# GPRASP1 deletion in mice abrogates adverse side effects associated with chronic stimulation of Beta2-adrenoceptor

Alaa Abu-Helo, François Daubeuf, Thibaud Tranchant, Christine Lehalle, Khadija Elhabazi, Gabrielle Zeder-Lutz, Valérie Kugler, Claire Lugnier, Nelly Frossard, Sandra Lecat, et al.

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► To cite this version:

Alaa Abu-Helo, François Daubeuf, Thibaud Tranchant, Christine Lehalle, Khadija Elhabazi, et al. GPRASP1 deletion in mice abrogates adverse side effects associated with chronic stimulation of Beta2-adrenoceptor. Biomedicine and Pharmacotherapy, 2025, 187, pp.118073. 10.1016/j.biopha.2025.118073. hal-05217592

HAL Id: hal-05217592

https://cnrs.hal.science/hal-05217592v1

Submitted on 21 Aug 2025

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Distributed under a Creative Commons Attribution 4.0 International License
GPRASP1 deletion in mice abrogates adverse side effects associated with chronic stimulation of Beta2-adrenoceptor

Alaa Abu-Helo<sup>a,1,2</sup>, François Daubeuf<sup>b,2</sup>, Thibaud Tranchant<sup>a,2</sup>, Christine Lehalle<sup>b</sup>, Khadija Elhabazi<sup>a</sup>, Gabrielle Zeder-Lutz<sup>a</sup>, Valérie Kugler<sup>a</sup>, Claire Lugnier<sup>c</sup>, Nelly Frossard<sup>d,3</sup>, Sandra Lecat<sup>a,3</sup>, Frédéric Simonin<sup>a,*3,4</sup>

ARTICLE INFO

Keywords:

G protein-coupled receptors

Beta2-adrenergic receptor

Beta2-adrenergic agonist

GPRASP1/GASP1

Bronchial hyperresponsiveness

Beta2-adverse events

A B S T R A C T

GPCR associated sorting protein 1 (GPRASP1) interacts with numerous GPCRs including the Beta2-adrenoceptor (B2AR) and has been proposed to be involved in adaptations associated with chronic stimulation of those re- ceptors. In clinic, long acting B2AR agonists (LABAs) such as formoterol are used in the treatment of asthma as potent bronchodilators but with adverse side effects including the development of tolerance and airway hyperresponsiveness upon chronic administration. Here, we investigated the role of GPRASP1 on B2AR activity and on B2AR agonists-associated side effects *in vitro* and *in vivo*. To this purpose, we set-up a model of chronic formoterol administration in mouse leading to B2AR down-regulation as well as to the development of airway hyperreactivity and bronchodilator tolerance and studied the phenotype of GPRASP1 knockout animals. We show in cells that GPRASP1 expression has no impact on agonist-induced B2AR down-regulation but strongly modulate B2AR-associated signalling. Moreover, wild-type mice chronically treated with formoterol developed airway hyperresponsiveness to methacholine and bronchodilator tolerance to formoterol that were absent in GPRASP1 KO mice while B2AR down-regulation was similar in both genotypes. These adverse side effects were correlated with an increase in the number of cells and in collagen levels in the lungs of wild-type but not of GPRASP1 KO mice. Collectively, our data show that GPRASP1 is critically involved in adaptations to chronic activation of B2AR that leads to lung tissue remodelling, development of bronchial hyperresponsiveness and bronchodilator tolerance to B2AR agonist formoterol and could therefore represent an interesting target to limit side effects associated with LABAs.

1. Introduction

G-protein coupled receptors (GPCRs) belong to one of the largest family of proteins encoded by the human genome [1]. These seven

transmembrane domain receptors, which bind a variety of ligands [2], are involved in the regulation of many physiological processes and are targeted by more than 33 % of currently commercially available drugs [3,4]. However, chronic administration of GPCR agonists often leads to

Abbreviations: BTAR, Beta-adrenoceptors; B1AR, Beta1-adrenoceptors; B2AR, Beta2-adrenoceptor; GPCRs, G-protein coupled receptors; GPRASP1, GPCR-associated sorting protein 1; IBMX, 3-Isobutyl-1-Methylxanthine; LABAs, long acting B2AR agonists; MCh, methacholine.

* Correspondence to: Biotechnologie et Signalisation Cellulaire, UMR7242-BSC, 300 Blvd Sebastien Brant, 67412, Illkirch, France.

E-mail addresses: aabuhelo@philadelphia.edu.jo (A. Abu-Helo), daubeuf@unistra.fr (F. Daubeuf), tranchant.thibaud@wanadoo.fr (T. Tranchant), lehalle@unistra.fr (C. Lehalle), gabrielle.zeder-lutz@unistra.fr (G. Zeder-Lutz), valerie.kugler@unistra.fr (V. Kugler), claire.lugnier@unistra.fr (C. Lugnier), nelly.frossard@unistra.fr (N. Frossard), sandra.lecat@unistra.fr (S. Lecat), simonin@unistra.fr (F. Simonin).

<footnote><sup>a</sup> Biotechnologie et Signalisation Cellulaire, UMR 7242, Centre National de la Recherche Scientifique, Université de Strasbourg, Illkirch, France</footnote>

<footnote><sup>b</sup> Plateforme de Chimie Biologique Intégrative de Strasbourg, UAR 3286, Centre National de la Recherche Scientifique, Université de Strasbourg, Illkirch, France</footnote>

<footnote><sup>c</sup> Section de Structures Biologiques, Pharmacologie et Enzymologie, CNRS/Unistra, CRBS, UR, Strasbourg 3072, France</footnote>

<footnote><sup>d</sup> Laboratoire d'Innovation Thérapeutique, Faculté de Pharmacie, UMR 7200, Centre National de la Recherche Scientifique, Université de Strasbourg, Illkirch, France</footnote>

<footnote n="1">¹ Present address: University of Philadelphia, Faculty of Science, Department of Biotechnology and Genetic Engineering, Jarash Road, 20 KM out of Amman, Amman Jordan P.O.Box: 1 Amman - Jordan 19392</footnote>

<footnote n="2">² co-first authors</footnote>

<footnote n="3">³ co-senior authors</footnote>

<footnote n="4">⁴ orcid.org/0000-0003-2852-0860</footnote>
the development of tolerance and paradoxical effects.

Signalling pathways modulated by GPCRs are tightly regulated by numerous interacting proteins some of which are not fully functionally characterized yet. GPCR-associated Sorting Protein 1 (GPRASP1 also reported as GASP1) has been found by us and others as a cytosolic protein that can bind the cytoplasmic C-terminal tail of numerous GPCRs [5-7], the interaction taking place at least with helix 8 of the receptors [7]. The property to interact with the C-tail of GPCRs has been further extended in vitro to other members of the GPRASP/ARMCX protein family particularly those homologues that contain multiple repeats of the unique GASP motif in their central domain [7-11]. At the cellular level, GPRASP1 has been proposed to be involved in the post-endocytic sorting of different GPCRs towards lysosomes and their degradation including delta-opioid receptor, US28, dopaminergic D₂ and D₃, GPR55, CXCR4 and cannabinoid CB₁ receptors [5,12-17]. The role of GPRASP1 in vivo with respect to GPCR signalling and degradation is currently being investigated. In mouse models in which GPRASP1 expression is blunted by gene deletion, the drastic phenotype identified so far is a reduction in the adaptations following chronic activation of GPCRs such as a diminution of analgesic tolerance to repeated agonist activation of CB₁ receptors [18] and a diminution of the hyper-locomotor activity following repeated cocaine treatment that leads to sustain activation of dopaminergic systems [19,20]. These phenotypes have been proposed to be directly linked to the molecular role of GPRASP1 in sorting GPCRs toward lysosomes and degradation [18,20,21]. However, conflicting results have been reported concerning the involvement of GPRASP1 in the down-regulation of dopaminergic receptors after chronic treatment with cocaine [19,20], indicating that the role of GPRASP1 needs further investigation.

