Functional impact of intramolecular cleavage and dissociation of adhesion G protein–coupled receptor GPR133 (ADGRD1) on canonical signaling
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100798
ISSN1083-351X
AutoresJoshua D. Frenster, Gabriele Stephan, Niklas Ravn-Boess, Devin Bready, Jordan Wilcox, Bjoern Kieslich, Caroline Wilde, Norbert Sträter, Giselle R. Wiggin, Ines Liebscher, Torsten Schöneberg, Dimitris G. Placantonakis,
Tópico(s)Cell Adhesion Molecules Research
ResumoGPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM. GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM. The adhesion family of G protein–coupled receptors (GPCRs) has attracted increasing interest in the recent years for essential functions in health and disease (1Morgan R.K. Anderson G.R. Arac D. Aust G. Balenga N. Boucard A. Bridges J.P. Engel F.B. Formstone C.J. Glitsch M.D. Gray R.S. Hall R.A. Hsiao C.C. Kim H.Y. Knierim A.B. et al.The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.Ann. N. Y. Acad. Sci. 2019; 1456: 5-25Crossref PubMed Scopus (5) Google Scholar, 2Langenhan T. Adhesion G protein-coupled receptors-candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2019; 126 Suppl 6: 5-16PubMed Google Scholar). The large extracellular N termini of adhesion GPCRs contain a GPCR autoproteolysis-inducing domain, which is thought to catalyze autoproteolytic cleavage at the GPCR proteolysis site (GPS) marked by the tripeptide sequence H-L/I-∗-S/T (∗ denotes the cleavage site) (3Lin H.H. Stacey M. Yona S. Chang G.W. GPS proteolytic cleavage of adhesion-GPCRs.Adv. Exp. Med. Biol. 2010; 706: 49-58Crossref PubMed Scopus (24) Google Scholar, 4Arac D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Sudhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (226) Google Scholar). After this intramolecular cleavage, adhesion GPCRs are generally believed to exist as noncovalently bound heterodimers of their extracellular N-terminal fragment (NTF) and transmembrane-spanning C-terminal fragment (CTF) (5Gray J.X. Haino M. Roth M.J. Maguire J.E. Jensen P.N. Yarme A. Stetler-Stevenson M.A. Siebenlist U. Kelly K. CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation.J. Immunol. 1996; 157: 5438-5447PubMed Google Scholar, 6Krasnoperov V. Bittner M.A. Holz R.W. Chepurny O. Petrenko A.G. Structural requirements for alpha-latrotoxin binding and alpha-latrotoxin-stimulated secretion. A study with calcium-independent receptor of alpha-latrotoxin (CIRL) deletion mutants.J. Biol. Chem. 1999; 274: 3590-3596Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The recent demonstration of a tethered internal agonist, also known as the Stachel sequence, immediately C-terminal to the GPS, has given rise to the hypothesis that NTF–CTF dissociation facilitates the conformational changes needed for the Stachel sequence to initiate receptor activation (7Liebscher I. Schon J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Coster M. Simon K.U. Rothemund S. Monk K.R. Schoneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2014; 9: 2018-2026Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). However, the mechanism of receptor activation mediated by autoproteolytic cleavage and NTF–CTF dissociation is not generalizable to all members of the adhesion GPCR family. Indeed, several adhesion GPCRs have not yet been observed to undergo intramolecular cleavage, and cleavage is not necessarily required for their activity (2Langenhan T. Adhesion G protein-coupled receptors-candidate metabotropic mechanosensors and novel drug targets.Basic Clin. Pharmacol. Toxicol. 2019; 126 Suppl 6: 5-16PubMed Google Scholar, 8Wilde C. Fischer L. Lede V. Kirchberger J. Rothemund S. Schoneberg 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 (57) Google Scholar, 9Vallon M. Essler M. Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin alpha(v)beta3 to glycosaminoglycans.J. Biol. Chem. 2006; 281: 34179-34188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Promel S. Frickenhaus M. Hughes S. Mestek L. Staunton D. Woollard A. Vakonakis I. Schoneberg T. Schnabel R. Russ A.P. Langenhan T. The GPS motif is a molecular switch for bimodal activities of adhesion class G protein-coupled receptors.Cell Rep. 2012; 2: 321-331Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Bohnekamp J. Schoneberg T. Cell adhesion receptor GPR133 couples to Gs protein.J. Biol. Chem. 2011; 286: 41912-41916Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In addition, in some adhesion GPCRs, cleavage occurs in selective cellular contexts but not others (4Arac D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Sudhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (226) Google Scholar, 12Iguchi T. Sakata K. Yoshizaki K. Tago K. Mizuno N. Itoh H. Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a G alpha 12/13 and rho pathway.J. Biol. Chem. 2008; 283: 14469-14478Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Hsiao C.C. Cheng K.F. Chen H.Y. Chou Y.H. Stacey M. Chang G.W. Lin H.H. Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97.FEBS Lett. 2009; 583: 3285-3290Crossref PubMed Scopus (28) Google Scholar, 14Yang L.Y. Liu X.F. Yang Y. Yang L.L. Liu K.W. Tang Y.B. Zhang M. Tan M.J. Cheng S.M. Xu Y.C. Yang H.Y. Liu Z.J. Song G.J. Huang W. Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.Acta Pharmacol. Sin. 2017; 38: 56-68Crossref PubMed Scopus (4) Google Scholar). Finally, cleavage- and Stachel-independent signaling have been reported for several adhesion GPCRs (8Wilde C. Fischer L. Lede V. Kirchberger J. Rothemund S. Schoneberg 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 (57) Google Scholar, 10Promel S. Frickenhaus M. Hughes S. Mestek L. Staunton D. Woollard A. Vakonakis I. Schoneberg T. Schnabel R. Russ A.P. Langenhan T. The GPS motif is a molecular switch for bimodal activities of adhesion class G protein-coupled receptors.Cell Rep. 2012; 2: 321-331Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Kishore 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 (64) Google Scholar, 16Salzman G.S. Zhang S. Gupta A. Koide A. Koide S. Arac D. Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 10095-10100Crossref PubMed Scopus (35) Google Scholar, 17Patra C. van Amerongen M.J. Ghosh S. Ricciardi F. Sajjad A. Novoyatleva T. Mogha A. Monk K.R. Muhlfeld C. Engel F.B. Organ-specific function of adhesion G protein-coupled receptor GPR126 is domain-dependent.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 16898-16903Crossref PubMed Scopus (65) Google Scholar). These observations emphasize the need to study mechanisms of activation for adhesion GPCRs on an individual basis and in physiologically relevant biological contexts. We previously described that GPR133 (ADGRD1), a member of the adhesion family of GPCRs, is expressed in, and required for growth of, glioblastoma (GBM), an aggressive primary brain malignancy (18Bayin 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; 5e263Crossref PubMed Scopus (28) Google Scholar, 19Frenster 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; 2vdaa053PubMed Google Scholar, 20Frenster 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 (2) Google Scholar). In heterologous expression systems, N-terminally truncated CTF constructs of GPR133 generate significantly more G protein–mediated signaling than the full-length receptor (7Liebscher I. Schon J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Coster M. Simon K.U. Rothemund S. Monk K.R. Schoneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2014; 9: 2018-2026Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Nonetheless, there has not been any prior study of the extent of GPR133 cleavage or the NTF–CTF association. Here, we demonstrate that GPR133 is almost entirely cleaved in patient-derived GBM cells and that cleaved GPR133 has a higher basal activity than an uncleavable GPR133 point mutant. While the cleaved CTF and NTF remain noncovalently bound to each other within the secretory pathway, we demonstrate that the NTF dissociates from the CTF once reaching the plasma membrane (PM). Using a fusion protein of the N terminus from human protease-activated receptor 1 (hPAR1) receptor and the CTF of GPR133, we show that acutely induced dissociation of the NTF and thus liberation of the CTF at the PM increases canonical signaling of GPR133. These findings favor an NTF–CTF dissociation model for activation of GPR133 signaling. Previous reports suggested canonical signaling by GPR133 is mediated via coupling to GαS, resulting in an increase of intracellular cAMP (11Bohnekamp J. Schoneberg T. Cell adhesion receptor GPR133 couples to Gs protein.J. Biol. Chem. 2011; 286: 41912-41916Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We independently confirmed that expression of GPR133 in HEK293T cells is associated with robust increase in cAMP levels as detected by a cAMP response element–Luciferase reporter, but not other known GPCR signaling pathways (Fig. 1A). To test whether intramolecular cleavage has implications for canonical signaling, we generated an H543R mutant GPR133 carrying a point mutation at the −2 residue of the GPS cleavage site (11Bohnekamp J. Schoneberg T. Cell adhesion receptor GPR133 couples to Gs protein.J. Biol. Chem. 2011; 286: 41912-41916Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) (Fig. 1Bi and ii and Fig. S1, A and B). This mutation is known to abolish GPR133 cleavage but still permits cAMP signaling (7Liebscher I. Schon J. Petersen S.C. Fischer L. Auerbach N. Demberg L.M. Mogha A. Coster M. Simon K.U. Rothemund S. Monk K.R. Schoneberg T. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.Cell Rep. 2014; 9: 2018-2026Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 11Bohnekamp J. Schoneberg T. Cell adhesion receptor GPR133 couples to Gs protein.J. Biol. Chem. 2011; 286: 41912-41916Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We used homogenous time-resolved fluorescence (HTRF) assays to measure the cAMP levels produced by the cleaved WT and uncleaved mutant receptor in HEK293T cells. While overexpression of either receptor variant significantly raised cAMP levels above background, indicating a high baseline signaling activity, overexpression of the uncleavable H543R mutant GPR133 increased cAMP levels only to ∼60% of the cAMP levels obtained with WT GPR133 (Fig. 1C). This difference in signaling intensity could not be explained by differences in expression levels of the constructs as assessed by ELISA (Fig. 1D). Our previous work has demonstrated that GPR133 is expressed in human GBM (19Frenster 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; 2vdaa053PubMed Google Scholar), as well as our own patient-derived GBM cultures, where it is required for tumor growth (18Bayin 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; 5e263Crossref PubMed Scopus (28) Google Scholar, 20Frenster 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 (2) Google Scholar). We thus sought to confirm our findings in the disease-relevant context of patient-derived GBM cultures and obtained similar results (Fig. 1, F and G). These findings suggest that autoproteolytic cleavage might promote receptor activation. To assess the extent of intramolecular cleavage of GPR133, we overexpressed WT GPR133 and the H543R point mutant in HEK293T cells and analyzed whole-cell lysates by Western blot. Multiplexed fluorescent staining with both a commercial antibody (HPA042395, Sigma) against the GPR133 CTF and our own previously described mouse monoclonal antibody against the GPR133 NTF (18Bayin 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; 5e263Crossref PubMed Scopus (28) Google Scholar, 19Frenster 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; 2vdaa053PubMed Google Scholar) detected separate and distinct bands for the CTF (∼25 kDa) and the NTF (∼75 kDa), respectively (Fig. S1C; red arrows mark the CTF, green arrows mark the NTF). Affinity-purifying the receptor fragments to reduce nonspecific background staining confirmed these distinct CTF and NTF bands with increased clarity (Fig. 1E; red arrows mark the CTF, and green arrows mark the NTF). For the WT receptor, we also identified a faint band at 110 kDa (blue arrow), which we hypothesized to represent the uncleaved WT GPR133, as well as a faint band around 48 kDa (red arrow), possibly a dimer of the CTF (Fig. 1E and Fig. S1C). In contrast, the H543R-mutated GPR133 was detected as a single full-length band (∼110 kDa, yellow arrow), consistent with its cleavage deficiency (Fig. 1E and Fig. S1C; yellow arrows). Because the extent of intramolecular cleavage of adhesion GPCRs has been reported as cell type specific (4Arac D. Boucard A.A. Bolliger M.F. Nguyen J. Soltis S.M. Sudhof T.C. Brunger A.T. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.EMBO J. 2012; 31: 1364-1378Crossref PubMed Scopus (226) Google Scholar, 12Iguchi T. Sakata K. Yoshizaki K. Tago K. Mizuno N. Itoh H. Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a G alpha 12/13 and rho pathway.J. Biol. Chem. 2008; 283: 14469-14478Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Hsiao C.C. Cheng K.F. Chen H.Y. Chou Y.H. Stacey M. Chang G.W. Lin H.H. Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97.FEBS Lett. 2009; 583: 3285-3290Crossref PubMed Scopus (28) Google Scholar, 14Yang L.Y. Liu X.F. Yang Y. Yang L.L. Liu K.W. Tang Y.B. Zhang M. Tan M.J. Cheng S.M. Xu Y.C. Yang H.Y. Liu Z.J. Song G.J. Huang W. Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.Acta Pharmacol. Sin. 2017; 38: 56-68Crossref PubMed Scopus (4) Google Scholar), we next interrogated GPR133 cleavage in our patient-derived GBM cultures. Indeed, the same cleavage pattern was confirmed in four separate patient-derived GBM cell cultures (affinity-purified receptor fragments in Fig. 1H and whole-cell lysates in Fig. S1D). It is important to comment on the discrepancies between expected and observed molecular weights (MWs) of the uncleaved receptor, NTF, and CTF. The expected MW of the uncleaved receptor without the signal peptide is 93 kDa, whereas those of the NTF and CTF are expected to be 57 kDa (without the signal peptide) and 36 kDa, respectively. The shifts in the observed MW of the uncleaved