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• W1971775650 abstract "The recently described protocadherin gene clusters encode cadherin-related proteins, which are highly expressed in the vertebrate nervous system. Here, we report biochemical studies addressing proteolytic processing of γ-protocadherins. These type-I transmembrane proteins are cleaved by a metalloproteinase in vivo, generating a soluble extracellular fragment and a carboxyl-terminal fragment associated with the cellular membrane. In addition, we show that the carboxyl-terminal fragment is a substrate for further cleavage mediated by presenilin. Consequently, accumulation of the fragment is found when γ-secretase is inactivated either by the specific presenilin-inhibitor L685,458 or in double mutant murine embryonic fibroblasts lacking both presenilin genes. The γ-secretase-generated carboxyl-terminal fragment is largely unstable but accumulates when proteasomal degradation is inhibited. Interestingly, the proteolytic fragment generated by γ-secretase can localize to the nucleus. This is the first report providing experimental evidence for a cell surface receptor signaling function of protocadherins regulated by proteolytic events. The recently described protocadherin gene clusters encode cadherin-related proteins, which are highly expressed in the vertebrate nervous system. Here, we report biochemical studies addressing proteolytic processing of γ-protocadherins. These type-I transmembrane proteins are cleaved by a metalloproteinase in vivo, generating a soluble extracellular fragment and a carboxyl-terminal fragment associated with the cellular membrane. In addition, we show that the carboxyl-terminal fragment is a substrate for further cleavage mediated by presenilin. Consequently, accumulation of the fragment is found when γ-secretase is inactivated either by the specific presenilin-inhibitor L685,458 or in double mutant murine embryonic fibroblasts lacking both presenilin genes. The γ-secretase-generated carboxyl-terminal fragment is largely unstable but accumulates when proteasomal degradation is inhibited. Interestingly, the proteolytic fragment generated by γ-secretase can localize to the nucleus. This is the first report providing experimental evidence for a cell surface receptor signaling function of protocadherins regulated by proteolytic events. The cadherin superfamily comprises a large number of cell adhesion molecules with ectodomains composed of multiple Ca2+-binding cadherin domains (1Angst B.D. Marcozzi C. Magee A.I. J. Cell Sci. 2001; 114: 625-626Crossref PubMed Google Scholar). Classical cadherins like E- or N-cadherin possess five such domains, which typically engage in homophilic binding. The highly conserved cytoplasmic domain binds to catenins, which establish a link to the actin cytoskeleton and thus promote cadherin-mediated cell adhesion (2Aberle H. Schwartz H. Kemler R. J. Cell. Biochem. 1996; 61: 514-523Crossref PubMed Scopus (714) Google Scholar). The juxtamembrane region of classical cadherins interacts with another cytoplasmic Armadillo repeat protein, p120, which is involved in the control of cadherin levels at the cell surface (3Davis M.A. Ireton R.C. Reynolds A.B. J. Cell Biol. 2003; 163: 525-534Crossref PubMed Scopus (567) Google Scholar, 4Peifer M. Yap A.S. J. Cell Biol. 2003; 163: 437-440Crossref PubMed Scopus (82) Google Scholar). Alternatively, the same domain can bind to presenilin, the catalytically active component of the γ-secretase complex (5Baki L. Marambaud P. Efthimiopoulos S. Georgakopoulos A. Wen P. Cui W. Shioi J. Koo E. Ozawa M. Friedrich Jr., V.L. Robakis N.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2381-2386Crossref PubMed Scopus (156) Google Scholar, 6Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (770) Google Scholar). Recent reports further showed that indeed both E-cadherin (7Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Baki L. Wen P. Efthimiopoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (616) Google Scholar) and N-cadherin (8Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) undergo proteolytic processing events involving γ-secretase activity. For both proteins, a first cleavage is executed by a matrix metalloproteinase (MMP) 1The abbreviations used are: MMP, matrix metalloproteinase; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; CBP, CREB-binding protein; CTF, carboxyl-terminal fragment; NTF, amino-terminal fragment; MEF, mouse embryo fibroblast(s); WT, wild type.1The abbreviations used are: MMP, matrix metalloproteinase; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; CBP, CREB-binding protein; CTF, carboxyl-terminal fragment; NTF, amino-terminal fragment; MEF, mouse embryo fibroblast(s); WT, wild type. activity, shedding a soluble cadherin amino-terminal fragment off the membrane. The remaining, membrane-associated, carboxyl-terminal part undergoes a presenilin-dependent intramembrane cleavage, which releases the cytoplasmic portion into the cytosol. The processing of E-cadherin is proposed to modulate its adhesive properties (5Baki L. Marambaud P. Efthimiopoulos S. Georgakopoulos A. Wen P. Cui W. Shioi J. Koo E. Ozawa M. Friedrich Jr., V.L. Robakis N.