In vitro, one of the most documented GPCR interacting with GPRASP1 or with the central domain of GPRASP1 is B2AR, with demonstrations ranging from co-immunoprecipitation in cells [9,22] to surface plasmon resonance and AlphaScreen assay using purified proteins [9,11]. The B2AR is also one of the most characterized GPCRs in vitro and numbers of drugs are targeting it. In particular, B2AR agonists are commonly used in asthma therapy as potent bronchodilators. However, detrimental adaptations following chronic activation of B2AR in patients with asthma have been suspected. Indeed, during the last two decades of the twentieth century, a resurgence of death related to asthma was observed, which has been linked to chronic use of these compounds in monotherapy, in particular with long acting B2AR agonists (LABAs; [23-31]). A meta-analysis of various clinical trials confirmed that patients treated for four weeks or more with the LABA formoterol had a significantly higher risk for serious adverse events [32]. Chronic activation of B2AR was shown to lead to a reduction in the number of receptors in lungs in the rat [33-35]. Using a mouse model [36] or examining asthmatic patients [37], adverse effects associated with chronic administration of LABAs were proposed to be related to the development of airway hyperresponsiveness induced by chronic activation of B2AR but molecular events underlying this phenomenon are still unclear (see [29]).

In the present study, we therefore sought to investigate the role played by GPRASP1 in the degradation of activated B2AR as well as in adaptations following its chronic activation by using both cellular assays and GPRASP1 KO mice.

# 2. Material and methods

## 2.1. Material reagents, vectors and cells

Isoproterenol hydrochloride was purchased from Tocris Bioscience. The phosphodiesterase inhibitor 3-Isobutyl-1-Methylxanthine (IBMX), formoterol fumarate dihydrate was from Sigma-Aldrich. D-Luciferin was from Synchem UG & Co. DMEM, foetal calf serum, penicillin, streptomycin, glutamine and trypsin-EDTA (GIBCO) were purchased from Life technologies. The plasmid encoding the GloSensor™-22F was from

Promega and the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene plasmid # 48138). Mouse anti-phospho-CREB (Ser133) was from Santa Cruz Biotechnology (cat# RRID:AB-1125727 Santa Cruz, CA, USA) while Rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) polyclonal antibodies were from Cell Signalling Technology Inc (cat# RRID:AB-331646). Secondary antibodies (peroxidase-conjugated F(ab')2 fragment donkey anti-rabbit IgG and peroxidase-conjugated F(ab')2 fragment goat anti-mouse IgG) were from Jackson ImmunoResearch Laboratories. Immobilon-P PVDF membrane and Immobilon Western chemiluminescent HRP substrate were from Merck Millipore. HEK293 cells (ATCC Cat# CRL-1573, RRID:CVCL-0045) were cultured in MEM supplemented with 10% fetal calf serum (GIBCO), 100 U/ml penicillin, 100 µg/ml streptomycin and Glutamine at 37°C in a humidified incubator with 5% CO₂.

## 2.2. Cloning and stable expression in HEK293 cells of SP-GFP-human B2AR and mCherry-GPRASP1

The human B2AR sequence was subcloned into pCEP4 Hygro-EGFP by the Ligation Independent Cloning technique leading to a construct allowing the expression of B2AR fused at its N-terminus with EGFP fluorophore preceded by a signal peptide for translocation of the fusion protein through the membrane of the endoplasmic reticulum [38]. The human GPRASP1 sequence was subcloned into pcDNA3.1 neo-mCherry by the Ligation Independent Cloning technique leading to a construct allowing the expression of full length GPRASP1 fused with mCherry at its N-terminus. Constructs were verified by DNA sequencing before transfection by the calcium phosphate technique.

## 2.3. Functional assays in HEK293 cells

### 2.3.1. Fluorescence measurement of agonist-induced degradation of GFP-B2AR

HEK293 cells stably expressing the GFP-tagged human B2AR and mCherry GPRASP1 or not were stimulated during increasing periods of time (from 2 h to 18 h) with 10 µM isoproterenol. At the end of the incubation time, cells were harvested with PBS-EDTA (5 mM) buffer, pH 7.4, resuspended in ice-cold 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.3 % digitonin and incubated at 4 °C for 15 min. Cells were then washed and resuspended in PBS-EDTA and kept on ice until GFP fluorescence measurement with FACS Calibur cytometer (Becton Dickinson).

### 2.3.2. cAMP accumulation

cAMP accumulation assays were performed with adaptation from previous work [39]. HEK293 cells were transiently transfected with 172 ng/cm² of the pGloSensor™-22F plasmid (Promega) using Lipofectamine2000, and, when indicated together with different amount of pcDNA3-GPRASP1 vector, according to the manufacturer instructions. The assays were performed 48 hours post-transfection in the presence of 100 µM of the phosphodiesterase inhibitor IBMX (except for transient GPRASP1 expression, for which the transfection was performed 24 h before cAMP measurement). Cells were loaded with HEPES / BSA buffer (10 mM HEPES, 137.5 mM NaCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 6 mM KCl, 5.6 mM Glucose, 0.4 mM NaH2PO4, 0.1 % BSA) containing 1 mM luciferin for two hours and then stimulated with 1 µM isoproterenol. Luminescence signal was monitored during 28 min at 25 °C (1 read / 2 min). Results were expressed as percentage of HEK293 cells maximal response.

### 2.3.3. CRE reporter gene assay

HEK293 cells were transfected with the pSOM-luc reporter plasmid and increasing amounts GPRASP1 construct as previously described [40]. After 24 h, cells were split into poly-L-lysine coated 12-well plates and serum-starved for 18 hours prior stimulation over a time range of 2 hours with 1 µM of isoproterenol. Cells were then washed twice with
ice-cold PBS and lysed in 100 µl of Luciferase Cell Lysis Buffer (25 mM Tris-phosphate pH 7.8, 2 mM DTT, 10% Glycerol, 1% Triton X-100). Luciferase activity was measured using a luciferase assay buffer containing HEPES/BSA buffer (see composition above), 1 mM ATP and 1 mM luciferin. An aliquot (20 µl) of each sample was mixed with 100 µl of luciferase assay Buffer and the emitted light was measured in a luminescence counter (Victor Light, Perkin Elmer). Values were expressed in percentage of maximum light unit emission by the control cells.

