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Activation of the adhesion G protein–coupled receptor GPR133 by antibodies targeting its N-terminus

2022; Elsevier BV; Volume: 298; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2022.101949

ISSN

1083-351X

Autores

Gabriele Stephan, Joshua D. Frenster, Ines Liebscher, Dimitris G. Placantonakis,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein–coupled receptor involved in raising cytosolic cAMP levels, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. Our previous work suggested that dissociation of autoproteolytically generated N-terminal and C-terminal fragments of GPR133 at the plasma membrane correlates with receptor activation and signaling. To promote the goal of developing biologics that modulate GPR133 function, we investigated the effects of antibodies against the N-terminus of GPR133 on receptor signaling. Here, we show that treatment of HEK293T cells overexpressing GPR133 with these antibodies increased cAMP levels in a concentration-dependent manner. Analysis of culture medium following antibody treatment further indicated the presence of complexes of these antibodies with the autoproteolytically cleaved N-terminal fragments of GPR133. In addition, cells expressing a cleavage-deficient mutant of GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage dependent. Finally, we demonstrate the antibody-mediated stimulation of WT GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that the intramolecular cleavage in the N-terminus modulates receptor activation and signaling. We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein–coupled receptor involved in raising cytosolic cAMP levels, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. Our previous work suggested that dissociation of autoproteolytically generated N-terminal and C-terminal fragments of GPR133 at the plasma membrane correlates with receptor activation and signaling. To promote the goal of developing biologics that modulate GPR133 function, we investigated the effects of antibodies against the N-terminus of GPR133 on receptor signaling. Here, we show that treatment of HEK293T cells overexpressing GPR133 with these antibodies increased cAMP levels in a concentration-dependent manner. Analysis of culture medium following antibody treatment further indicated the presence of complexes of these antibodies with the autoproteolytically cleaved N-terminal fragments of GPR133. In addition, cells expressing a cleavage-deficient mutant of GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage dependent. Finally, we demonstrate the antibody-mediated stimulation of WT GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that the intramolecular cleavage in the N-terminus modulates receptor activation and signaling. Adhesion G protein–coupled receptors (aGPCRs) represent the second largest subfamily within the GPCR superfamily (1Krishnan A. Nijmeijer S. de Graaf C. Schiöth H.B. Classification, nomenclature, and structural aspects of adhesion GPCRs.Handbook Exp. Pharmacol. 2016; 234: 15-41Crossref PubMed Scopus (25) Google Scholar, 2Hamann J. Aust G. Araç D. Engel F.B. Formstone C. Fredriksson R. Hall R.A. Harty B.L. Kirchhoff C. Knapp B. Krishnan A. Liebscher I. Lin H.H. Martinelli D.C. Monk K.R. et al.International union of basic and clinical pharmacology. XCIV. Adhesion G protein-coupled receptors.Pharmacol. Rev. 2015; 67: 338-367Crossref PubMed Scopus (252) Google Scholar) and have been implicated in numerous physiological processes and disease mechanisms (3Langenhan T. Adhesion G protein-coupled receptors-Candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2020; 126: 5-16Crossref PubMed Scopus (22) Google Scholar, 4Kaczmarek I. Suchý T. Prömel S. Schöneberg T. Liebscher