receptor and NTF are due to glycosylation, as demonstrated later in the article. The shift in the MW of the CTF from 36 kDa (expected) to 25 kDa (observed) is likely explained by increased SDS loading on the helical hydrophobic transmembrane segments of the CTF, as previously reported for other transmembrane proteins (21Rath 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 (525) Google Scholar). Overall, these findings suggest that GPR133 is almost entirely cleaved in human GBM and HEK293T cells. To understand mechanisms underlying the increased signaling generated by WT GPR133 compared with the uncleavable mutant receptor, we first analyzed their trafficking to the PM through the secretory pathway. Using confocal microscopy and indirect immunofluorescent staining under nonpermeabilizing conditions, we detected both the WT and the H543R-mutated GPR133 at the PM of cells (Fig. 2Ai–iii and Fig. S2A). Similarly, under permeabilizing conditions, the WT and the H543R-mutated GPR133 demonstrated analogous staining patterns in both intracellular organelles of the secretory pathway, as well as at the PM (Fig. 2Bi–iii and Fig. S2B). To confirm these findings biochemically, we used subcellular fractionation and Western blot analysis to separately interrogate three fractions enriched for (1) cytosol, with some endoplasmic reticulum (ER) contamination, (2) nucleus, ER, and the Golgi apparatus (Nuc/ER/Golgi), and (3) the PM. It is noteworthy that while the first two fractions showed enrichment for distinct subcellular compartments/organelles, the PM fraction was highly specific, as demonstrated by absence of staining for any non-PM compartment markers by Western blot (Fig. 2E). Both the cleaved NTF and CTF of the WT receptor, as well as the uncleaved H543R mutant, were prominently detected in the PM fraction (Fig. 2, C and D, red arrowheads), consistent with our microscopy data. Collectively, these findings suggested that intramolecular cleavage of GPR133 is not required for subcellular trafficking to the PM and that the observed difference in signaling intensities between the cleaved and uncleaved GPR133 variants is not likely to be caused by subcellular trafficking defects. While interrogating the distribution of GPR133 across the subcellular fractions, it became apparent that the cleaved WT GPR133 NTF and the H543R full-length GPR133 undergo an MW shift from lower weight bands in the fractions containing proteins from the early secretory pathway toward higher MW bands in the PM fraction (Fig. 2, C and D, green and red arrowheads, respectively). Because there are nine N-linked glycosylation sites predicted within the NTF (Fig. S1A), we hypothesized that these observed size shifts are due to different extents of glycosylation as the receptor matures through the secretory pathway. To test this hypothesis, we treated the different subcellular fractions with an enzymatic deglycosylation mix (containing PNGase F, O-glycosidase, α2-3,6,8,9 neuraminidase A, β1-4 galactosidase S, and β-N-acetylhexosaminidasef). Indeed, upon deglycosylation, the different MW isoforms of cleaved WT NTF and full-length H543R GPR133 shifted to the same predicted MW independent of their subcellular fraction of origin (Fig. 3A, green, red, and blue arrowheads demarking immaturely glycosylated, maturely glycosylated, and deglycosylated bands, respectively; deglycosylated whole-cell lysates are shown in Fig. S3A for reference), confirming that these bands represent the same protein with different extents of glycosylation. When staining WT GPR133 using the anti-CTF antibody, we detected a band in the Nuc/ER/Golgi fraction shifting from ∼100 kDa to ∼70 kDa upon deglycosylation (Fig. 3Ai, green and blue arrowheads, furthest left). This pattern mimics the immaturely glycosylated uncleaved H543R GPR133, leading to our hypothesis that this band is the low-abundance uncleaved form of WT GPR133 we had previously observed in Western blots from whole-cell lysates (Fig. 1, E and H, and Fig. S1, C and D). The faster-than-expected mobility of this uncleaved deglycosylated form likely results from increased SDS binding on the helical transmembrane segments of the receptor (21Rath 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 (525) Google Scholar). To test whether this uncleaved and immaturely glycosylated form of WT GPR133 is a stable form of the receptor or a transient state during receptor maturation, we blocked protein synthesis with cycloheximide. Protein synthesis block indeed abolished this uncleaved form of GPR133, supporting the hypothesis that the uncleaved WT GPR133 is a short-lived transition state (Fig. 3B, green and blue arrowheads). To determine whether this short-lived full-length WT GPR133 