### 2.3.4. Phospho-ERK1/2 and Phospho-CREB analysis

About 150,000 cells were seeded in poly-L-lysine coated 12-well plates. After an overnight serum starvation, cells were stimulated with 1 µM isoproterenol and directly lysed in 2X Laemmli buffer. The amount of protein was determined with Bradford assay on an extra well. Typically, 40 µg of total proteins were separated on 10% polyacrylamide gel and analysed by Western blot. Immobilon-P PVDF membranes were incubated overnight with anti-phospho-p44/p42 MAPK (ERK1/2, Thr202/Tyr204; 1:5000) or anti-phospho-CREB-1 (10E9, 1:500) antibodies. Anti-rabbit or mouse IgG horseradish peroxidase conjugate were used as secondary antibodies. Protein loading homogeneity was confirmed by stripping, washing and reprobing membranes with monoclonal antibody against actin (1:100 000). Signal was developed using the Immobilon Western chemiluminescent HRP substrate detection kit (Millipore) and acquired on a LAS 4000 camera. Bands were quantified using ImageQuant software (GE-Healthcare). Results were expressed as percentage of HEK293 cells maximal response.

## 2.4. Animals

All animal care and experimental procedures complied with the French Animals Scientific Procedures act 2013-118, the European Communities Council Directive (2010/63/EU) and were approved by the French "Ministère de l'Enseignement Supérieur et de la recherche" and ethical committee (CREMEAS, CEEA 35) according to the APAFiS procedure (agreement number #14866-2018042708392769). All mice used in this study were GPRASP1 KO male mice and their wild-type littermates (8-10 weeks old) obtained from intercrosses between F1 heterozygous females and wild-type males (50% 129S2/svPas X 50% C57BL/6 J). Engineering and characterization of GPRASP1 KO line is described in [19]. Mice were housed at three per cage, in an environment maintained at 22 °C, under a normal 12/12-h light/dark cycle (lights on from 08:00-20:00 h) with food and water available ad libitum.

### 2.5. Formoterol administration

Formoterol (400 µg/kg) or vehicle (2-hydroxypropyl-β-cyclodextrine 1.25% in PBS) were administered subcutaneously (s.c., 4 ml/kg) or intranasally (i.n., 1 ml/kg) under anaesthesia with 50 mg/kg ketamine and 3.33 mg/kg xylazine (i.p.).

## 2.6. Measurement of airway resistance by the forced oscillation technique (Flexivent®)

Airway responsiveness to methacholine (MCh) was assessed using the forced oscillation technique (Flexivent®, SCIREQ) as previously described [41,42]. Mice were anesthetized with an intraperitoneal injection of xylasine (Rompun®; 15 mg/kg), followed 10 min later by an intraperitoneal injection of pentobarbital sodium (54 mg/Kg). The trachea was exposed, and an 18-gauge metal needle was inserted into the trachea. Airways were connected to a computer-controlled small animal ventilator, and quasi-sinusoidally ventilated with a tidal volume of 10 ml/Kg at a frequency of 150 breaths/min and a positive end-expiratory pressure of 2 cm H₂O to achieve a mean lung volume close to that during spontaneous breathing. After measurement of the baseline, each mouse was challenged for 10 sec with an aerosol (PBS or

methacholine, MCh 50 mg/ml) generated with an in-line nebulizer and administered directly through the ventilator. For calculation of the effect of MCh, the peak response was used as the mean of the three maximal values and integrated for calculation of airway resistance (R). Airway resistance was expressed as cm H₂O.s.ml⁻¹ [43].

Tolerance to the bronchodilator effect of formoterol was assessed in mice chronically treated (for 28 or 35 days) with formoterol as compared with placebo-treated mice 24 h after the last i.n. administration of formoterol. The effect of acute treatment with formoterol (i.n., 400 µg/kg) on airway resistance R to MCh was measured 15 min after its administration that was performed 10 min after xylasine anesthesia and before the i.p. injection of pentobarbital.

## 2.7. Measurement of airway responsiveness by whole body plethysmography

Airway responsiveness to aerosolized MCh at increasing concentrations (0–0.3 M) was measured by whole body plethysmography (Emka Technologies) in vigil and unrestrained mice, using the enhanced pause (penh) as an index of airway obstruction as previously reported [43]. Briefly, mice were stabilized in the plethysmograph chamber for 30 min until stable baseline, and then exposed to aerosolized saline (30 sec) as a control. Then, mice were challenged every 20 min with increasing doses of aerosolized MCh for 30 sec each, and airway responses were recorded for 20 min.

## 2.8. Preparation of lung total lysates

The left lung (with one lobe) of each mouse was lysed using an ultra-Turrax® (IKA) homogenizer in 0.6 ml ice-cold 20 mM Tris-HCl pH 7.5, 5 mM EGTA, 150 mM NaCl, complete-EDTA free Protease inhibitor, Phostop phosphatase inhibitor cocktails (Roche Diagnostics). Lysates were adjusted with 0.1% Triton X-100 and 1% Tween20 and vortexed in the cold room for 20 min. Unsoluble material was removed by a 10 min centrifugation at 14,000 g at 4 °C. The proteins concentration of the supernatant was measured using the Bradford assay (yield around 5–7 mg/ml), then lung total lysates were stored at –80 °C as aliquots.

## 2.9. Western blot of lung total lysates

Homogenates of lung total lysates were mixed with 4x Laemmli buffer (50 mM Tris pH 6.8, 6 M urea, 6% beta-mercaptoethanol, 3% SDS), denatured 5 min at 95 °C, loaded on a 10% SDS-PAGE gel (14 µg/ lane). Protein samples were transferred to immobilon PVDF membranes. Membranes were blocked for 1 h (using 5% non-fat dried milk in PBS, pH 7.2, Tween 0.1 %), probed overnight with affinity-purified rabbit (in- house polyclonal) anti-GPRASP1 ([7]; 1:10,000). Anti-rabbit IgG horseradish peroxidase conjugate was used as secondary antibody (1:10, 000). Signal was developed using the Immobilon Western chemilumi- nescent HRP substrate detection kit (Millipore) and acquired on a LAS 4000 camera. Bands were quantified using ImageQuant software (GE-Healthcare).

## 2.10. Preparation of lung total membranes

The right lung (with multiple lobes) of each mouse was lysed using an ultra-Turrax® (IKA) homogenizer in 3 ml ice-cold 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.25 M sucrose. After a 500 g spin for 10 min at 4 °C, the supernatant S1 was collected and the pellet P1 underwent a second lysis in 3 ml ice-cold 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.25 M sucrose. The lysate was spun at 1100 g for 10 min at 4 °C and the supernatant S2 was collected and mixed with the S1 supernatant. The lung total membranes were recovered from a 100,000 g spin of 30 min at 4 °C. The membrane pellet was washed with 25 ml 50 mM of Tris-HCl pH 7.4, 1 mM EDTA, 0.25 M sucrose and the membranes were collected by a second 100,000 g spin of 30 min at 4 °C. The membrane pellet was
suspended in 0.75 ml ice-cold 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.25 M sucrose and homogenized by 10 passages through a 2 ml potter, then the final volume was adjusted to 1.5 ml. The protein concentration was measured using the Bradford assay (yield around 1–2 mg/ml) and total membranes were stored at –80 °C as aliquots.