I. Thor D. The relevance of adhesion G protein-coupled receptors in metabolic functions.Biol. Chem. 2021; 403: 195-209Crossref PubMed Scopus (3) Google Scholar, 5Gad A.A. Balenga N. The emerging role of adhesion GPCRs in cancer.ACS Pharmacol. Translational Sci. 2020; 3: 29-42Crossref PubMed Scopus (23) Google Scholar). Adhesion GPCRs are structurally characterized by an intracellular C-terminus, a seven transmembrane segment domain and a large extracellular N-terminus (2Hamann J. Aust G. Araç D. Engel F.B. Formstone C. Fredriksson R. Hall R.A. Harty B.L. Kirchhoff C. Knapp B. Krishnan A. Liebscher I. Lin H.H. Martinelli D.C. Monk K.R. et al.International union of basic and clinical pharmacology. XCIV. Adhesion G protein-coupled receptors.Pharmacol. Rev. 2015; 67: 338-367Crossref PubMed Scopus (252) Google Scholar, 6Scholz N. Langenhan T. Schöneberg T. Revisiting the classification of adhesion GPCRs.Ann. New York Acad. Sci. 2019; 1456: 80-95Crossref PubMed Scopus (14) Google Scholar, 7Hayflick J.S. A family of heptahelical receptors with adhesion-like domains: A marriage between two super families.J. Recept. Signal. Transduct. Res. 2000; 20: 119-131Crossref PubMed Scopus (26) Google Scholar). While distinct functional domains within the N-terminus are thought to mediate receptor-specific interactions with adjacent cells or the extracellular matrix (2Hamann J. Aust G. Araç D. Engel F.B. Formstone C. Fredriksson R. Hall R.A. Harty B.L. Kirchhoff C. Knapp B. Krishnan A. Liebscher I. Lin H.H. Martinelli D.C. Monk K.R. et al.International union of basic and clinical pharmacology. XCIV. Adhesion G protein-coupled receptors.Pharmacol. Rev. 2015; 67: 338-367Crossref PubMed Scopus (252) Google Scholar), almost all aGPCRs share a conserved GPCR autoproteolysis-inducing (GAIN) domain within the N-terminus. This domain catalyzes intramolecular autoproteolytic cleavage at the GPCR proteolysis site (GPS) within the N-terminus, resulting in an N-terminal fragment (NTF) and a C-terminal fragment (CTF) (8Araç D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Südhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (251) Google Scholar). A prevalent hypothesis in the field is that binding of ligands from adjacent cells or the extracellular matrix to the N-terminus, as well as mechanical stimuli, induce conformational changes or NTF-CTF dissociation (3Langenhan T. Adhesion G protein-coupled receptors-Candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2020; 126: 5-16Crossref PubMed Scopus (22) Google Scholar, 9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 10Stoveken H.M. Hajduczok A.G. Xu L. Tall G.G. Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 6194-6199Crossref PubMed Scopus (138) Google Scholar, 11Petersen S.C. Luo R. Liebscher I. Giera S. Jeong S.J. Mogha A. Ghidinelli M. Feltri M.L. Schöneberg T. Piao X. Monk K.R. The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with laminin-211.Neuron. 2015; 85: 755-769Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Monk K.R. Hamann J. Langenhan T. Nijmeijer S. Schöneberg T. Liebscher I. Adhesion G protein-coupled receptors: From in vitro pharmacology to in vivo mechanisms.Mol. Pharmacol. 2015; 88: 617-623Crossref PubMed Scopus (39) Google Scholar). These events, in turn, enable the Stachel sequence (9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 13Demberg L.M. Rothemund S. Schöneberg T. Liebscher I. Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2.Biochem. Biophys. Res. Commun. 