Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m402974200
ISSN1083-351X
AutoresHsi-Hsien Lin, G. Chang, John Q. Davies, Martin Stacey, James Harris, Siamon Gordon,
Tópico(s)Adenosine and Purinergic Signaling
ResumoPost-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu↓Ser518 cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P+1 cleavage site (Ser518) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P-2 His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases. Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu↓Ser518 cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P+1 cleavage site (Ser518) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P-2 His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases. Site-specific limited proteolysis plays an important role in a diverse range of biological processes such as the blood coagulation cascade (1Krem M.M. Cera E.D. Trends Biochem. Sci. 2002; 27: 67-74Google Scholar), determination of cell fate (2Selkoe D. Kopan R. Annu. Rev. Neurosci. 2003; 26: 565-597Google Scholar), ligand-induced receptor activation (3Coughlin S.R. Nature. 2000; 407: 258-264Google Scholar), release of cell-associated growth factors (4Hooper J.D. Clements J.A. Quigley J.P. Antalis T.M. J. Biol. Chem. 2001; 276: 857-860Google Scholar), tissue remodeling (5Stamenkovic I. J. Pathol. 2003; 200: 448-464Google Scholar), and apoptosis (6Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Google Scholar). These activities are usually carried out by specific proteolytic enzymes, some of which are themselves activated by limited proteolysis (7Anderson E.D. Molloy S.S. Jean F. Fei H. Shimamura S. Thomas G. J. Biol. Chem. 2002; 277: 12879-12890Google Scholar). A detailed understanding of the proteolytic mechanisms is not only critical for the functional studies of these biological processes but may also provide means for possible intervention and regulation. Of the various modes of proteolytic reactions, self-catalyzed proteolysis, or autoproteolysis, has been recognized as an essential step in the proper folding, trafficking, and activation of several endoproteases such as furin (7Anderson E.D. Molloy S.S. Jean F. Fei H. Shimamura S. Thomas G. J. Biol. Chem. 2002; 277: 12879-12890Google Scholar, 8Anderson E.D. VanSlyke J.K. Thulin C.D. Jean F. Thomas G. EMBO J. 1997; 16: 1508-1518Google Scholar, 9Creemers J.W. Vey M. Schafer W. Ayoubi T.A. Roebroek A.J. Klenk H.D. Garten W. Van de Ven W.J. J. Biol. Chem. 1995; 270: 2695-2702Google Scholar) and other subtilisin-like proprotein convertases (10Elagoz A. Benjannet S. Mammarbassi A. Wickham L. Seidah N.G. J. Biol. Chem. 2002; 277: 11265-11275Google Scholar) that are involved in the activation of many secretory protein precursors. Autoproteolysis is also involved in the activation of a novel group of hydrolytic enzymes, the N-terminal nucleophile hydrolases (Ntn-hydrolases) 1The abbreviations used are: Ntn-hydrolase, N-terminal nucleophile hydrolase; Ntn, N-terminal nucleophile; EGF, epidermal growth factor; EGFP, enhanced green fluorescence protein; EMR2, the epidermal growth factor module-containing mucin-like receptor 2; GPCR, G-protein-coupled receptor; GPS, GPCR proteolytic site; HA, hydoxylamine; LNB-TM7, long N-terminal family B GPCR-related 7TM receptor; Ab, antibody; mAb, monoclonal antibody; REJ, receptor for egg jelly; TM7, seven-transmembrane; WT, wild type; ER, endoplasmic reticulum; CM, conditioned medium; CL, cell lysate; GPS-receptor, GPS motif-containing receptor; GFP, green fluorescent protein. 