## 2.11. Radioligand binding assay

Binding experiments were adapted from [33,44] with the following modifications. 50 µg of total membrane proteins of the right lung were incubated in 500 µl total volume using ice-cold 50 mM Tris-HCl pH 7.4 binding buffer. Total Beta-adrenoceptors (BTAR) were detected using [³H]-CGP-12177 (4-[3-[(1,1-Dimethylethyl)amino]-2-hydroxypropoxy] -1,3-dihydro-2H-benzimidazol-2-one hydrochloride) at a final concentration ranging from 0.065 nM to 2.2 nM for K_d measurements. The mixture was incubated for 30 min at 37 °C and the bound material was collected on pre-soaked (10 min in ice-cold 50 mM Tris-HCl pH 7.4, 0.1 % PEI) GF/B filters (Whatmann) using a Brandel harvester. The filters were immediately rinsed 4 times with ice-cold binding buffer. Non-specific binding was measured with excess of the non-selective Beta-adrenoceptor antagonist alprenolol (2 µM final concentration). For K_i measurements, binding was measured as the difference between total binding of 2 nM [³H]-CGP-12177 in competition with the antagonists CGP-20712 or ICI-118551 at concentrations ranging from 0.05 nM to 5 µM. Specific BTAR binding was measured as the difference between total binding of 1.5 nM [³H]-CGP-12177 and binding with an excess of the non-selective BTAR antagonist alprenolol (2 µM final concentration). B2AR binding was measured as the difference between total binding of 1.5 nM [³H]-CGP-12177 and binding with an excess of the Beta1-adrenergic receptor (B1AR) selective antagonist CGP-20712 (1 µM final concentration), B1AR binding was measured as the difference between total binding of 1.5 nM [³H]-CGP-12177 and binding with an excess of the B2AR selective antagonist ICI-118551 (0.5 µM final concentration). Radioactivity was quantified using a liquid scintillation Beckman counter. Assays were performed in monoplicates for K_d measurements (n = 5) and in duplicates for K_i evaluations and for Bmax measurements of B2AR and B1AR (n = 5).

## 2.12. Histological examination

The left lungs were fixed (4 % formaldehyde) and paraffin-embedded. Six µm sections were cut, mounted on Superfrost glass slides (Fisher Scientific) and stained with Haematoxylin-Eosin for epithelium thickness measurements (H&E) or Masson's trichrome staining for collagen visualization as previously described [45]. Smooth muscle was labelled by immunohistochemistry against alpha-smooth muscle actin (αSMA, Abcam, ab5694, 400 ng/ml). Smooth muscle thickness, tissue density, the number of cells labelled with DAPI and tissue area were measured with the *Cell dimension software* coupled to a DP72 camera (Olympus).

## 2.13. Measurement of alpha-smooth muscle actin, collagen and MPO in lung homogenate

Lung right lobes were homogenized (ultra-Turrax®, IKA) in Aqua B (Braun) centrifuged (10,000 g, 10 min, 4 °C) and the supernatants were collected and stored for alpha-smooth muscle actin, collagen and MPO measurements. Alpha-smooth muscle actin (αSMA) was measured by direct ELISA on supernatant proteins (0.5 mg/ml) coated in plate (Nunc, 456537) with anti-αSMA antibody (Abcam, ab5694, 200 ng/ml). Neutrophil myeloperoxidase (MPO) activity was used as an indirect measurement of tissue neutrophil infiltration in lung supernatant with the hexadecyltrimethyl ammonium bromide (HTAB) substrate and reported to total protein in lung homogenate (BCA) [46]. Lung collagen in lung supernatant was measured using Sircol™ colorimetric assay (Bio-color Ltd).

## 2.14. Data and statistical analysis

Data were analyzed and graphs were created using GraphPad Prism software (San Diego, CA, USA) and represented as means ± SEM. Statistical analyses were performed using GraphPad Prism software. Comparisons for *in vitro* experiments were performed using two-way (Figs. 1 and 2) or one-way ANOVA (Fig. 3) followed by Bonferroni Post-hoc test between groups when appropriate. Comparisons on airway responsiveness to MCh (Fig. 4) and on airway remodelling (Figs. 5 and 6) were performed using two-way and one-way ANOVA (respectively) followed by Tukey's post-hoc test between groups. P < 0.05 was considered statistically significant.

# 3. Results

## 3.1. GPRASP1 does not modify the kinetic and extent of degradation of B2AR *in vitro*

In order to study the role played by GPRASP1 in agonist-stimulated B2AR trafficking and signalling, we first used HEK293 cells for which we and others have previously shown that they respond to B2AR agonists, indicating endogenous expression of the receptor [47–49]. Interestingly, HEK293 cells do not express GPRASP1 but express GPRASP2 as shown by Western blot using our in-house validated polyclonal antibody [7,11, 19]. Since GPRASP1 has been proposed to mediate post-endocytic sorting of GPCRs towards lysosomes, we first tested the effect of heterologous expression of GPRASP1 on agonist-activated B2AR degradation. To quantify B2AR receptors, we used HEK293 cells stably expressing N-terminally GFP-tagged B2AR receptor (allowing a finely tuned detection of the receptor) in the presence or not of N-terminally mCherry-tagged GPRASP1. B2AR quantities following different time points of isoproterenol agonist exposure were monitored by measuring GFP fluorescence using flow cytometry on digitonin-permeabilised cells. As shown in Fig. 1A, we observed that the total GFP fluorescence of cells decreased in a time-dependent manner following stimulation with 1 µM isoproterenol leading to more than 40 % diminution of the GFP fluorescence after 18 h exposure to agonist as compared with non-activated cells indicating a substantial degradation of B2AR (Fig. 1A bold line). Similar results were obtained with cells co-expressing mCherry-GPRASP1 (Fig. 1A dashed line) indicating that GPRASP1 is not involved in the degradation of agonist-activated B2AR in this cellular context.

## 3.2. GPRASP1 increases B2AR-mediated cAMP production in vitro and potentiates further B2AR downstream signalling

We next investigated the impact of GPRASP1 heterologous expression on endogenous B2AR signalling (Fig. 1B) in HEK293 cells including modulation of cAMP level and phosphorylation of ERK and CREB (Fig. 1C-F). We monitored cAMP production upon 1 µM isoproterenol stimulation in living cells using the reporter permuted luciferase Glo-Sensor™-22F (see Material and methods) that produces a luminescent signal proportional to cAMP quantities. Wild-type HEK293 cells with endogenous B2AR expression and HEK293 cells stably expressing mCherry-GPRASP1 were compared. We observed for both cell lines similar kinetics of isoproterenol-induced increase in cAMP production, but efficacy of isoproterenol was significantly increased (about two-fold) in cells overexpressing GPRASP1 (Fig. 1C), suggesting that GPRASP1 potentiates the Gs signalling pathway activated by B2AR.