2015; 464: 743-747Crossref PubMed Scopus (68) Google Scholar, 14Demberg L.M. Winkler J. Wilde C. Simon K.U. Schön J. Rothemund S. Schöneberg T. Prömel S. Liebscher I. Activation of adhesion G protein-coupled receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.J. Biol. Chem. 2017; 292: 4383-4394Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Müller A. Winkler J. Fiedler F. Sastradihardja T. Binder C. Schnabel R. Kungel J. Rothemund S. Hennig C. Schöneberg T. Prömel S. Oriented cell division in the C. elegans embryo is coordinated by G-protein signaling dependent on the adhesion GPCR LAT-1.PLoS Genet. 2015; 11e1005624Crossref Scopus (59) Google Scholar, 16Kishore A. Purcell R.H. Nassiri-Toosi Z. Hall R.A. Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and Bai1 (ADGRB1).J. Biol. Chem. 2016; 291: 3385-3394Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Wilde C. Fischer L. Lede V. Kirchberger J. Rothemund S. Schöneberg T. Liebscher I. The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.FASEB J. 2016; 30: 666-673Crossref PubMed Scopus (69) Google Scholar, 18Scholz N. Guan C. Nieberler M. Grotemeyer A. Maiellaro I. Gao S. Beck S. Pawlak M. Sauer M. Asan E. Rothemund S. Winkler J. Prömel S. Nagel G. Langenhan T. et al.Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons.Elife. 2017; 6e28360Crossref Scopus (78) Google Scholar, 19Favara D.M. Liebscher I. Jazayeri A. Nambiar M. Sheldon H. Banham A.H. Harris A.L. Elevated expression of the adhesion GPCR ADGRL4/ELTD1 promotes endothelial sprouting angiogenesis without activating canonical GPCR signalling.Sci. Rep. 2021; 11: 8870Crossref PubMed Scopus (4) Google Scholar), a tethered internal agonist peptide sequence immediately distal to the GPS, to activate signaling (3Langenhan T. Adhesion G protein-coupled receptors-Candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2020; 126: 5-16Crossref PubMed Scopus (22) Google Scholar, 20Vizurraga A. Adhikari R. Yeung J. Yu M. Tall G.G. Mechanisms of adhesion G protein-coupled receptor activation.J. Biol. Chem. 2020; 295: 14065-14083Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, the exact activation mechanisms likely differ among members of the aGPCR family and are not well characterized. Our group recently demonstrated part of the mechanism that mediates the activation of GPR133 (ADGRD1), a member of group V of aGPCRs (2Hamann J. Aust G. Araç D. Engel F.B. Formstone C. Fredriksson R. Hall R.A. Harty B.L. Kirchhoff C. Knapp B. Krishnan A. Liebscher I. Lin H.H. Martinelli D.C. Monk K.R. et al.International union of basic and clinical pharmacology. XCIV. Adhesion G protein-coupled receptors.Pharmacol. Rev. 2015; 67: 338-367Crossref PubMed Scopus (252) Google Scholar) implicated in the pathogenesis of glioblastoma (GBM) (21Bayin N.S. Frenster J.D. Kane J.R. Rubenstein J. Modrek A.S. Baitalmal R. Dolgalev I. Rudzenski K. Scarabottolo L. Crespi D. Redaelli L. Snuderl M. Golfinos J.G. Doyle W. Pacione D. et al.GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.Oncogenesis. 2016; 5: e263Crossref PubMed Scopus (35) Google Scholar, 22Frenster J.D. Inocencio J.F. Xu Z. Dhaliwal J. Alghamdi A. Zagzag D. Bayin N.S. Placantonakis D.G. GPR133 promotes glioblastoma growth in hypoxia.Neurosurgery. 2017; 64: 177-181Crossref PubMed Scopus (8) Google Scholar), an aggressive brain malignancy (23Dolecek T.A. Propp J.M. Stroup N.E. Kruchko C. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2005-2009.Neuro-oncology. 2012; 14: v1-49Crossref PubMed Scopus (1273) Google Scholar). The N-terminus of GPR133, which contains a pentraxin (PTX) domain, undergoes autoproteolytic cleavage almost immediately after protein synthesis (24Frenster J.D. Stephan G. Ravn-Boess N. Bready D. Wilcox J. Kieslich B. Wilde C. Sträter N. Wiggin G.R. Liebscher I. Schöneberg T. Placantonakis D.G. Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.J. Biol. Chem. 2021; 296100798Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). However, NTF and CTF stay noncovalently bound to each other until they are trafficked to the plasma membrane, where their dissociation occurs and correlates with increased signaling mediated by Gαs, resulting in activation of adenylate cyclase and elevation in cAMP levels (21Bayin N.S. Frenster J.D. Kane J.R. Rubenstein J. Modrek A.S. Baitalmal R. Dolgalev I. Rudzenski K. Scarabottolo L. Crespi D. Redaelli L. Snuderl M. Golfinos J.G. Doyle W. Pacione D. et al.GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.Oncogenesis. 2016; 5: e263Crossref PubMed Scopus (35) Google Scholar, 24Frenster J.D. Stephan G. Ravn-Boess N. Bready D. Wilcox J. Kieslich B. Wilde C. Sträter N. Wiggin G.R. Liebscher I. Schöneberg T. Placantonakis D.G. Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.J. Biol. Chem. 2021; 296100798Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 25Bohnekamp J. Schöneberg T. Cell adhesion receptor GPR133 couples to Gs protein.J. Biol. Chem. 2011; 286: 41912-41916Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 26Gupte J. Swaminath G. Danao J. Tian H. Li Y. Wu X. Signaling property study of adhesion G-protein-coupled receptors.FEBS Lett. 2012; 586: 1214-1219Crossref PubMed Scopus (73) Google Scholar, 27Fischer L. Wilde C. Schöneberg T. Liebscher I. Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133).BMC genomics. 2016; 17: 609Crossref PubMed Scopus (11) Google Scholar). Our finding that dissociation of NTF and CTF correlates with increased signaling is in accordance with the previous observation that the CTF of GPR133, when expressed without the NTF, demonstrates hyperactive signaling relative to its full-length counterpart (9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Collectively, our data suggest that the cleaved but noncovalently associated NTF-CTF holoreceptor is signaling competent, but its dissociation at the plasma membrane enables full activation of receptor signaling. Here, we demonstrate that antibodies targeting epitopes outside of the GAIN domain of the N-terminus of GPR133 increase receptor-mediated Gαs signaling and cAMP levels. Preventing specific antibody binding by deleting the targeted epitope abolishes the effect. The antibody-mediated activation is dependent on receptor cleavage, because antibodies fail to modulate signaling of a cleavage-deficient GPR133 mutant (H543R). These findings suggest that GPR133 function can be modulated by antibodies, and likely other biologics as well, which can be used as molecular tools in the study of receptor activation but also as therapeutic platforms in the context of GBM and possibly other malignancies, where GPR133 plays important roles. To test whether GPR133 signaling is modulated by antibodies binding to the extracellular N-terminus, we transfected Human embryonic kidney 293T (HEK293T) cells with GPR133 tagged with an N-terminal hemagglutinin (HA) and a C-terminal FLAG epitope (Fig. 1A). Overexpression of tagged GPR133 was verified by Western blot analysis of whole cell lysates 48 h after transfection (Fig. 1B). As expected (9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 24Frenster J.D. Stephan G. Ravn-Boess N. Bready D. Wilcox J. Kieslich B. Wilde C. Sträter N. Wiggin G.R. Liebscher I. Schöneberg T. Placantonakis D.G. Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.J. Biol. Chem. 2021; 296100798Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 27Fischer L. Wilde C. Schöneberg T. Liebscher I. Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133).BMC genomics. 