1The abbreviations used are: Ntn-hydrolase, N-terminal nucleophile hydrolase; Ntn, N-terminal nucleophile; EGF, epidermal growth factor; EGFP, enhanced green fluorescence protein; EMR2, the epidermal growth factor module-containing mucin-like receptor 2; GPCR, G-protein-coupled receptor; GPS, GPCR proteolytic site; HA, hydoxylamine; LNB-TM7, long N-terminal family B GPCR-related 7TM receptor; Ab, antibody; mAb, monoclonal antibody; REJ, receptor for egg jelly; TM7, seven-transmembrane; WT, wild type; ER, endoplasmic reticulum; CM, conditioned medium; CL, cell lysate; GPS-receptor, GPS motif-containing receptor; GFP, green fluorescent protein. (11Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Google Scholar). The Ntn-hydrolases are activated from an inactive proenzyme by self-mediated hydrolysis of an internal peptide bond via an N → O or N → S acyl shift between a specific nucleophilic residue and its preceding amino acid (12Perler F.B. Xu M.Q. Paulus H. Curr. Opin. Chem. Biol. 1997; 1: 292-299Google Scholar, 13Perler F.B. Nat. Struct. Biol. 1998; 5: 249-252Google Scholar). The newly generated N-terminal nucleophile then acts as a single enzymatic active site to attack its specific protein substrates. One unique feature of the Ntn-hydrolases is that a single nucleophilic residue is used as the reactive nucleophile for both the autoproteolytic and enzymatic activity (14Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nat. Struct. Biol. 1995; 2: 1102-1108Google Scholar, 15Saarela J. Laine M. Tikkanen R. Oinonen C. Jalanko A. Rouvinen J. Peltonen L. J. Biol. 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Riikonen A. Oinonen C. Rouvinen R. Peltonen L. EMBO J. 1996; 15: 2954-2960Google Scholar, 22Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar), the proteasome (23Kisselev A.F. Songyang Z. Goldberg A.L. J. Biol. Chem. 2000; 275: 14831-14837Google Scholar), γ-glutamyltranspeptidase (24Suzuki H. Kumagai H. J. Biol. Chem. 2002; 277: 43536-43543Google Scholar), and Taspase1 (25Hsieh J.J. Cheng E.H. Korsmeyer S.J. Cell. 2003; 115: 293-303Google Scholar). In addition to these proteolytic enzymes, several other proteins such as hedgehog proteins (26Lee J.J. Ekker S.C. von Kessler D.P. Porter J.A. Sun B.I. Beachy P.A. Science. 1994; 266: 1528-1537Google Scholar, 27Porter J.A. von Kessler D.P. Ekker S.C. Young K.E. Lee J.J. Moses K. Beachy P.A. Nature. 1995; 374: 363-366Google Scholar), inteins (28Perler F.B. Cell. 1998; 92: 1-4Google Scholar), and nucleoporins (29Teixeira M.T. Fabre E. Dujon B. J. Biol. Chem. 1999; 274: 32439-32444Google Scholar, 30Rosenblum J.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11370-11375Google Scholar) also belong to the Ntn-hydrolase family. In recent years, a proteolytic motif known as the G protein-coupled receptor (GPCR) proteolytic site (GPS) (31) has been identified in over 40 cell surface receptors (see, on the World Wide Web, smart.embl-heidelberg.de/). As suggested in its denotation, the GPS motif is primarily found in members of the class B2 GPCRs (32Harmar A.J. Genome Biol. 2001; 2: 3013Google Scholar) or the LNB-TM7 receptors (33Stacey M. Lin H.H. Gordon S. McKnight A.J. Trends Biochem. Sci. 2000; 25: 284-289Google Scholar) that contain a large N-terminal cell adhesion-like extracellular domain coupled to a secretin receptor-like seven-pass transmembrane (TM7) domain. Examples include Flamingo (34Usui T. Shima Y. Shimada Y. Hirano S. Burgess R.W. Schwarz T.L. Takeichi M. Uemura T. Cell. 1999; 98: 585-595Google Scholar), latrophilin (31Krasnoperov V.G. Bittner M.A. Beavis R. Kuang Y. Salnikow K.V. Chepurny O.G. Little A.R. Plotnikov A.N. Wu D. Holz R.W. Petrenko A.G. Neuron. 