We next compared further downstream pathways associated with B2AR activation between these two cell lines. In lysates from cells stimulated by 1 µM isoproterenol at different time points, we analysed by Western blotting the level of activation of the MAPKinase pathway by following ERK1/2 phosphorylation (Fig. 1D and E) and of the Gs pathway by following the cAMP Responsive Element Binding protein (CREB) phosphorylation (Fig. 1D and F). As shown in Figs. 1D and E, in time-course experiments on ERK1/2 phosphorylation, we observed that
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Effect of mCherry-GPRASP1 stable expression on activated B2AR degradation and signalling in HEK293 cells. (A) Cells stably over-expressing a GFP-tagged B2AR ± mCherry-GPRASP1 were used. GFP-fluorescence was monitored by flow cytometry in digitonin-permeabilized cells after different times of exposure to isoproterenol (1 µM). Non stimulated cells were taken as 100 % of GFP fluorescence to normalize median fluorescence obtained. Data are shown as means ± SEM of three independent experiments. (B) Scheme illustrating the signalling pathways investigated in C, D, E and F and Fig. 2. ISO= isoproterenol, AC= Adenylate cyclase, ARRB = Beta-arrestin, CRE = cAMP responsive element (C) HEK293 cells stably expressing or not mCherry-GPRASP1 and transiently expressing the GloSensor-22F (to detect cAMP accumulation) were subjected to 1 µM isoproterenol stimulation. Luminescence was monitored in real time (every two min) at 25 °C. Data are shown as means ± SEM of three independent experiments. Comparison of the full kinetics was made by two-way ANOVA. \*\* p < 0.01. (D) HEK293 cells stably overexpressing or not mCherry-GPRASP1 were serum-starved for 18 h and then stimulated with 1 µM isoproterenol for different periods of time. Cell lysates were subjected to SDS-page and Western-blot analysis. Representative immunoblots of one experiment are shown (n = 3). (E) Time-course of MAPK ERK1/2 phosphorylation evaluated by Western blot and normalized to actin expression level. Maximal response obtained in HEK293 cells was chosen as 100 %. Data are shown as means ± SEM of 3 independent experiments. Comparisons were made by two-way ANOVA followed by Bonferroni's test. \* p < 0.05, \*\* p < 0.01. (F) Time-course of CREB phosphorylation evaluated by Western blot and normalized to actin expression level. Maximal response obtained in control HEK293 cells was chosen as 100 %. Data are shown as means ± SEM of 3 independent experiments. Comparisons were made by two-way ANOVA followed by Bonferroni's test. \* p < 0.05, \*\*\* p < 0.001.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Effect of transient GPRASP1 expression on B2AR signalling through Gs coupling. (A) HEK293 were transiently transfected with increasing amounts (0.1 µg, 0.5 µg and 1 µg) of a plasmid encoding wild-type GPRASP1 and a constant amount of the GloSensor-22F. After 24 h, cells were incubated with isoproterenol (1 µM) and cAMP was monitored as described in Material and Methods. Basal (NS) and maximal responses (Iso) are shown as means ± SEM of 3 experiments performed in triplicates. Comparisons were made by two-way ANOVA followed by Bonferroni's test. \*\*\* p < 0.001 (B) HEK293 were transiently transfected with 1 µg or 1.5 µg of a plasmid encoding GPRASP1 and the pSOMluc reporter vector encoding the luciferase under a CRE-driven promotor. Luminescence signal from cell lysates after activation with 1 µM isoproterenol, indicating CRE-driven transcription, is plotted as a % of the maximal response of luminescence upon isoproterenol activation of mock transfected cells. Basal (NS) and maximal responses (Iso) are shown as means ± SEM of 3 experiments performed in triplicates. Comparisons were made by two-way ANOVA followed by Bonferroni's test. \*\* p < 0.01, \*\*\* p < 0.001. A representative Western blot using anti-GPRASP1 in-house polyclonal antibodies illustrating the increasing amount of GPRASP1 expressed in cell lysates depending on the GPRASP1 expression vector quantities transfected is shown below.</figcaption></figure>

HEK-mCherry-GPRASP1 cells displayed an earlier peak than control HEK293 cells (5 min for mCherry-GPRASP1 expressing cells versus 15 min for HEK293 cells (Fig. 1D and E). Moreover, the P-ERK1/2 response efficacy was remarkably increased for the GPRASP1 expressing cells reaching a peak value of around 250 % of the maximal response observed in HEK293 cells. Similarly, for the phosphorylation of CREB, GPRASP1 expressing cells reached a peak of maximal phosphorylation at 5 min (Fig. 1D and F), whereas this peak occurred between 5 min and 15 min in control HEK293 cells. In addition, the P-CREB maximal peak value was doubled in the mCherry-GPRASP1 expressing cells as compared to control wild-type HEK293 cells.

To confirm the effect of GPRASP1 expression on downstream B2AR signalling pathways, we studied the effect of increasing amount of GPRASP1 using transient transfection of HEK293 cells with different quantities of vector encoding untagged-GPRASP1 (Fig. 2). We observed that wild-type GPRASP1 dose-dependently increased the efficacy of isoproterenol-induced cAMP production (Fig. 2A), reaching cAMP levels of around 160 % of control values comparable to those observed with the mCherry-GPRASP1 expressing cell line. We then examined the impact of GPRASP1 expression on isoproterenol-induced CRE-driven transcription by using a luciferase reporter gene assay. Isoproterenol induced an increase in luminescence as compared with non-stimulated cells indicating CRE-dependent activation of gene transcription (Fig. 2B). GPRASP1 increased dose-dependently the basal level of CRE-driven luciferase gene transcription from 50 % to around 80 % (Fig. 2B, non-stimulated cells) and isoproterenol stimulation triggered a further significant increase of the luminescent signal when GPRASP1 was expressed, reaching up to 180 % in cells expressing the higher amount of GPRASP1 as compared to mock-transfected cells (Fig. 2B). Taken together, our data indicates that in cells, GPRASP1 can modulate agonist-induced B2AR signalling but not its degradation.

