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

Adhesion G protein-coupled receptors (aGPCRs) are critical for a variety of biological and pathophysiological processes, including modulation of metabolic (Olaniru and Persaud, 2019) and immune responses (Lin et al., 2017) to regulatory roles in the central and peripheral nervous systems (Folts et al., 2019). In addition, several lines of evidence suggest key functions for aGPCRs in cancer where they may, for example, relay cellular signaling in response to surrounding mechanical cues (reviewed in Scholz, 2018).

Mechanosensory roles may prove to be a general feature of these receptors, in relation to the overall structure of aGPCRs and the mode of activation that was recently identified (Langenhan, 2019). In aGPCRs, a large extracellular domain, termed the N-terminal fragment (NTF), is linked to the transmembrane 7 domain (7TM), which is referred to as the C-terminal fragment (CTF) of the protein. The NTF and CTF fragments are linked by GPCR autoproteolysis-inducing (GAIN) domain containing an autocatalytic cleavage site (GPS) motif that is activated during receptor processing. Following proteolytic cleavage, the NTF and CTF remain associated via non-covalent interactions during trafficking to the cytoplasmic membrane (Araç et al., 2012). Groundbreaking work by Liebscher et al., 2014 and Stoveken et al., 2015 has shown that the CTF, in the absence of the NTF, possesses basal signaling activity and that the 15–27 most N-terminal amino acids of the CTF act as a tethered activating ligand, reminiscent of the activation mode of the thrombin receptor (Seeley et al., 2003). In light of these data, it is conceivable that interactions of the NTF with extracellular matrix (ECM) components, or with membrane components of neighboring cells, serve as an anchor for the receptor and that binding of an additional ligand, or generation of tension (e.g., shear stress, stiffness of the ECM), changes the structural conformation, thereby releasing or altering the conformation of the NTF, resulting in exposure of the tethered agonist and activation of the receptor.

Several studies have shown constitutive basal activity of CTF constructs and activation of full-length aGPCRs with exogenous synthetic peptides (which we refer to as GPCR-activating peptides [GAPs]) corresponding to their cognate tethered agonist sequence (Liebscher et al., 2014; Demberg et al., 2015; Müller et al., 2015; Stoveken et al., 2015; Wilde et al., 2016; Brown et al., 2017; reviewed in Bassilana et al., 2019). Specifically, release of the NTF concomitant to receptor activation has been shown for GPR56/ADGRG1 upon binding to the ECM laminin 111 and transglutaminase 2 (Luo et al., 2014) and for EMR2/ADGRE2 bound to the ECM dermatan sulfate and subjected to mechanical cues (vibration) (Boyden et al., 2016; Le et al., 2019; Naranjo et al., 2020). However, it is important to note that some adhesion GPCRs such as CELSR1/ADGRC1, GPR115/ADGRF4, and GPR111/ADGRF2 lack a consensus GPS site and do not show evidence of cleavage. Further, the biological functions of GPR114/ADGRG5 and LAT1 (LPHN1/ADGRL1) are independent of cleavage at the GPS site, suggesting that these receptors may be activated by other structural changes (Langenhan, 2019; Scholz et al., 2017; Beliu et al., 2021).

In order to gain insight into the mechanisms of receptor activation at the level of the TM domains, several studies analyzed activation motifs and conserved functional elements within the TMs from various GPCR classes (Peeters et al., 2016; Arimont et al., 2019). For example, Nazarko et al., 2018 performed a comprehensive mutagenesis screen of LPHN1/ADGRL1. For the tethered agonist peptide, a beta strand conformation was suggested followed by a turn element that may be key in receptor activation (detailed in Vizurraga et al., 2020). However, only recently a first study explored in greater detail the modalities of the tethered agonist-mediated activation for GPR64/ADGRG2 (Sun et al., 2021). This study highlighted the importance of two residues within extracellular loop (ECL)2 and the tryptophan (W) toggle switch in TM6, showing reduced binding of an optimized peptide upon mutation. These functional data were recently complemented by cryo-EM structures of several aGPCRs in the presence of their activating tethered peptide (Qu et al., 2022; Xiao et al., 2022; Ping et al., 2022; Barros-Álvarez et al., 2022).

