Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane region

TLDR: In this paper, the authors investigated the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation.

Abstract: The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic 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 transmembrane7 (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. Grant supportThis work was supported in part by HL131634 (JPB) from the National Heart, Lung and Blood Institute of the National Institutes of Health.

Figures

Figure 6. Engineering the predicted mouse GPR116 binding site (mBS) into human GPR116 leads to increased peptide activation. A. Snake plot of the mouse GPR116 CTF highlighting mouse-specific amino acids in the putative binding site in blue and pink. Amino acids responsible for mGPR116-specific aspects of receptor activation are shown in pink. The Ct tail was truncated for visualization purposes as indicated by double hash lines. B. Stable HEK293 populations for m/hCTFbax and hCTFbax mBS were stimulated with GAP14 and analysed for the induction of calcium transients. A representative graph of 9 repeat experiments is shown. C. Characterization of the key amino acids transmitting the effects of the mouse binding site sequence. hCTFbax single mutants were compared to hCTFbax and hCTFbax mBS in calcium transient assays. Stable HEK293 populations were stimulated with GAP14 (n=3 independent experiments each performed in duplicate).

Figure 5. Evaluation of the role of ECL amino acids not conserved between human and mouse GPR116. A.

Figure 8. hGPR116 7TM homology model. A. Homology model of human GPR116 7TM. The cloud of black dots defines the putative main pocket of the hGPR116 CTF as identified by SiteMap and corresponding to the CRF1R agonist binding site. The mouse-specific amino acids included in the potential antagonist binding site are displayed as spheres and include A1254 and V1258, which generate a stronger activation response upon mutation to the mouse sequence. The residue G1011 involved in stronger mouse GPR116 activation is at the top of TM1 (not shown). Amino acid numbering with respect to the mouse CTF is shown. B. Focus on ECL2 highlighting putative key amino acids for GPR116 activation and possible interactions with the tethered agonist drawn on top of the 7TM structure. C. Zoom around opposite side of ECLs 2 and 3 highlighting an amino acid at the top of TM6, T1240, important for receptor activation.

Figure 4. Identification of key ECL amino acids involved in GPR116 activation by the tethered agonist. A. Snake plot model of mouse GPR116 CTF. ECL residues individually mutated in the alanine scan of mouse GPR116 CTF constructs tested for functional activity are colored in blue or pink; the five residues shown in pink were identified as strongly modulating receptor activity. The two cysteine residues forming the disulfide bridge are shown in green. The Ct tail was truncated for visualization purposes as indicated by double hash lines. B. Alanine scan of ECLs in mCTFbax, a CTF construct without basal activity. Calcium transient assays were performed in transiently transfected HEK293 cells with exogenous GAP14 as the stimulus. mCTFbax mutants that were completely inactive following GAP14 stimulation are shown. Data are from 3 experiments, each performed in quadruplicates. See Suppl Fig4 for further alanine scan data, including mutants with no effect. C. Expression of the mCTFbax mutants not activated by GAP14, in transiently transfected HEK293 cells. Proteins were detected by Western blot using an anti-FLAG antibody. D. Membrane expression of the mGPR116 full-length (mFL) ECL mutants assessed by V5 tag immunocytochemistry upon stable expression in HEK293. E. Expression of mFL ECL mutants stably expressed in HEK293 cells. Constructs were detected by Western blot using an anti-V5 antibody; the major band (~30kD) corresponds to the cleaved CTF and the minor bands at high molecular weight (~185kDa) correspond to the full-length receptor. The mFL WT stably transfected clone 3C is not V5 tagged. F. GAP14-induced calcium transients of mFL mutants for 5 key ECL residues, stably expressed HEK293 cells, as compared to the WT mGPR116 3C clone. Data shown are from two independent experiments performed with biological quadruplicates. G. Mutation of key ECL residues in the mCTFbax construct to amino acids with functional relevance. mCTFbax and mutants thereof were stably expressed in HEK293 cells and GAP14-induced calcium transients were measured. Data are from 3 experiments each performed in quadruplicate.