### 3.3. Down-regulation of B2AR receptors induced by chronic formoterol is similar in WT and GPRASP1 KO mice

Our *in vitro* results prompted us to investigate further the role of GPRASP1 on B2AR functions *in vivo*. We examined the role of GPRASP1 on agonist-induced B2AR degradation by comparing wild-type and GPRASP1 KO mice in a model of bronchial hyperresponsiveness induced by daily subcutaneous administration of the LABA formoterol for 28 days. We first verified that GPRASP1 is expressed in lung extracts by Western blotting using our validated in-house polyclonal antibodies ([7, 11,19]; Fig. S1). In a second step, we quantified the total number of Beta-adrenoceptor binding sites (BTAR) in lung membrane preparations of WT and GPRASP1 KO mice with the radiolabelled non-selective BAR antagonist CGP-12177. As shown in Fig. 3A, saturation curves obtained with lung membranes of WT and GPRASP1 KO mice treated with vehicle were very similar with a total number of β-adrenoceptors binding sites (Bmax) of 800 ± 38 and 888 ± 32 fmol/mg of proteins with Kd values of 0.25 ± 0.04 and 0.27 ± 0.04 nM (respectively). In the same experiment, we observed that chronic formoterol treatment induced a strong decrease of the BTAR (approximately 40 %) both in WT and GPRASP1 KO mice while Kd values were unchanged as compared to non-treated animals (0.25 ± 0.02 and 0.23 ± 0.04 nM). We then evaluated the proportion of B1AR and B2AR present in lung membranes from WT and GPRASP1 KO animals by performing competition experiments with non-radiolabelled selective B1AR antagonist CGP-20712 or selective B2AR antagonist ICI-118551 in presence of a saturating concentration of [³H]CGP-12177. Preliminary dose-response competition curves performed with these two compounds on saline-treated WT and GPRASP1 KO lung membranes showed that 1/3 of BTAR binding sites could be displaced by increasing quantities of CGP-20712 (Fig. S2A) while 2/3 of BTAR binding sites could be displaced by increasing quantities of ICI-118551 (Fig. S2B). Ki values for both ligands were similar between WT and KO animals (CGP-20712: 18 ± 7 nM and 9 ± 2 nM, respectively; ICI-118551: 0.78 ± 0.006 nM and 1.05 ± 0.05 nM, respectively), indicating that GPRASP1 deletion did not affect the affinity of these ligands for their receptor. In animals that were chronically treated with formoterol for 28 days, we observed that the down-regulation of BTAR (Fig. 3A and B) was specifically due to a decrease of almost 2/3 of the B2AR (Fig. 3C) while there was no significant reduction in the number of B1AR binding sites (Fig. 3D). No difference was observed between lung membranes from WT and GPRASP1 KO mice. Altogether, these data clearly show that chronic formoterol treatment in mice lead to a strong and specific down-regulation of B2AR that is not affected by the absence of GPRASP1 indicating that this protein is not critically involved in agonist-induced degradation of B2AR.
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effect of chronic administration of formoterol on the number of Beta-adrenoceptor binding sites in lung membranes of WT and GPRASP1 KO mice. WT and GPRASP1 KO were administered subcutaneously (s.c.) with 400 µg/kg of formoterol or vehicle once daily for 28 consecutive days. The day after the last formoterol administration, lungs were collected and total membranes were prepared, washed and stored before being submitted to analysis in binding experiments (A) Saturation binding curves of [³H]CGP-12177 from lung membranes (50 µg of proteins) of WT (black) and GPRASP1 KO (blue) mice chronically treated with formoterol (plain symbols) or vehicle (open symbols). Specific binding was measured as the difference between total binding of [³H]CGP-12177 and binding with an excess of the non-selective beta-adrenoceptor antagonist alprenolol (2 µM final concentration). Values are shown as means ± SEM of single measurements from lung membranes of 5 saline- and 10 formoterol-treated animals. Comparisons were made by one-way ANOVA followed by Bonferroni's test. \*\*\*P < 0.001 between formoterol and saline treated animals of the same genotype at saturating concentrations of [³H]CGP-12177 of 1 and 2.5 nM (B) BTAR present in lung membranes (50 µg of proteins) of WT (black circles) versus GPRASP1 KO (blue triangles) upon chronic subcutaneous administration of vehicle (open symbols) or of formoterol (closed symbols) in binding experiments with a saturating concentration of [³H]CGP-12177 (1.5 nM). (C) B2AR present in lung membranes of WT (black circles) versus GPRASP1 KO (blue triangles) upon chronic subcutaneous administration of vehicle (open symbols) or of formoterol (closed symbols) in binding experiments with a saturating concentration of [³H]CGP-12177 (1.5 nM) in presence of the selective B1AR antagonist CGP-20712 (1 µM). (D) B1AR present in lung membranes of WT (black circles) versus GPRASP1 KO (blue triangles) upon chronic subcutaneous administration of vehicle (open symbols) or of formoterol (closed symbols) in binding experiments with a saturating concentration of [³H]CGP-12177 in presence of the selective B2AR antagonist ICI-118551 (0.5 µM). Data in B, C and D are shown as individual values with means ± SEM of duplicate measurements from lung membranes of 5 animals. Comparisons were made by one-way ANOVA followed by Bonferroni's test. \*\*\*P < 0.001 as compared to WT saline animals; \#\#\# P < 0.001 as compared to KO saline animals.</figcaption></figure>

**3.4. GPRASP1 KO mice are protected from airway hyperresponsiveness induced by chronic subcutaneous administration of formoterol**

We next examine the consequences of daily formoterol administration (400 µg/kg, sc) on the respiratory function of WT and GPRASP1 KO mice using the forced oscillation technique (FlexiVent®). As shown in Fig. 4A, in mice chronically administered with vehicle, acute administration of the muscarinic M3 agonist methacholine (MCh) induced a bronchoconstriction, as revealed by an increase in airway resistance $R_t$, that was similar in WT and GPRASP1 KO animals (black open circles and blue open triangle). As expected [36], in WT animals daily subcutaneous injections of formoterol for 28 days led to an increased response to acute MCh revealing the development of bronchial hyperresponsiveness induced by chronic activation of B2AR receptors (black plain circles versus black open circles). This phenomenon was completely absent in GPRASP1 KO mice (blue plain triangles versus blue open triangles), indicating that the expression of GPRASP1 is mandatory for the development of bronchial hyperresponsiveness induced by chronic activation of B2AR receptors with formoterol. Together with our observations on lung membranes (Fig. 3), these results suggest that GPRASP1 could play a role in B2AR signalling rather than in sorting of the receptors towards lysosomes and degradation.

**3.5. Airway hyperresponsiveness and tolerance induced by chronic intranasal formoterol is absent in GPRASP1 KO mice**

According to these previous results, we decided to investigate in detail the consequences of chronic formoterol on lung function following daily intranasal (instead of subcutaneous) administrations, which more closely mirrors the situation observed in humans who develop bronchial hyperresponsiveness due to chronic LABA inhalation during asthma treatment. In a first step, we observed with FlexiVent®

that acute bronchodilator activity of intranasal formoterol (400 µg/kg) was similar in WT and GPRASP1 KO mice (Fig. S3). We then performed daily intranasal administrations of formoterol in WT and GPRASP1 KO mice for 35 days and measured the enhanced pause (PenH) as a characteristic of bronchial reactivity of the animals using whole-body barometric plethysmography on vigil animals at day 28 (Fig. 4B) followed by the forced oscillation technique on anesthetized animals on day 35 (Fig. 4C). Both techniques revealed that chronic formoterol induced the development of a significant bronchial hyperreactivity to MCh (0.3 M and 50 mg/ml, respectively) in WT animals that was strongly attenuated in GPRASP1 KO mice. In the same model, we next investigated the ability of acute formoterol to reduce MCh bronchoconstriction after chronic intranasal formoterol treatment (Fig. 4D). Both in WT and GPRASP1 KO vehicle-treated mice (open symbols), acute intranasal administration of formoterol (400 µg/kg, grey histograms) 15 min before MCh significantly prevented the bronchoconstriction induced by MCh. In mice treated with chronic formoterol (plain circles), acute intranasal administration of formoterol (grey histograms) failed to prevent bronchoconstriction induced by MCh in WT animals (black plain circles) while the bronchodilator activity of formoterol was present in GPRASP1 KO mice (blue plain triangles) to the same level that was observed in animals chronically administered with vehicle (blue open triangles). These data indicate that tolerance to the bronchodilator activity of formoterol develops in WT mice chronically treated with intranasal formoterol while GPRASP1 KO are protected from tolerance and respond as on the first day. As observed in our previous experiments, BTARs were similarly down-regulated in lung membranes of WT and GPRASP1 KO mice following chronic formoterol (Fig. S4). Together, these results indicate that GPRASP1 is critically involved in the adaptations associated with chronic LABA treatment, including the development of bronchial hyperresponsiveness and bronchodilator tolerance, but not in B2AR down-regulation.
<figure><img src="image_6.png" /></figure>