2016; 17: 609Crossref PubMed Scopus (11) Google Scholar), staining with an anti-FLAG antibody detected the CTF (blue arrow, ∼25 kDa), staining with an anti-HA antibody detected bands representing the maturely and immaturely glycosylated NTF (green arrows, ∼95/75 kDa), and both antibodies detected small amounts of the full-length uncleaved receptor (red arrows, ∼110 kDa). The band sizes of the full-length receptor, detected with the anti-FLAG and anti-HA antibodies, and the NTF, as detected by the anti-HA antibody, as well as the shift from the expected molecular weight of the CTF (∼36 kDa) to the observed molecular weight (blue arrow, ∼25 kDa), are in agreement with our previous findings (24Frenster J.D. Stephan G. Ravn-Boess N. Bready D. Wilcox J. Kieslich B. Wilde C. Sträter N. Wiggin G.R. Liebscher I. Schöneberg T. Placantonakis D.G. Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.J. Biol. Chem. 2021; 296100798Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Indeed, we previously showed that glycosylation increases the apparent molecular weight of the NTF (24Frenster J.D. Stephan G. Ravn-Boess N. Bready D. Wilcox J. Kieslich B. Wilde C. Sträter N. Wiggin G.R. Liebscher I. Schöneberg T. Placantonakis D.G. Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.J. Biol. Chem. 2021; 296100798Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The size shift of the CTF is most likely caused by increased loading of SDS to hydrophobic transmembrane regions of the CTF (28Rath A. Glibowicka M. Nadeau V.G. Chen G. Deber C.M. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 1760-1765Crossref PubMed Scopus (549) Google Scholar). Moreover, both antibodies detected bands >260 kDa (gray arrows), presumably representing aggregates of the receptor. We used a homogeneous time resolved fluorescence (HTRF)-based assay to quantify cAMP concentrations after expression of GPR133 (Fig. 1C). In agreement with previously published data (24Frenster J.D. Stephan G. Ravn-Boess N. Bready D. Wilcox J. Kieslich B. Wilde C. Sträter N. Wiggin G.R. Liebscher I. Schöneberg T. Placantonakis D.G. Functional impact of intramolecular cleavage and dissociation of adhesion G protein-coupled receptor GPR133 (ADGRD1) on canonical signaling.J. Biol. Chem. 2021; 296100798Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 25Bohnekamp J. Schöneberg T. Cell adhesion receptor GPR133 couples to Gs protein.J. Biol. Chem. 2011; 286: 41912-41916Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Fischer L. Wilde C. Schöneberg T. Liebscher I. Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133).BMC genomics. 2016; 17: 609Crossref PubMed Scopus (11) Google Scholar), intracellular cAMP levels increased significantly in HEK293T cells overexpressing GPR133 relative to cells transfected with the empty vector (p < 0.001, t test). We then treated the HEK293T cells with either a mouse monoclonal antibody (8E3E8) that we raised against the PTX domain of GPR133 (Fig. 1Di) (21Bayin N.S. Frenster J.D. Kane J.R. Rubenstein J. Modrek A.S. Baitalmal R. Dolgalev I. Rudzenski K. Scarabottolo L. Crespi D. Redaelli L. Snuderl M. Golfinos J.G. Doyle W. Pacione D. et al.GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.Oncogenesis. 2016; 5: e263Crossref PubMed Scopus (35) Google Scholar, 29Frenster J.D. Kader M. Kamen S. Sun J. Chiriboga L. Serrano J. Bready D. Golub D. Ravn-Boess N. Stephan G. Chi A.S. Kurz S.C. Jain R. Park C.Y. Fenyo D. et al.Expression profiling of the adhesion G protein-coupled receptor GPR133 (ADGRD1) in glioma subtypes.Neurooncol. Adv. 2020; 2: vdaa053PubMed Google Scholar), a commercial anti-HA antibody (Fig. 1Ei), or a commercial anti-FLAG antibody (Fig. 1Fi). A range of antibody concentrations was tested. Since these antibodies were stored in solution containing NaN3, increasing concentrations of NaN3 (0.015 mM, 0.06 mM, 0.15 mM) served as a control. To verify binding of the antibodies to GPR133, we performed an ELISA under nonpermeabilizing conditions (Fig. 1, Dii, Eii, Fii). Optical density increased proportionally with increasing concentrations of the