is cleaved directly after synthesis in the ER, or later in the Golgi, we treated cells with brefeldin A (BFA), which interrupts ER-to-Golgi transport. The effectiveness of BFA to prevent transport along the secretory pathway was confirmed by confocal microscopy (Fig. S3B). BFA treatment did not lead to an accumulation of the uncleaved WT GPR133 isoform (Fig. 3Ci, green arrowheads), suggesting cleavage happens immediately after protein synthesis. However, BFA treatment did result in the elimination of maturely glycosylated NTF in the Nuc/ER/Golgi fraction 14 h after its addition to the medium (Fig. 3Cii, red arrowheads). Finally, we treated the subcellular fractions with endoglycosidase H (EndoH), which removes the immature high mannose glycosylation of proteins within the ER but not mature glycosylation of proteins that have reached the Golgi. Indeed, we observed that the uncleaved WT GPR133 band was sensitive to EndoH-mediated deglycosylation without an additional deglycosylation effect conferred by a complete deglycosylation mix containing PNGase, suggesting that it represents an immaturely glycosylated protein localizing to the ER (Fig. 3D). These data suggest a model in which newly synthesized WT GPR133 carrying immature glycosylation gets intramolecularly cleaved within the ER before trafficking to the Golgi, where it acquires mature glycosylation. WT GPR133 reaches the PM as a fully cleaved and maturely glycosylated protein. To investigate whether the cleaved GPR133 CTF and NTF remain noncovalently bound to each other, we created GPR133 constructs carrying either C-terminal or N-terminal Twin-Strep-tags for affinity purification (Fig. 4A). When purifying the cleaved receptor from whole-cell lysates, identical stoichiometries of the CTF and NTF were eluted, independent of whether they are purified using the N-terminal or C-terminal affinity tag (Fig. 4B). While this finding suggested that the NTF and CTF remain noncovalently associated at the whole-cell level, we proceeded to dissect whether this association varied depending on the location of GPR133 along the secretory pathway. To this end, we quantified the relative distribution of each receptor fragment (WT CTF, WT NTF, H543R full length as detected with the CTF antibody, H543R full length as detected with the NTF antibody) across the three subcellular fractions mentioned above (Fig. 4C). The subcellular distribution did not differ between the cleaved WT CTF and the uncleaved H543R mutant using either the C-terminal or N-terminal antibody, supporting the previous observation that cleavage does not affect the trafficking of the receptor's transmembrane segment. However, the cleaved WT NTF was significantly underrepresented at the PM when compared with the WT CTF or the H543R uncleaved receptor (Fig. 4C). We note that, although the WT NTF appears to be overrepresented in the Nuc/ER/Golgi fraction compared with the WT CTF, this is likely a mathematical artifact arising from reduced representation of the WT NTF in the PM fraction. This finding suggested that either the cleaved NTF traffics within the cells independently of the CTF or, more likely, the NTF dissociates from the CTF at the PM and is thus less abundant in that fraction. To test these two models, we repeated the purification of the receptor using its C-terminal affinity tag and compared the Nuc/ER/Golgi fraction against the PM fraction as input. We detected a significantly lower NTF-to-CTF ratio at the PM than the Nuc/ER/Golgi fraction (Fig. 4Di–iii). Furthermore, we were able to purify and detect the soluble NTF from precleared cell culture supernatants, while not detecting any associated CTFs (Fig. 4E and Fig. S4, A and B). These findings suggested that, while the cleaved NTF and CTF are noncovalently bound in the secretory pathway, they do partially dissociate at the PM. To gather additional evidence for such NTF–CTF dissociation at the PM, we assayed protein decay of the two fragments after blocking protein synthesis with cycloheximide in HEK293T cells overexpressing either the cleaved WT or uncleaved H543R mutant receptor. Whole-cell lysates of different cycloheximide chase time points were analyzed by Western blot using our CTF- and NTF-targeting antibodies, and intensities were plotted as a function of time (Fig. 4Fi and ii and Fig. S4C). No significant difference was detected between the decay curves and half-life times of uncleaved H543R GPR133 and the WT CTF, indicating that the above described signaling intensity differences were not caused by differences in protein stability. However, the cleaved WT GPR133 NTF decayed at a significantly faster rate than the WT CTF. Although these da
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