1997; 18: 925-937Google Scholar, 35Sugita S. Ichtchenko K. Khvotchev M. Sudhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Google Scholar), Ig-hepta (36Abe J. Fukuzawa T. Hirose S. J. Biol. Chem. 2002; 277: 23391-23398Google Scholar), HE-6 (37Obermann H. Samalecos A. Osterhoff C. Schroder B. Heller R. Kirchhoff C. Mol. Reprod. Dev. 2003; 64: 13-26Google Scholar), and the EGF-TM7 receptors (38McKnight A.J. Gordon S. J. Leukocyte Biol. 1998; 63: 271-280Google Scholar). However, the GPS motif is not exclusively restricted to the TM7 proteins. Receptors with one- or 11-pass TM configurations such as suREJ1 (39Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar), suREJ3 (40Mengerink K.J. Moy G.W. Vacquier V.D. J. Biol. Chem. 2002; 277: 943-948Google Scholar), and polycystin-1 (41Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Google Scholar, 42Ponting C.P. Hofmann K. Bork P. Curr. Biol. 1999; 9: 585-588Google Scholar) also contain the consensus GPS motif, which is characterized by a Cys-rich segment of approximate 50 amino acids located proximal to the first TM domain. Proteolytic cleavage at the GPS motif generates an extracellular (α) and a TM (β) subunit that associate noncovalently on the cell surface as a heterodimer. This has led to a notion that these adhesion GPCRs might couple extracellular adhesion events to intracellular signaling via the α- and β-subunits, respectively (43Stacey M. Chang G.W. Sanos S.L. Chittenden L.R. Stubbs L. Gordon S. Lin H.H. J. Biol. Chem. 2002; 277: 29283-29293Google Scholar, 44Stacey M. Chang G.W. Davies J.Q. Kwakkenbos M.J. Sanderson R.D. Hamann J. Gordon S. Lin H.H. Blood. 2003; 102: 2916-2924Google Scholar). Furthermore, the highly conserved GPS motif-associated proteolysis suggests that this unique cleavage process is likely to be mediated by a common proteolytic mechanism and might be important for the function or regulation of the receptor. Indeed, the proteolysis of polycystin-1 has been found to be essential for its normal biological activity, since several autosomal dominant polycystic kidney disease-associated point mutations were shown to disrupt the GPS cleavage and the function of polycystin-1 (41Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Google Scholar). The epidermal growth factor (EGF)-like module containing mucin-like hormone receptor 2 (EMR2) is a human myeloid-restricted EGF-TM7 receptor whose extracellular domain consists of tandem repeats of EGF-like modules followed by a Ser/Thr-rich stalk and a GPS motif (45Lin H.H. Stacey M. Hamann J. Gordon S. McKnight A.J. Genomics. 2000; 67: 188-200Google Scholar, 46Kwakkenbos M.J. Chang G.W. Lin H.H. Pouwels W. de Jong E.C. van Lier R.A. Gordon S. Hamann J. J. Leukocyte Biol. 2002; 71: 854-862Google Scholar). Our previous studies on the proteolysis of EMR2 have located the precise cleavage site to a conserved tripeptide (His-Leu↓Ser518) sequence and demonstrated that the cleavage requires not only the GPS motif but also other extracellular domains in the stalk region (47Chang G.W. Stacey M. Kwakkenbos M.J. Hamann J. Gordon S. Lin H.H. FEBS Lett. 2003; 547: 145-150Google Scholar). In the present study, we elucidate the molecular basis for the cleavage of EMR2 and demonstrate that no protease is required for the proteolytic reaction. Instead, we show that EMR2 is cleaved by a self-catalyzed process characteristic of the autoproteolytic reaction commonly employed by the Ntn-hydrolases (11Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Google Scholar). Materials—All chemicals and reagents were obtained from Sigma unless otherwise specified. The EMR2 stalk-specific 2A1 mAb (mouse IgG1) was a gift from Dr J. Hamann (University of Amsterdam, The Netherlands). Anti-Myc (mouse IgG1) and rabbit anti-GFP polyclonal Ab (Living Colors A.v. peptide antibody) were from Invitrogen and Clontech, respectively. Rabbit polyclonal Ab against the 300-kDa mannose 6-phosphate receptor was provided by Dr. D. Werling (RVC, London). Mouse