In this study, we set out to investigate the molecular mechanisms of tethered agonist-induced activation of the aGPCR GPR116/ADGRF5. GPR116 is expressed in several tissues and cell types, including alveolar type II epithelial (AT2) cells in the lung. One of the primary roles of GPR116 is in the regulation of pulmonary surfactant as revealed by global knock-out of the gene in mice (Bridges et al., 2013; Yang et al., 2013; Fukuzawa et al., 2013; Niaudet et al., 2015). Cell-specific deletion of GPR116 in AT2 cells of adult mice, or deletion of the downstream signaling proteins GNAQ/GNA11, recapitulated the increased pulmonary surfactant phenotype observed in the global knock-out mouse model (Brown et al., 2017). With respect to putative ligands, GPR116 has been proposed to associate with surfactant protein D (SFTPD) via its NTF (Fukuzawa et al., 2013). However, evidence supporting a direct role for SFTPD in the activation of GPR116 is lacking. Additionally, FNDC4 has recently been shown to activate GPR116-mediated Gs signaling in adipocytes (Georgiadi et al., 2021). Whether FNDC4 modulates Gq-mediated signaling via GPR116 in pulmonary AT2 cells remains to be determined.

In order to evaluate the role of the GPS cleavage and the tethered agonist for in vivo activation and functionality of GPR116, we generated knock-in mice in which autocatalytic cleavage at the GPS site is abolished. This approach has been employed for other aGPCRs in Caenorhabditis elegans and in vitro systems, using mutations immediately upstream or downstream of the GPS cleavage site (Kishore et al., 2016; Prömel et al., 2012; Peeters et al., 2015). In addition, we generated synthetic peptides corresponding to the tethered ligand sequence and mutations within the extracellular loops (ECLs) of GPR116 to identify key amino acid residues involved in receptor activation. Finally, we developed a GPR116 homology model based on recently published cryo-EM structures of ADGRF1/GPR110 (Qu et al., 2022). Together, our findings provide additional insight into the molecular mechanisms underlying the activation of GPR116/ADGRF5.

While great strides have been made in delineating the mechanisms of activation of aGPCRs in vitro, the mode of activation of adhesion GPCRs in vivo, including GPR116, is not well understood. Through generation and characterization of a cleavage-deficient mutant, H991A, we show that cleavage at the GPS site is required for GPR116 activity and modulation of pulmonary surfactant levels in vivo. These studies validate a tethered agonist-mediated activation mode for GPR116 in alveolar type 2 epithelial cells and alveolar homoeostasis. This is, to our knowledge, the first in vivo demonstration of the key role of the GPS cleavage and the unmasking of a tethered agonist in a mammalian system.

The tethered agonist sequence of each aGPCR family member is highly conserved between species, which explains cross-activation among orthologs (Liebscher et al., 2014; Brown et al., 2017). In this study, we identified F995, L998, and M999 as key amino acids in the tethered agonist sequence required for GPR116 activation. These three amino acids are highly conserved in the ECDs of multiple aGPCR CTFs (Supplementary file 1) and have been highlighted for other receptors such as GPR126/ADGRG6, GPR133/ADGRD1, GPR110/ADGRF1, GPR56/ADGRG1 (Liebscher et al., 2014; Stoveken et al., 2015), suggesting that they play a critical structural role for activation in the aGPCR family. In this sense, these data may explain some of the promiscuous activities observed within and between aGPCR subfamilies (Demberg et al., 2017).

Other studies aimed at analyzing the critical residues comprising the tethered agonist are consistent with our identification of phenylalanine at position 3 (F995) as the most N-terminal residue playing a key role in aGPCR activation (GPR64/ADGRG2, Azimzadeh et al., 2019 and Sun et al., 2021; GPR126/ADGRG6, Liebscher et al., 2014; Lphn3/ADGRL3, Mathiasen et al., 2020). Similarly, amino acids at positions 6 and 7 (L998 and M999), albeit not strictly conserved as leucine and methionine residues in all aGPCR tethered ligands, are key for GPR116 activation. It is noteworthy that additional residues within the tethered agonist that were dispensable for GPR116 activity are critical for other aGPCRs, possibly explaining the specificity for activation exhibited by some receptors. For example, alanine substitution of the first amino acid within the tethered agonist (usually a threonine) can lead to a loss of activity (Liebscher et al., 2014) while substitution with a hydrophobic residue can increase the activity of the peptide (Sun et al., 2021). This is consistent with our observations with the GAP10-Pro1 peptide, further supporting the concept that position 1 can modulate the strength of molecular interactions between ligand and its binding domain during aGPCR activation.