**Fig. 4.** Chronic administration of formoterol increases airway hyperresponsiveness to methacholine in WT but not in GPRASP1 KO mice. (A) Airway resistance (R) was assessed by FlexiVent technique in response to aerosolized PBS and MCh (50 mg/ml) on WT and GPRASP1 KO mice treated once daily for 28 consecutive days with formoterol (400 µg/kg) or vehicle by subcutaneous route. (B) Airway reactivity (Penh) to increasing doses of methacholine (MCh, 0–0.3 M) measured by whole body plethysmography in vigil and unrestrained WT and GPRASP1 KO mice treated once daily for 28 consecutive days with formoterol (400 µg/kg) or vehicle by intranasal route. (C) Airway resistance (R) was measured by FlexiVent technique in response to aerosolized PBS and MCh (50 mg/ml) on WT and GPRASP1 KO mice treated once daily for 35 consecutive days with formoterol (400 µg/kg) or vehicle by intranasal route. (D) Acute intranasal administration of formoterol (400 µg/kg, grey histograms) or vehicle (white histograms) was performed between xylasine and pentobarbital anaesthesia to measure acute bronchodilator activity on airway resistance (R) assessed by FlexiVent technique in response to aerosolized PBS and MCh (50 mg/ml). WT or GPRASP1 KO vehicle-treated mice (open symbols) were compared to mice chronically treated for 35 days with formoterol (400 µg/kg, plain symbols). Data are shown in A, C and D as individual values with means ± SEM of n = 5–10 mice per group. Comparisons were made by two-way ANOVA followed by Tukey's test. \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001, ns = non-significant. Data shown in B are means ± SEM of n = 5–10 mice per group. Comparisons were made by two-way ANOVA followed by Tukey's test. \#P < 0.05, \#\#P < 0.001 vs WT-Veh group \*P < 0.05 vs WT-For group.

### 3.6. GPRASP1 KO mice are protected from lung remodelling induced by chronic formoterol

In order to study the origin of the protective effect of the absence of GPRASP1 on the adverse effects associated with chronic formoterol, we investigated the potential lung remodelling, which may result from chronic formoterol after 35 days of daily intranasal administration. As shown in Fig. 5A and B, histological analysis of lungs epithelium of WT and GPRASP1 KO mice did not show any significant difference in the bronchial epithelium thickness between vehicle and formoterol-treated mice. Similarly, we did not observe any effect of chronic formoterol treatment on bronchial smooth muscle density analysed by IHC (anti-αSMA) on lung sections of WT and GPRASP1 KO animals (Fig. 5C) and αSMA levels measured by ELISA in lung homogenates (Fig. 5D).

In agreement with the histopathological analysis, measurement of the MPO activity in lung homogenates confirmed the absence of a neutrophilic infiltration and inflammation in response to the daily intranasal administration of vehicle or formoterol in WT and GPRASP1 KO mice (Fig. 6A). However, DAPI nucleus labelling of cells in lung tissue sections revealed a significant increase in the total number of cells in WT but not GPRASP1 KO mice following chronic intranasal

formoterol administration (Fig. 6B).

Finally, we observed collagen accumulation around bronchi and blood vessels on tissue sections stained with Masson's trichrome in WT but not GPRASP1 KO treated with formoterol (Fig. 6C). This observation was confirmed by the measurement of total collagen in lung homogenates by a collagen colorimetric assay (Fig. 6D) that revealed a significant remodelling of the extracellular matrix in WT mice chronically treated with formoterol while GPRASP1 KO mice were protected.

Overall, histopathological analysis indicated that tissue remodelling associated with chronic administration of formoterol was absent in GPRASP1 KO as compared to WT mice thus confirming the mandatory role of GPRASP1 in adaptations associated with chronic activation of B2AR.

### 4. Discussion and conclusion

In this study, we have observed that GPRASP1 can dose-dependently increase acute B2AR cAMP and MAPK signalling in HEK-293 cells but has no effect on agonist-stimulated receptor degradation. This latter observation was further confirmed *in vivo* by measurements of B2AR binding sites in lungs of WT or GPRASP1 KO mice chronically treated
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Epithelium thickness and smooth muscle are not affected by chronic intranasal administrations of formoterol in WT mice and GPRASP1 KO mice. Histological and biochemical analysis of lungs of WT and GPRASP1 KO animals having received a daily dose of formoterol (400 µg/kg, i.n.) or vehicle for 35 days. (A) Representative Haematoxylin-Eosin (H&E) stained lung sections used for epithelium thickness measurements. (B) Epithelium thickness measured on lung sections and expressed in µm. (C) Smooth muscle density identified by anti-αSMA immunohistochemistry measured on lung sections and expressed as the measure of labelled smooth muscle surface normalized to the length of the bronchial perimeter (in µm²/µm). (D) Alpha-smooth muscle actin measured by ELISA and reported by mg of total proteins in lung homogenates. Data are shown as individual values with means ± SEM of n = 7–9 mice per group.</figcaption></figure>

with formoterol using two different routes of administration, subcutaneous and intranasal, for 28 and 35 days, respectively. In both cases, we could show a similar reduction of B2AR binding sites in WT and GPRASP1 KO mice. We further observed that although acute formoterol displayed similar bronchodilator effect in WT and GPRASP1 deficient animals, chronic administration of this compound induced bronchial hyperresponsiveness and tolerance in WT but not in GPRASP1 KO mice. Furthermore, we have combined these physiological measurements to biochemical and histological analyses performed on the lungs of animals collected at the end of the 35 days of formoterol treatment and observed that the development of bronchial hyperresponsiveness and tolerance to chronic formoterol is associated with an increase in the total number of cells and in collagen deposition in WT mice that was not observed in GPRASP1 KO mice. This study is the first one that allowed to identify GPRASP1 as a novel key player in the development of these side effects by combining measurements of most parameters described in the literature including B2AR numbers, airway hyperresponsiveness, tolerance to β2 agonists, tissue remodeling and inflammation (see Table S1).

In the present study, we observed that stimulation of B2AR by systemic daily injection of formoterol for 28 days led to a reduction of around 60 % of B2AR binding sites in lungs of both WT and GPRASP1 KO mice. This result is in agreement with our *in vitro* observations and clearly show that GPRASP1 is not necessary for down-regulation of B2AR despite its proposed function in triggering degradation of different GPCRs [5]. The reduction that we observed in WT mice is similar to the reduction in the quantities of B2AR in lungs of rats following systemic isoproterenol, albuterol or salmeterol treatment for 7 consecutive days ([33–35], see Table S1). This down-regulation could arise from both degradation in lysosomes of the receptor at the protein level and a reduction of the level or the translation of its messenger RNA (via let-7f

miRNA; [50]). In the three studies that evaluated the level of B2AR mRNA after a week of systemic activation of the receptor (see Table S1), two of them observed a reduction in quantities at mRNA and protein levels [33–35] but one did not detect any change at the mRNA level [34]. Therefore, in our study, we can propose that GPRASP1 is not necessary for receptor degradation, in as much as lysosomal degradation of B2AR is in part responsible for the reduction in the quantities of B2AR in lungs following their chronic activation. In addition, we found that 35 days of intranasal formoterol in GPRASP1 KO mice did not reduce the capacity of an acute dose of formoterol, 24 h after the last administration, to diminish bronchoconstriction by MCh, whereas in WT mice, tolerance to the bronchodilator effect of formoterol had fully developed. This absence of agonist tolerance observed in GPRASP1 KO mice as a result of prolonged treatment with formoterol indicates that the remaining B2AR binding sites are sufficient to mediate bronchodilation, suggesting the existence of a significant reserve (or spared) of B2AR in the lung as proposed previously [51–53]. It also clearly implicates GPRASP1 in the development of tolerance and mechanistically dissociates this phenomenon from receptor degradation.