mAbs against ERGIC-53 (mouse IgG1) and proteindisulfide isomerase (1D3, IgG1) were provided by Drs. H. P. Hauri (University of Basel, Switzerland) and David Vaux (Sir William Dunn School of Pathology, Oxford), respectively. Secondary antibodies used are Cy3-conjugated F(ab′)2 donkey anti-mouse IgG (Jackson Immunoresearch) and Alexa-Fluor 647-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Leiden, The Netherlands). Cell Culture—All culture media were supplemented with 10% heat-inactivated fetal calf serum, 2 mm l-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin. All cells were incubated at 37 °C in a 5% CO2, 95% humidity incubator. Human embryonic kidney 293T cells were grown in Dulbecco's modified Eagle's medium and CHO-K1 cells in Ham's F-12 medium. EMR2 expression constructs were transfected into cells cultured in 100-mm dishes using LipofectAMINE™ (Invitrogen) as previously described (44Stacey M. Chang G.W. Davies J.Q. Kwakkenbos M.J. Sanderson R.D. Hamann J. Gordon S. Lin H.H. Blood. 2003; 102: 2916-2924Google Scholar, 48Lin H.H. Stacey M. Saxby C. Knott V. Chaudhry Y. Evans D. Gordon S. McKnight A.J. Handford P. Lea S. J. Biol. Chem. 2001; 276: 24160-24169Google Scholar). Generation of the EMR2 Expression Constructs and EMR2 Fusion Proteins—The EMR2 fusion proteins employed in this report are depicted in Fig. 1A. All expression constructs were generated using standard molecular biology methods. In brief, the cDNA fragments encoding the EMR2 extracellular domain or the full-length EMR2 protein were subcloned in frame into appropriate expression vectors upstream of the protein tags via selected restriction sites. The vectors used are pcDNA3.1/myc-HIS (Invitrogen), pEGFP-N1 (Clontech), and pcDNA3.1/mFc vector as previously described (43Stacey M. Chang G.W. Sanos S.L. Chittenden L.R. Stubbs L. Gordon S. Lin H.H. J. Biol. Chem. 2002; 277: 29283-29293Google Scholar, 44Stacey M. Chang G.W. Davies J.Q. Kwakkenbos M.J. Sanderson R.D. Hamann J. Gordon S. Lin H.H. Blood. 2003; 102: 2916-2924Google Scholar). The EMR2 site-directed mutants were made according to the protocols suggested by the manufacturer (GeneEditor Mutagenesis System; Promega). For the construction of the endoplasmic reticulum (ER)-restricted expression vector, a cDNA fragment encoding the KDEL ER retention signal was amplified by PCR using pCMV/myc/ER (Clontech) as a template. The cDNA fragment was then subcloned immediately after the EMR2-EGFP sequence. All expression constructs were subjected to DNA sequencing to confirm their identities. EMR2 fusion proteins were produced by transient transfection of cells. 48–72 h post-transfection, the EMR2 fusion protein was collected from conditioned medium (CM) or total cell lysate (CL) of transfected cells. Briefly, CM was spun at 2,000 rpm at 4 °C for 20 min followed by 100,000 rpm at 4 °C for 20 min. The supernatant was collected and stored at -80 °C. Total cell lysates were collected in cell lysis buffer (20 mm Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 5 mm MgCl2, 100 mm NaCl, 1 mm sodium orthovanadate, 1 mm AEBSF, 5 mm Levamisole, 1× complete™ (Roche Applied Science) protease inhibitors) at 4 °C. Protein concentration was determined by a Dc Protein Analysis Kit (Bio-Rad). For the purification of soluble EMR2-mFc fusion proteins, human embryonic kidney 293T cells were transfected with 40 μg of DNA/175-cm2 flask using calcium phosphate precipitation as previously described (43Stacey M. Chang G.W. Sanos S.L. Chittenden L.R. Stubbs L. Gordon S. Lin H.H. J. Biol. Chem. 2002; 277: 29283-29293Google Scholar, 44Stacey M. Chang G.W. Davies J.Q. Kwakkenbos M.J. Sanderson R.D. Hamann J. Gordon S. Lin H.H. Blood. 