With respect to the 7TM, our mutational analysis of the extracellular surface of GPR116 identified ECL2 and top of TM6/ECL3 as the structural components most involved in receptor activation. Mutation of key residues may prevent GPR116 from transitioning to an active state or may prevent interaction with the tethered agonist peptide. We identified Y1158, R1160, W1165, and L1166 in ECL2 and T1240 at the TM6/ECL3 interface to be the key residues involved in GPR116 activation. Interestingly, we noted that W1165 and L1166, which immediately follow the cysteine involved in the disulfide bridge linking the top of TM3 and ECL2, comprise a CWL triad that is key for receptor activation. The tryptophan residue is globally conserved in aGPCRs, and the leucine (or sometimes isoleucine) is frequently present in this position (Supplementary file 1). Residues within ECL2 have also been implicated in ligand binding and activation of classical GPCRs (Wheatley et al., 2012). In particular, the CW motif in the ECL2 is conserved in family B, suggesting commonalities in the activation mechanism between aGPCRs and family B GPCRs. In particular, the (C)WF in ECL2 is involved in CRF1 binding to CRF1R (Gkountelias et al., 2009). In PAR1, a (C)HD sequence is important for receptor activation by the peptide, further highlighting the relevance of the ECL2 amino acids following the disulfide bridge, not only for activation per se but also ligand binding (OBrien et al., 2001). Within the aGPCR family, Sun et al., 2021 and Gad et al., 2021 also identified the (C)WI triad in ECL2 and residues in TM6 as being involved in GPR64/ADGRG2 activation by the tethered agonistic peptide. The tyrosine at position 1158 is located six amino acids upstream of the CWL triad and it is conserved in most aGPCRs, except in the ADGRA and ADGRG subfamily members, and ADGRV1 (Supplementary file 1). It is noteworthy that the CWL cluster is also less strictly conserved in these ADGRs. It is possible that a tyrosine at a position further upstream in the ECL2 may functionally replace Y1158 in these receptors, depending on the flexibility of the ECL. With regard to position 1240, a role for ECL3 in aGPCR activation has already been suggested for disease-associated mutations of GPR56/ADGRG1 (Chiang et al., 2017; Kishore and Hall, 2017; Luo et al., 2014). The mutation L640R (corresponding to H1250 in GPR116) in ECL3 affects collagen 3-mediated activation of GPR56 signaling, likely by inducing a conformation that locks the receptor in an inactive state. While the mutational work presented here highlighted features in line with previously published studies on aGPCR activation, interpretation of the functional data is limited and addressing the role of the identified key amino acids in GPR116 activation was further investigated in a homology model.

Indeed, recently, nine cryo-EM structures of aGPCRs, not including GPR116, were published (Barros-Álvarez et al., 2022; Ping et al., 2022; Qu et al., 2022; Xiao et al., 2022). These structures highlight, among other aspects, that the stachel is indeed buried into the 7TM domain, consistent with our data, with a key role of hydrophobic amino acids in positions 3, 4, 5, 7 of the tethered agonist and that the ECL2 can also bend into the barrel with variations depending on the receptor. The agonist peptide is surrounded by ECL2 and TMs on one side, and ECL3 and TMs on the other side. These structures confirm the key role ECL2, and it will be interesting to compare aGPCR activation with that of GPR52, recently shown to involve residues within ECL2 (Lin et al., 2020). Although the tryptophan following the cysteine bridge is critical for receptor activation in all aGPCRs, additional essential residues appear distinct in the various studied receptors.

The homology model of human GPR116 confirms that amino acids in ECL2 are important for receptor activation, even if it is surprising that only three residues facing the extracellular side (two in ECL2, one in ECL3) are involved in interactions with the tethered peptide. Based on this model, we could address a role for amino acids identified as key for activation, with some residues being involved in interactions mediating the activation and others being involved in receptor or peptide conformation and stabilization. In particular, the C-terminal part of the peptide (S1000 to S1008) forms intramolecular interactions stabilizing the peptide and Y1158-L1166 in ECL2 may participate in receptor stabilization than activation.

Relative to receptor activation, we identified key residues conserved in the ADGR family such as the tryptophan following the ECL2 cysteine involved in the disulfide bridge or the methionine conserved in most family members at position 7 in the tethered peptide. The homology model also highlighted GPR116-specific residues and interactions, such as R1160 and P1001 conserved in ADGRFs. Further, the species comparison studies highlighted that the mouse sequence-specific amino acids T1254 and A1258 in TM7 strengthen receptor activation by the peptide. The GPR116 homology model shows that the corresponding amino acids in the human sequence, A1254 and V1258, do form contacts with F995 of the tethered agonist peptide. This supports the possibility that the mouse variants induce stronger activation by the peptide, for example, via H bonds by the threonine. Further, it supports our finding that exchanging F995 with proline generates a mouse-specific peptide that is active on a human GPR116 construct exhibiting the mouse sequence at these two positions. The details of such interactions, including the glycine to lysine exchange at position 1011, cannot be predicted using this human GPR116 model and will require further studies.