extracellular antibodies (8E3E8: F (1,16) = 41.31, p < 0.0001; anti-HA: F (1,16) = 284.6, p < 0.0001; two-way ANOVA) compared to the NaN3 control. The anti-FLAG antibody, which recognizes the intracellular FLAG epitope, also showed a slight concentration-dependent increase (F (1,16) = 17.09, p = 0.0008; two-way ANOVA). However, this ELISA signal was dramatically smaller than with the 8E3E8 and anti-HA antibodies. The increase in optical density after applying the FLAG antibody might be due to partial permeabilization of the plasma membrane during fixation of the cells. To test the effect of antibodies on GPR133 signaling, we quantified intracellular cAMP levels following stimulation of HEK293T cells transfected with the empty vector or GPR133. We found significant concentration-dependent increases in cAMP following the treatment with 8E3E8 (F (1,32) = 6.509, p = 0.0157, two-way ANOVA) and anti-HA antibodies (F (1,32) = 6.997, p = 0.0125, two-way ANOVA), but not the anti-FLAG antibody (F (1,32) = 0.1115, p = 0.7407, two-way ANOVA) (Fig. 1, Diii, Eiii, Fiii; raw cAMP levels in nM are shown in Fig. S1). We observed a 1.4-fold increase in cAMP levels following treatment with 1.8 μg/ml 8E3E8 (fold change in cAMP levels relative to the untreated empty vector condition: GPR133 + NaN3 = 11.2 ± 1.5 versus GPR133 + 8E3E8 = 16.1 ± 1.9) and a 1.7-fold increase following treatment with 10 μg/ml anti-HA (fold change in cAMP levels relative to untreated empty vector condition: GPR133 + NaN3 = 14.0 ± 2.0 versus GPR133 + anti-HA = 24.4 ± 4.9). Signaling did not increase following treatment with 10 μg/ml anti-FLAG (fold change in cAMP levels relative to untreated empty vector condition: GPR133 + NaN3 = 13.9 ± 2.4 versus GPR133 + anti-FLAG = 15.3 ± 3.1). These findings suggest that antibodies targeting the NTF of GPR133 outside of the GAIN domain increase receptor signaling in HEK293T cells. Next, we compared the effect of the antibodies on HEK293T cells overexpressing GPR133 with the receptor's natural activation mechanism, by using a Stachel-derived soluble peptide (9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) and DMSO-containing medium as a control (Fig. 2; raw cAMP levels in nM are shown in Fig. S2). The soluble 13 amino acid-long peptide (p13) mimics the endogenous agonistic Stachel sequence, thereby specifically activating GPR133 (9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Solubility of the Stachel-derived peptide in aqueous solution was confirmed by measuring the absorbance of peptide solutions in different concentrations (Fig. S3). Indeed, peak absorbance at 195 nm increased in concentration-dependent fashion with increasing concentrations of p13, suggesting p13 is soluble at the concentrations used in our experiments. Dynamic light scattering measurements further validated the solubility of p13 (Fig. S4). As described previously (9Liebscher I. Schön J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Cöster M. Simon K.U. Rothemund S. Monk K.R. Schöneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2015; 10: 1021Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), treatment with p13 resulted in a concentration-dependent increase of GPR133 signaling (F (1,12) = 19.8, p = 0.0009, two-way ANOVA) (Fig. 2A). We observed a 1.5-fold increase in cAMP levels following treatment with a submaximal concentration of p13 (0.25 mM) relative to DMSO, which is comparable to the magnitude of increase following antibody stimulation. We then stimulated GPR133 with increasing concentrations of 8E3E8 alone (Fig. 2B) or in combination with 0.25 mM p13 (Fig. 2C). Different concentrations of NaN3 were used as a control. Treatment with 0.25 mM p13 increased the baseline of the 8E3E8 (or NaN3) concentration-response curve (Fig. 