2003; 102: 2916-2924Google Scholar). The medium was replaced with 25 ml of serum-free Opti-MEM I 16–18 h post transfection and incubated for a further 72 h. Conditioned medium was collected, spun, and passed through a 0.45-μm filter, followed by Protein A-Sepharose 4 Fast Flow (Amersham Biosciences) column purification as previously described (43Stacey M. Chang G.W. Sanos S.L. Chittenden L.R. Stubbs L. Gordon S. Lin H.H. J. Biol. Chem. 2002; 277: 29283-29293Google Scholar, 44Stacey M. Chang G.W. Davies J.Q. Kwakkenbos M.J. Sanderson R.D. Hamann J. Gordon S. Lin H.H. Blood. 2003; 102: 2916-2924Google Scholar). Immunoprecipitation, Western Blotting, and Other Protein Analysis—EMR2 fusion proteins were immunoprecipitated from CM or CL using appropriate Abs and/or protein A/G beads. Briefly, CM (1 ml) and CL (100 μg) were either incubated with protein A/G-Sepharose directly or precleared with irrelevant Ab and protein A/G-Sepharose, followed by subsequent incubation with appropriate primary Ab and protein A/G-Sepharose, respectively. After extensive washes, the immunopurified proteins were subjected to in vitro cleavage reaction or glycosidase treatment as described. For the glycosidase treatment, the proteins were incubated with 1 unit of PNGase F (Roche Applied Science), 1 unit of endoglycosidase H (Roche Applied Science), or 0.5 milliunits of O-glycosidase (Roche Applied Science) plus 1.0 milliunit of neuraminidase in 20 mm sodium phosphate buffer, pH 7.0, at 37 °C for 20 h prior to Western blot analysis. For Western blotting, proteins were denatured in reducing sample buffer, subjected to electrophoresis in 8 or 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp.), and probed with 2A1 mAb, anti-Myc, or anti-GFP Ab. Following extensive washes, the blots were incubated with appropriate horseradish peroxidase-conjugated second Ab for ECL detection (Amersham Biosciences). The fluorescence intensity of EMR2-GFP fusion proteins was determined by a FLUOstar Galaxy fluorescence plate reader (BMG LabTechnologies Ltd., Aylesbury, UK) using an excitation wavelength at 485 nm and emission wavelength at 520 nm. Immunofluorescence Confocal Microscopy—Transfected cells grown on glass coverslips in 24-well tissue culture plates were fixed with 4% paraformaldehyde in phosphate-buffered saline, blocked, and permeabilized in blocking buffer (phosphate-buffered saline with 0.5% bovine serum albumin, 0.1% Triton X-100, and 1% normal donkey or goat serum) for 20 min at room temperature. Cells were then incubated sequentially for 1 h at room temperature with primary antibodies (5–10 μg/ml) and appropriate secondary antibody (5–10 μg/ml) diluted in the same blocking buffer with extensive washing in between incubations. Cells were then mounted onto glass slides with fluorescent mounting medium (Dako, Cambridgeshire, UK). Immunofluorescence was analyzed on a Bio-Rad Radiance 2000 laser-scanning confocal microscope. The resulting images were processed in Adobe® Photoshop® 6.0. In Vitro Cleavage of EMR2 Proteins—Immunoprecipitated EMR2 fusion proteins or those in CM and CL were incubated in cleavage buffer (50 mm Tris, pH 7.5, 20 mm NaCl, 1 mm EDTA) with or without 250 mm NH2OH at 37 °C unless otherwise specified. At various time points, samples were withdrawn and analyzed by Western blotting. For the biochemical characterization of EMR2 autoproteolysis, samples were incubated in the cleavage buffer containing protease inhibitors or other test reagents such as EDTA, as indicated in the throughout. For the demonstration of intramolecular cleavage, EMR2-H516S-mFc fusion protein was first purified by Protein A chromatography as described above. Purified proteins were then incubated at different concentrations (0.2 and 1.0 mg/ml) in cleavage buffer alone at 37 °C. At various time points, samples were withdrawn, subjected to SDS-PAGE, and stained in Simply Blue™ Safe-Stain (Invitrogen). The intensity of the uncleaved precursor protein band (∼110 kDa) was determined from the image captured by a Gel Doc 2000 gel documentation system (Bio-Rad). Proteolytic Cleavage at the Highly Conserved GPS Motif Occurs in the ER—We and others have previously shown that the proteolytic cleavage of the GPS motif-containing receptors (GPS-receptors) is a TM-independent process and that the GPS motif is necessary but not sufficient for proteolysis to occur (41Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Google Scholar, 47Chang G.W. Stacey M. Kwakkenbos M.J. Hamann J. Gordon S. Lin H.H. FEBS Lett. 2003; 547: 145-150Google Scholar, 49Krasnoperov V. Lu Y. Buryanovsky L. Neubert T.A. Ichtchenko K. Petrenko A.G. J. Biol. Chem. 2002; 277: 46518-46526Google Scholar). To further investigate this unique proteolytic process, we first compared the GPS motifs of all GPS-receptors that are known to be processed (Fig. 1B). The GPS motif is evolutionary conserved and widely present in cell surface receptors, including members of the LNB-TM7 or class B2 GPCRs as well as receptors with one- or 11-pass TM configuration such as suREJ1 (39Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Google Scholar), polycystin-1 (41Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16981-16986Google Scholar, 42Ponting C.P. Hofmann K. Bork P. Curr. Biol. 1999; 9: 585-588Google Scholar), and suREJ3 (40Mengerink K.J. Moy G.W. Vacquier V.D. J. Biol. Chem. 2002; 277: 943-948Google Scholar). The GPS motif is always located at the membrane-proximal region, ∼20–30 residues from the first TM domain. The cleavage site tripeptide is highly conserved: the P-2 residue is His, the P-1 residue is Leu (or Ile in the Drosophila Flamingo protein (34Usui T. Shima Y. Shimada Y. Hirano S. Burgess R.W. Schwarz T.L. Takeichi M. Uemura T. Cell. 1999; 98: 585-595Google Scholar)), and the P+1 residue is either Ser or Thr. N-terminal to the cleavage site are two invariable Trp residues and four constrained Cys residues believed to form two intramolecular disulfide bridges. One exception to the rule is polycystin-1 that contains only two Cys residues. C-terminal to the cleavage site are 6–8 small, hydrophobic residues that have been shown to be important both for proteolysis and noncovalent association of the cleaved subunits (47Chang G.W. Stacey M. Kwakkenbos M.J. Hamann J. Gordon S. Lin H.H. FEBS Lett. 2003; 547: 145-150Google Scholar). Overall, these features indicate that there is an ordered and complex structure surrounding the GPS cleavage site and suggest that all GPS-receptors probably undergo the same proteolytic process. To unveil the proteolytic mechanism at the GPS, we first investigated the subcellular compartment in which the proteolysis takes place. Previous pulse-chase experiments examining the cleavage of CD97, latrophilin/CL1 (calcium-independent receptor for latrotoxin (CIRL)/latrophilin), ETL, and Ig-Hepta have shown that the GPS proteolysis occurs very early (within 10–15 min) during protein biogenesis and suggested that it might occur in the ER (36Abe J. Fukuzawa T. Hirose S. J. Biol. Chem. 2002; 277: 23391-23398Google Scholar, 49Krasnoperov V. Lu Y. Buryanovsky L. Neubert T.A. Ichtchenko K. Petrenko A.G. J. Biol. Chem. 2002; 277: 46518-46526Google Scholar, 50Gray J.X. Haino M. Roth M.J. Maguire J.E. Jensen P.N. Yarme A. Stetler-Stevenson M.A. Siebenlist U. Kelly K. J. Immunol. 