Figure 7. Characterization of a mouse-specific agonistic peptide. A. GAP10-Pro1 only activates mouse GPR116. Full-length mouse and human GPR116 (mFL clone 3C and hFL clone A6, respectively), mCTFbax and hCTFbax, all stably expressed in HEK293 cells, were stimulated with GAP14, GAP10 and GAP10-Pro1. Calcium transients are measured, in biological duplicates, as a signaling readout. B. Identification of the key amino acids mediating the mouse-specific effects in the mouse binding site. The hCTFbax construct, the hCTFbax mBS chimera and the single point mutants A1254T and V1258A in hCTFbax were stimulated with GAP10 or GAP10-Pro1 upon stable expression in HEK293 cells. Their activation was measured in a calcium transient assays, in biological duplicates. Data are representative of at least two independent experiments with identical results. C. Calcium transients evoked in HEK293 cells stably expressing hCTFbax or the mutated construct G1011K upon stimulation with GAP10 or GAP10-Pro1. Data are representative of three independent experiments with identical results.

Full text

1
Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in
extracellular loop 2 of the transmembrane region
Authors
James P. Bridges
1
, Caterina Safina
2
, Bernard Pirard
2
, Kari Brown
1
, Alyssa Filuta
1
, Rochdi
Bouhelal
2
, Sejal Patel
4
, Klaus Seuwen
2
, William E. Miller
3
, Marie-Gabrielle Ludwig
2,*
1
Department of Pediatrics, Perinatal Institute, Section of Pulmonary Biology, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH, USA, 45229.- present address: Department of Medicine,
Division of Pulmonary, Critical Care and Sleep Medicine, National Jewish Health, Denver, CO,
USA, 80206
2
Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland
3
Department of Molecular Genetics, Biochemistry and Microbioloby, University of Cincinnati
College of Medicine, Cincinnati, OH 45267
4
Novartis Institutes for Biomedical Research, Cambridge, USA
* Corresponding author; email: marie-gabrielle.ludwig@novartis.com
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2
Grant support: This work was supported in part by HL131634 (JPB) from the National Heart,
Lung and Blood Institute of the National Institutes of Health.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.01.438115doi: bioRxiv preprint

3
Abstract
The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not
been completely deciphered. We set out to investigate the physiologic 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 transmembrane7 (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.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 2, 2021. ; https://doi.org/10.1101/2021.04.01.438115doi: bioRxiv preprint

4
Introduction
Adhesion G Protein-Coupled Receptors (aGPCRs) are involved in a variety of
pathophysiological processes, from development of the brain and the musculoskeletal system to
modulation of metabolism (Olaniru et al., 2019), immune responsiveness and angiogenesis.
Important roles in the central and peripheral nervous system have been described (reviewed in
Folts et al., 2019), exemplified by the genetic linkage of GPR56/ADGRG1 variants with bilateral
frontoparietal polymicrogyria (BFPP) pathology. The capacity of aGPCRs to modulate
inflammatory responses has been shown in several contexts (Lin et al., 2017), with leading data
on apoptotic cell sensing and phagocytosis (EMR2/ADGRE2, BAI1/ADGRB1), activation of mast
cells (EMR2 genetic link to vibratory urticaria) or NK cells (GPR56), and resistance to listeriosis
(CD97/ADGRE5). In addition, several lines of evidence suggest key roles for aGPCRs in cancer
where they may, for example, relay cellular signaling of mechanical cues (reviewed in Scholz,
2018).
A specific aspect of aGPCR biology is their mechanosensory role, an aspect that may
prove to be a general feature of these receptors (Langenhan, 2019). These properties are related
to the structure of aGPCRs and their mode of activation that was recently identified. Several
methods of activating aGPCRs have been identified, including using endogenous ligands, peptide
agonists and small molecules (reviewed in Bassilana et al., 2019). What may be seen as the
primary activation mode of aGPCRs is activation via a tethered agonistic peptide (Liebscher et
al., 2014; Stoveken et al., 2015). Intracellular autocatalytic cleavage of aGPCRs at the GPS site
upstream of the seven transmembrane domain (7TM) generates the NTF and CTF (N-terminal
and C-terminal fragments, respectively), with the amino terminal 15 to 27 amino acids of the CTF
being referred to as the tethered agonist. The NTF and CTF then re-associate non-covalently
during trafficking to the cytoplasmic membrane such that the tethered agonist is buried within the
C-terminal portion of the NTF (Araç et al., 2012). Interactions of the NTF with extracellular matrix
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5
(ECM) components, or with neighboring cells, serves as an anchor for the receptor. Upon binding
of an additional ligand that changes the structural conformation, or generation of tension (e.g.
shear stress, stiffness of the ECM), the tethered agonist is released from the NTF thereby
activating the 7TM of the receptor. Several studies have shown constitutive basal activity of CTF
constructs and activation of full-length aGPCRs with exogenous synthetic peptides (GAP)
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). Some receptors such as CELSR1/ADGRC1, GPR115/ADGRF4,
GPR111/ADGRF2 do not possess a consensus GPS cleavage site and are not cleaved. Further,
the biological function of GPR114/ADGRG5 and LAT1 (LPHN1/ADGRL1) is not dependent on
the cleavage at the GPS site and such receptors may be activated by structural changes not
requiring the dissociation of the NTF and the CTF (Langenhan 2019; Scholz et al., 2017; Beliu et
al., 2021). In contrast, release of the NTF, concomitant to receptor activation, has been shown for
GPR56 upon binding to the ECM laminin 111 and transglutaminase 2 (Luo et al., 2014) and for
EMR2 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, the molecular details for the activation of aGPCRs by their tethered agonists
have not been well described; in part due to difficulty of generating 3D structures of GPCRs.
Multiple sequence alignments from previous reports revealed a 25-30% amino acid identity
between aGPCRs and class B1 GPCRs (Bjarnadóttir et al., 2007; Stacey et al., 2000). Available
3D structures of class B1 GPCRs (19 structures, 11 X-ray and 8 cryo-electron microscopy (cryo-
EM)) in the GPCR DB, www.gpcrdb.org as accessed on January 8
th
2020, have provided
templates for homology models which could be used to gain insights into the structure of the CTF
of aGPCRs. However, it remains unclear as to what extent this knowledge on class B receptors
will be directly applicable to the mechanism of aGPCR activation.
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Frequently asked questions