Regarding the mechanism of tolerance, it is often associated with receptor desensitization or tachyphylaxis mechanisms [54,55]. Previous studies in guinea pig [56] and rat [33,34] reported desensitization to bronchodilation following sustained β-adrenoreceptors activation (Table S1). The desensitization was proposed to be heterologous in rats due to both down-regulation of Gαs measured by Western blot and an increase in the activity of the phosphodiesterases PDE3 and PDE4 tested *ex vivo* [33,34]. The increase in PDE activity was also observed *ex vivo* in rabbit [57] but was not confirmed in human. Indeed, an *in vitro* study using human airway smooth muscle cells from asthmatic and non-asthmatic volunteers showed that although mRNA levels of PDE
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Lung remodelling in response to chronic intranasal administrations of formoterol in WT but not in GPRASP1 KO mice. Histological and biochemical analysis of lungs of WT and GPRASP1 KO animals having received a daily dose of formoterol (400 µg/kg, i.n.) or vehicle for 35 days. (A) Neutrophil myeloperoxidase (MPO) activity in lung homogenate (B) DAPI positive labelling was used to count cells on histologic lung slides. (C) Representative Masson's trichrome stained lung sections demonstrating collagen distribution (in blue) around bronchus and vessels. (D) Collagen in lung homogenate was measured by collagen colorimetric assay and represented as percent of collagen in total proteins. Data are shown as individual values with means ± SEM of n = 7–9 mice per group. Comparisons were made by one-way ANOVA followed by Tukey's test. \*P < 0.05 and \*\*P < 0.001, ns = non-significant.</figcaption></figure>

were altered upon chronic Beta-adrenoceptors activation there was no effect on PDE activity [58]. In this study, we tried to investigate the consequences of GPRASP1 knock down in human bronchial smooth muscle cells on the production of cAMP induced by B2AR activation with formoterol. Although the expression of GPRASP1 was reduced in cells transfected with GPRASP1 siRNA, we did not observe any significant impact on cAMP production induced by acute formoterol in those cells as compared to control cells (Fig. S5). Similarly, desensitization of the B2AR response observed after a 48 h treatment of cells with formoterol was similar in control and GPRASP1 siRNA-treated cells (Fig. S5), suggesting that GPRASP1 is not critically involved in B2AR desensitization in human bronchial smooth muscle cells.

GPRASP1 has been shown *in vitro* to interact with Gαs [16] and we have now observed in this study that GPRASP1 expression potentiates acute B2AR/Gαs/cAMP activity. Potentiation of cAMP synthesis triggered by GPRASP1 was also observed previously with the constitutively activated viral chemokine US28 receptor [14]. It is thus tempting to speculate that a role of the acute Gαs/AC/cAMP pathway in the development of tolerance to B2AR agonist might depend on GPRASP1. Our observations further question the novel paradigm that proposes to develop Gαs-biased B2AR agonists over the β-arrestin pathway that fail to evoke airway smooth muscle cell tachyphylaxis *in vitro* [54,59–61], as promising drugs to reduce tolerance and the development of bronchial hyperresponsiveness in the treatment of asthma. These compounds will undoubtedly need further evaluation in relevant animal models. In addition, our data suggest that GPRASP1 and/or GPRASP1 associated signalling events could be interesting to target to limit the development of bronchial hyperreactivity and tolerance upon chronic administration of LABAs.

At the same time than observing a potentiation of the Gs pathway, we also observed that GPRASP1 potentiates acute B2AR/MAPKinase activation *in vitro*. GPRASP1 has also been recently proposed to potentiate ERK phosphorylation via the IGFR1/AKT pathway [62]. Previous *in vitro* studies have suggested that the airway hyperresponsiveness observed in animals could be due to a modification in B2AR signalling in airway smooth muscle cells [57,63] or/and in human airway epithelial cells [64]. More specifically, it has been suggested that repeated B2AR agonist treatments could lead to the stimulation of β-arrestin/MAPKinase/ERK dependent signalling pathways, promoting inflammation and bronchial hyperresponsiveness [29,53,65,66]. In our model, examination of lung histological sections and measurement of myeloperoxidase in the lungs clearly showed that inflammation did not develop in animals that were chronically treated with formoterol. After careful analysis of the literature, inflammation was only observed in animal models when allergic-like conditions have been implemented together with agonists of B2AR chronic administration ([64,67–72], see Table S1). In these studies, the effect of B2AR chronic administration without allergen was not investigated except once, when an excess of mucin deposition was observed upon intratracheal administration of IL-13 and formoterol nebulization for 21 days [64]. However, formoterol nebulization alone did not produce any significant increase in mucin deposition [64]. Interestingly, a previous study of chronic activation of B2AR with inhaled salbutamol in mice also reported the development of bronchial hyperresponsiveness together with airway mucosal thickening due to proliferation of tracheal epithelial cells [36]. In this study, both phenomena were blocked by simultaneous injections of the selective MEK1/2 PD98059 inhibitor. In our model, the increase in the total number of cells and in collagen deposition was absent in
GPRASP1 KO mice. Proliferation induced by activation of the MAP ki-
nase pathway is well known and collagen secretion by a number of cells
in the lung notably during fibrosis is also known to be inhibited by
MEK1/2 inhibitors [73]. Thus, the effect of GPRASP1 on bronchial
hyperresponsiveness could be due to the exacerbated activation of the
MAP kinase signalling pathway that we have observed *in vitro*. Very
interestingly, a recent report identified an endosomally-localized non--
canonical signalling axis comprising Gαs, RAF and MAP kinase that re-
sults in endosomal ERK activity that propagates into the nucleus
following B2AR activation [74].

In conclusion, this study presents convergent *in vitro* and *in vivo* data indicating that GPRASP1 is not implicated in agonist-stimulated B2AR degradation but rather contributes to B2AR “adverse” signalling pathways that participate to the development of compensatory adaptations following chronic stimulation of B2AR. According to these observations we propose that developing biased B2AR agonists that do not recruit/activate GPRASP1 associated signalling or compounds that would block the interaction between B2AR and GPRASP1 might represent a promising strategy to reduce adverse side effects including bronchial hyperresponsiveness and tolerance associated with classical LABAs.

CRediT authorship contribution statement

**Simonin Frederic:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. **Abu-Helo Alaa:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Daubeuf françois:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Tranchant Thibaud:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Lehalle Christine:** Visualization, Validation, Methodology, Investigation. **Elhabazi Khadija:** Visualization, Validation, Methodology, Investigation. **Zeder-Lutz Gabrielle:** Visualization, Validation, Methodology, Investigation. **Kugler Valerie:** Validation, Methodology, Investigation. **Lugnier Claire:** Validation, Methodology, Investigation. **Frossard Nelly:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Conceptualization. **Lecat Sandra:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Conceptualization.

Funding

This research was funded by the CNRS, Region Grand-Est, LABEX
Medalis (ANR-10- LABX-0034, Programme d'investissement d'avenir),
graduate school of pain EURIDOL of the University of Strasbourg (ANR-
17-EURE-0022, Programme d'investissement d'avenir), IdEx Unistra
(ANR-10-IDEX-0002) and by the SFRI-STRAT'US project (ANR-20-SFRI-
0012) under the framework of the French Program "Investments for the
Future" given to the Strasbourg Drug Institute (IMS), as part of the
Interdisciplinary Thematic Institute (ITI) 2021-2028 program of the
University of Strasbourg, CNRS and Inserm.

Declaration of Competing Interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.biopha.2025.118073.

Data availability

Data will be made available on request.

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