2C) and blunted the dose-dependent increase in cAMP with increasing 8E3E8 concentrations (F (1,18) = 0.77, p = 0.3916, two way ANOVA). In contrast, treatment with both 8E3E8 and DMSO vehicle impaired the 8E3E8-induced increase in cAMP (F (1,18) = 4.0, p = 0.0597, two way ANOVA) (Fig. 2D), suggesting nonspecific effects of DMSO on antibody–GPR133 interactions. Similarly, an inactive control peptide (pCTRL), which does not increase cAMP production by GPR133 (F (1,18) = 1.7, p = 0.2113, two way ANOVA) (Fig. 2A), blocked the effects of 8E3E8 (F (1,18) = 1.3, p = 0.2728, two way ANOVA) (Fig. 2E). Collectively, these results indicate that cotreatment with a submaximal concentration of p13 blunts the dose-dependent agonistic effects of 8E3E8, which suggests that the antibody-induced increase in cAMP may be mediated by the endogenous Stachel agonist sequence. To ascertain the specificity of the activating effect of 8E3E8, we deleted the PTX domain (amino acids 79–276) of GPR133, which contains the epitope that 8E3E8 recognizes. The deletion is predicted to cause a 22 kDa decrease in molecular weight. Overexpression of HA-tagged GPR133 with the PTX deletion (HA-GPR133 ΔPTX) was confirmed by Western blot analysis of whole cell lysates (Fig. 3A). Staining with the PTX-recognizing 8E3E8 antibody detected HA-GPR133 but not HA-GPR133 ΔPTX, confirming the PTX deletion (Fig. 3A). Staining with the anti-HA antibody and a commercial antibody against the cytosolic C-terminus of GPR133 (anti-CTF) demonstrated the expected size shifts of the full-length receptor and the NTF after deletion of the PTX domain (Fig. 3A). Importantly, deletion of the PTX domain did not impair receptor cleavage. Immunofluorescent staining of HEK293T cells overexpressing either full-length HA-GPR133 or HA-GPR133 ΔPTX with an anti-HA antibody showed similar staining patterns, suggesting the subcellular localization and membrane trafficking of the mutant receptor is not altered (Fig. 3B). The baseline levels of cAMP were significantly reduced in HEK293T cells overexpressing HA-GPR133 ΔPTX compared to cells overexpressing HA-GPR133 (F (2,9) = 20.24, p = 0.0005, one-way ANOVA; Tukey's post hoc test: HA-GPR133 compared to HA-GPR133 ΔPTX, p = 0.0256) (Fig. 3Ci; raw cAMP levels in nM are shown in Fig. S5A). Of note, basal cAMP levels of HA-GPR133 are higher than the basal activity of GPR133 tagged with both the N-terminal HA and the C-terminal FLAG epitopes (Fig. 1), because we found that the C-terminal FLAG tag mildly reduces GPR133 signaling (data not shown). While treatment of cells overexpressing HA-GPR133 with either 10 μg/ml anti-HA or 1.8 μg/ml 8E3E8 activated receptor signaling equivalently (anti-HA = 1.5-fold, fold change in cAMP levels relative to untreated empty vector condition: HA-GPR133 + NaN3 = 41.1 ± 4.1 versus HA-GPR133 + anti-HA = 63.0 ± 8.0; 8E3E8 = 1.4-fold, fold change in cAMP levels relative to untreated empty vector condition: HA-GPR133 + NaN3 = 41.1 ± 4.1 versus HA-GPR133 + 8E3E8 = 59.2 ± 3.3), only treatment with anti-HA (2-fold, fold change in cAMP levels relative to untreated empty vector condition: HA-GPR133 ΔPTX + NaN3 = 20.1 ± 7.6 versus HA-GPR133 ΔPTX + anti-HA = 40.7 ± 15.8) but not 8E3E8 (fold change in cAMP levels relative to untreated empty vector condition: HA-GPR133 ΔPTX + NaN3 = 20.1 ± 7.6 versus HA-GPR133 ΔPTX + 8E3E8 = 18.5 ± 5.5) increased cAMP levels in cells expressing HA-GPR133 ΔPTX (F (2,18) = 9.490, p = 0.0015, two-way ANOVA; Tukey's post hoc test: HA-GPR133 control versus HA-GPR133 + α-HA, p = 0.0029; HA-GPR133 control versus HA-GPR133 + 8E3E8, p = 0.0126; HA-GPR133 ΔPTX control versus HA-GPR133 ΔPTX + α-HA, p = 0.0047; HA-GPR133 ΔPTX control versus HA-GPR133 ΔPTX + 8E

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