Q1. What have the authors contributed in "Activation of gpr116/adgrf5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane region" ?

A specific aspect of aGPCR biology is their mechanosensory role, an aspect that may be a general feature of these receptors this paper. 

Q2. What is the role of the methionine in the mCTF?

In order to rule out potential artifacts resulting from oxidation, the methionine corresponding to position 999 in the mCTF was replaced by norleucine (Nle). 

Q3. What is the role of the tethered agonist in GPR116?

While the endogenous ligand or the biological process(es) leading to displacement of the non-covalently linked N-terminal domain and subsequent GPR116 activation in vivo remains unknown, their 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. 

Q4. What are the key amino acids in the ECLs that form?

the five key amino acids in the ECLs that form suggested contact points for the tethered ligand were used to provide constraints and to include the ECLs in the modeling. 

Q5. What is the phenotype of a knock-in mouse?

A knock-in mouse expressing a noncleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. 

Q6. What did the mutated agonist amino acids show in the previous study?

In their previous study, mutation of the tethered agonist amino acids to alanines using sequential steps of 3 amino acids for each mutant highlighted the role of this N-terminal sequence in receptor activation. 

Q7. What is the significance of the alanine residues in the mCTFb?

In a related series of experiments the authors aimed at elucidating whether conservative aminoacid replacements within these key residues in mCTFbax would restore receptor activation of the single point alanine mutants. 

Q8. What is the likely role of the isoleucine in the aGPCR?

replacing L1166 with an isoleucine, as is observed in some aGPCRs, was not tolerated in GPR116, suggesting a specific role of this amino acid. 

Q9. What amino acid is responsible for the increased activity of the murine receptor?

In parallel, while analyzing the potential binding mode of a low molecular weight antagonistspecific for the murine receptor discovered in house at Novartis the authors began considering additional other amino acids deeper in the transmembrane domains of the receptor that might be responsible for the species differences in efficacy. 

Q10. What is the position of tyrosine in the aGPCR?

The tyrosine at position 1158 is located 6 amino acids upstream of the CWL cluster and itis conserved in most aGPCRs, except in the ADGRA and ADGRG subfamily members, and ADGRV1 (Suppl Fig11). 

Q11. What was the cDNA sequence of the H991A-V5 construct?

The H991A-V5 cDNA construct was generated by gene synthesis (Genewiz, South Plainfield, NJ), sequenced verified and cloned into pcDNA3.1+ for expression in cultured cells. 

Q12. What amino acid residues are involved in the hCTFbax receptor?

Similar to observations with the full-length receptors, the hCTFbax is less responsive to14GAP14 than the mCTFbax receptor (Fig5B).