While the model of the GPR116 activation that we present here is in line with other studies of aGPCRs and highlights aspects of tethered agonist:ECL2 interactions, it does have some limitations. First, further studies using cross-linking and binding experiments will be required to define the contact points between the tethered peptide and ECL2. Second, the potential role of the NTF, how it is positioned relative to the CTF, and how it may affect tethered agonist structure is not known. Modeling the highly flexible loops remains challenging, although some studies focused on modeling of GPCR ECLs are underway (Wink et al., 2019; Liessmann et al., 2021). In particular, ECL2 is the largest ECL in GPR116, as in many other GPCRs, and the disulfide bridge within TM3 does not introduce sufficient constraints to provide a more defined model. Even with adequate representation of the amino acid stereochemistry, significant degrees of freedom remain for the ECL conformation. Third, our studies evaluate the interaction of the tethered agonist with ECL2 in a context devoid of the NTF. In a setting of peptide- or antibody-based activation without NTF removal, exemplified recently by Mitgau et al., 2022 for GPR126, these tethered agonist/ECL interactions may be different.

Finally, it will be interesting to investigate whether the proposed FNCD4-mediated activation of GPR116, via binding to the NTF and subsequent G protein-coupling, also involves the tethered peptide sequence or another mechanism to induce activating structural changes in the 7TM.

Based on this study, our working model postulates that following unveiling of the tethered agonist, residues within this sequence interact with amino acids located in ECL2, ECL3, and TM7 to induce conformational changes, resulting in productive Gq/G11 engagement and activation. While the endogenous ligand or the biological process(es) leading to displacement and/or conformational change of the non-covalently linked N-terminal domain and subsequent GPR116 activation in vivo remain unknown, our data advance the mechanistic understanding of GPR116 activation in the context of pulmonary surfactant homeostasis and may facilitate the development of novel receptor modulators that can be used to treat clinically relevant lung diseases.

Source data for the modeling is provided (coordinates of the model).

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Author details

1. James P Bridges

   Department of Pediatrics, Perinatal Institute, Section of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States

   Present address

   Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, National Jewish Health, Denver, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4815-117X

2. Caterina Safina

   Novartis Institutes for Biomedical Research, Basel, Switzerland

   Contribution

   Resources, Investigation

   Competing interests

   is employed by and shareholder of Novartis Pharma AG

3. Bernard Pirard

   Novartis Institutes for Biomedical Research, Basel, Switzerland

   Contribution

   Conceptualization, Visualization, Methodology, Writing – review and editing

   Competing interests

   is employed by and shareholder of Novartis Pharma AG

4. Kari Brown

   Department of Pediatrics, Perinatal Institute, Section of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States

   Present address

   Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, National Jewish Health, Denver, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

5. Alyssa Filuta

   Department of Pediatrics, Perinatal Institute, Section of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States

   Present address

   Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, National Jewish Health, Denver, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

6. Ravichandran Panchanathan

   Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

7. Rochdi Bouhelal

   Novartis Institutes for Biomedical Research, Basel, Switzerland

   Contribution

   Conceptualization, Resources, Investigation

   Competing interests

   is employed by and shareholder of Novartis Pharma AG

8. Nicole Reymann

   Novartis Institutes for Biomedical Research, Basel, Switzerland

   Contribution

   Investigation

   Competing interests

   is employed by and shareholders of Novartis Pharma AG

9. Sejal Patel

   Novartis Institutes for Biomedical Research, Cambridge, United States

   Contribution

   Conceptualization, Resources, Writing – review and editing

   Competing interests

   is employed by and shareholder of Novartis Pharma AG

10. Klaus Seuwen

    Novartis Institutes for Biomedical Research, Basel, Switzerland

    Contribution

    Conceptualization, Resources, Formal analysis, Validation, Investigation, Writing – review and editing

    Competing interests

    is employed by and shareholder of Novartis Pharma AG

11. William E Miller

    Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, United States

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Validation, Investigation, Visualization, Writing – original draft, Project administration, Writing – review and editing

    Competing interests

    No competing interests declared

12. Marie-Gabrielle Ludwig

    Novartis Institutes for Biomedical Research, Basel, Switzerland

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Validation, Investigation, Visualization, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    marie-gabrielle.ludwig@novartis.com

    Competing interests

    is employed by and shareholder of Novartis Pharma AG

    "This ORCID iD identifies the author of this article:" 0000-0002-7799-4782

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