significantly from the data reported in this study. Although the study denotes the need for assays directly addressing the delineation of the binding pocket (such as binding assays among others), the data reported by the authors represent an initial assessment of structural requirements linked to the stabilization of an active conformational state. In the realm of GPCRs, this report is among very few recent studies highlighting the interplay between the agonist peptide and the second extracellular loop in aGPCRs, a description that unifies aGPCRs' structural features with the current knowledge on the binding and activation mode of GPCRs from other subfamilies.

    Although this study provides significant advancements for the field of aGPCRs it also contains noticeable scientific shortcomings, that would be good to attend to.

    Function of aGPCR cleavage in mammalian tissue physiology- Hint of the importance of aGPCRs' autoproteolysis is provided by the generation and analysis of the cleavage-deficient GPR116 H991A/H991A mouse line. However, a more complete in vivo functional strategy is needed in order to sort out a few important details. Of a contentious nature is the phenotype resulting from the replacement of Histidine991 by an Alanine. Although it is clear that this substitution leads to the loss of autoproteolytic activity, it is not clear if the actual substitution with alanine does not constitute the primary origin of the phenotypes observed in mice. In view of the high level of conservation of this amino acid within the GAIN domain of various GPR116 species as noted by the authors, one would have to try to discard the possibility of the loss of Histidine being the root cause of the changes and not the resulting loss of autoproteolytic activity. Also, a more detailed and systematic characterization of GPR116 H991A/H991A mouse line is warranted since the authors point to phenotypic similarities with GPR116 knock-out mice.

    Assessing agonist-receptor activation efficacy- As receptor constructs are expressed at varying levels throughout the study and because receptor activity is tightly correlated with cell membrane localization, it would have been helpful if the authors quantified receptor expression levels and incorporated this data as a normalizing criterion for their experiments throughout. This would greatly help to better understand the functional effects observed in the study.

    Correlating activation determinants with binding determinants- Peptide ligands are likely to possess multiple points of contact with their cognate GPCR, so the fact that single receptor point mutations are able to completely abolish receptor activation likely suggests that a critical activation network may have been perturbed rather than a complete loss of binding determinants from the receptor's ligand-binding pocket. Evidently, the reporter assay measuring calcium transients relates to the functional aspect of GPR116. This aspect represents the compound effect of various conformational states emanating from ligand interacting with the receptor's binding pocket and subsequent ligand-induced conformational transition allowing the receptor to couple to intracellular effectors. Thus, the mutational analysis presented here by the authors does not allow for a proper dissection of both events. Indeed, a given mutation could be impeding the receptor's transition to an active state while maintaining a proper ligand binding. As noted the C1088 mutation results in an unresponsive receptor probably because the active conformation cannot be properly reached and not necessarily because this residue is part of the binding pocket. Binding assays would be more appropriate to describe binding pocket determinants.

  7. Version published to 10.1101/2021.04.01.438115v1 on bioRxiv