1996; 157: 5438-5447Google Scholar, 51Nechiporuk T. Urness L.D. Keating M.T. J. Biol. Chem. 2001; 276: 4150-4157Google Scholar). To further confirm and locate the cleavage reaction in the ER, we took advantage of the specific ER retention signal, KDEL, and examined the proteolysis of KDEL-tagged EMR2-enhanced green fluorescence protein (EGFP) fusion proteins (Fig. 2). In addition to the wild type (WT) stalk, two other stalks containing a cleavage site-deficient S518A and a control S519A point mutation, respectively, were also used to demonstrate the specificity of the cleavage reaction. The KDEL-tagged fusion proteins were confirmed to localize in the ER by the following observations. First, whereas the soluble EMR2-EGFP fusion proteins (with no KDEL signal) were detected in both CM and the total CL, the KDEL-tagged fusion proteins were found only in the CL (Fig. 2A). Thus, the KDEL-tagged fusion proteins were expressed but retained inside the cells. Confocal immunofluorescence staining subsequently showed that the KDEL-tagged proteins co-localize with the ER-lumen resident proteins such as protein-disulfide isomerase and calreticulin (Fig. 2B) (data not shown). On the other hand, they showed only minimum co-localization with ERGIC-53, a mannose-specific membrane lectin involved in the transport of glycoproteins from the ER to the ER-Golgi intermediate compartment (52Hauri H.P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Google Scholar). No co-localization of the KDEL-tagged proteins with mannose 6-phosphate receptor that recycles between the trans-Golgi network and endosomes was found (53Boker C. von Figura K. Hille-Rehfeld A. J. Cell Sci. 1997; 110: 1023-1032Google Scholar) (Fig. 2B). Finally, since EMR2 is heavily glycosylated, the KDEL-tagged EMR2 proteins were subjected to glycosidase digestion. N-Linked glycosylation is initiated in the ER lumen as a high mannosyl oligosaccharide, which is then modified to a complex form in the cis-Golgi compartment. Peptide:N-glycosidase F recognizes and digests N-glycosylated proteins between Asn and GlcNAc, whereas endoglycosidase H cleaves only the high mannose oligosaccharides. O-Glycosylation mainly takes place in the cis-Golgi compartment, so sensitivity to O-glycosidase digestion could also help indicate the subcellular localization of proteins. Western blot analysis showed that the KDEL-tagged proteins were sensitive to both peptide:N-glycosidase F and endoglycosidase H but were resistant to O-glycosidase digestion, indicating that these proteins have not trafficked out of the ER compartment (Fig. 2C). When analyzed for proteolytic cleavage, the KDEL-tagged EMR2-EGFP fusion protein containing the WT stalk or a control S519A point mutant stalk was shown to be effectively cleaved to two subunits, whereas the cleavage site-deficient (S518A) stalk did not (Fig. 2C). The same results were observed in several cell lines including CHO-K1, COS-7, human embryonic kidney 293T, and NIH3T3 (data not shown). Together, these and earlier results indicate that the GPS proteolytic cleavage is likely to be carried out by a conserved proteolytic machinery in the ER. EMR2 Proteolysis Is an Autoproteolytic Reaction—In order to identify the proposed ER-located proteolytic machinery, we first aimed to define the GPS cleavage site specificity. EMR2-mFc fusion proteins provide an efficient way for specific purification and detection of the protein and have been used previously to characterize EMR2 proteolysis (47Chang G.W. Stacey M. Kwakkenbos M.J. Hamann J. Gordon S. Lin H.H. FEBS Lett. 2003; 547: 145-150Google Scholar). Thus, a series of the EMR2-mFc fusion proteins were generated, where the Ser518 cleavage site residue was individually mutated to 19 other amino acids. As shown in Table I, proteolytic cleavage was detected in only three point mutants; the S518C and S518T mutants displayed the same efficient pr
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