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2381-2386Crossref PubMed Scopus (156) Google Scholar, 7Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Baki L. Wen P. Efthimiopoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (616) Google Scholar). The N-cadherin carboxyl-terminal fragment seems to affect gene expression via binding to and inducing the degradation of CREB-binding protein, an essential co-factor for CREB-mediated transcription (8Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar).Protocadherins are cadherin-related adhesion molecules with six or seven extracellular cadherin motifs. Their cytoplasmic domains diverge from each other and from those of the classical cadherins, indicating different binding properties and intracellular functions (9Frank M. Kemler R. Curr. Opin. Cell Biol. 2002; 14: 557-562Crossref PubMed Scopus (216) Google Scholar). The recently described protocadherin α, β, and γ (Pcdhα, -β, and -γ) gene clusters together encode more than 50 protocadherin proteins. These clusters have attracted attention because of the unusual organization of arrayed genes, reminiscent of that of the immunoglobulin and T-cell receptor loci (10Wu Q. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3124-3129Crossref PubMed Scopus (95) Google Scholar, 11Wu Q. Zhang T. Cheng J.F. Kim Y. Grimwood J. Schmutz J. Dickson M. Noonan J.P. Zhang M.Q. Myers R.M. Maniatis T. Genome Res. 2001; 11: 389-404Crossref PubMed Scopus (199) Google Scholar). Large “variable” exons each comprise the coding information for a complete transmembrane protein, including ectodomain, transmembrane segment, and a short cytoplasmic tail. In the mouse, each of the 14 possible Pcdhα and 22 possible Pcdhγ transcripts is generated by alternative splicing of one of the variable exons to the three small exons, which together encode the constant portion, at the 3′- end of each of the clusters (12Tasic B. Nabholz C.E. Baldwin K.K. Kim Y. Rueckert E.H. Ribich S.A. Cramer P. Wu Q. Axel R. Maniatis T. Mol. Cell. 2002; 10: 21-33Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 13Wang X. Weiner J.A. Levi S. Craig A.M. Bradley A. Sanes J.R. Neuron. 2002; 36: 843-854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Thus, all Pcdhs α and Pcdhs γ share a family-specific constant domain that could transduce different extracellular information into a common intracellular signaling pathway or other executive functions. In contrast, Pcdhs of the β cluster lack a constant domain and thus represent single-exon genes (14Vanhalst K. Kools P. Vanden Eynde E. van Roy F. FEBS Lett. 2001; 495: 120-125Crossref PubMed Scopus (26) Google Scholar).The protocadherins derived from these gene clusters are predominantly expressed in the nervous system and have been found localized to synaptic junctions (15Kohmura N. Senzaki K. Hamada S. Kai N. Yasuda R. Watanabe M. Ishii H. Yasuda M. Mishina M. Yagi T. Neuron. 1998; 20: 1137-1151Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Functionally connected neurons are often characterized by the expression of a distinct set of cadherins, which may thus provide an adhesive code regulating neuronal differentiation and synaptogenesis (for reviews, see Refs. 16Shapiro L. Colman D.R. Neuron. 1999; 23: 427-430Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 17Redies C. Prog. Neurobiol. 2000; 61: 611-648Crossref PubMed Scopus (285) Google Scholar, 18Heyers D. Kovjanic D. Redies C. J. Comp. Neurol. 2003; 460: 155-166Crossref PubMed Scopus (24) Google Scholar). The large number of distinct protocadherins could contribute to the complexity of neuronal connections by significantly increasing the number of adhesive combinations (16Shapiro L. Colman D.R. Neuron. 1999; 23: 427-430Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 19Serafini T. Cell. 1999; 98: 133-136Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, the genetic loss of the entire Pcdh γ cluster has no obvious effect on synapse formation but is associated with severe neurological defects, including neuronal degeneration (13Wang X. Weiner J.A. Levi S. Craig A.M. Bradley A. Sanes J.R. Neuron. 2002; 36: 843-854Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). This indicates that Pcdh γ function also involves unknown signaling processes.Here we show that Pcdhs γ undergo endoproteolytic processing including metalloproteinase- and presenilin-dependent cleavages. In a final step, a short lived carboxyl-terminal fragment is generated by γ-secretase that can translocate to the nucleus, suggesting that Pcdhγ functions in the regulation of gene transcription.EXPERIMENTAL PROCEDURESCell Culture, Drug Treatments, and Transfections—Mouse embryonic fibroblasts, human embryonic kidney 293 cells, and COS-7 monkey fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 μm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin. The proteasome inhibitor lactacystin (Calbiochem) was used at 5 μm final concentration from a 5 mm stock solution prepared in dimethyl sulfoxide (Me2SO). The presenilin inhibitor L685,458 (Sigma) was dissolved in Me2SO (at 2 mm) and used at a final concentration of 2–10 μm. The metalloprotease inhibitor GM6001 (Biomol) was used at a final concentration of 10 μg/ml from a 1 mg/ml stock in Me2SO. Inhibitor treatments were performed for 12–16 h if not indicated otherwise.For transient transfections, cells were plated on 6-well plates (Costar) or on 35-mm cell culture dishes with a glass coverslip attached at the bottom and transfected with DNA (5 μg/well) using the calcium phosphate precipitation method for 293 cells or Polyfect reagent for COS-7 cells, according to the instructions of the manufacturer (Qiagen, Germany). After 48–72 h, expression was analyzed by Western blotting or fluorescence microscopy. Stable transfectants were obtained by culturing transfected cells in the presence of G418 (700 μg/ml; Invitrogen) or zeocyin (500 μg/ml; Invitrogen). Clonal cell lines were established using fluorescence-activated cell sorting technology and selection on 96-well plates (Greiner).cDNA Constructs and Antibodies—cDNA was obtained by reverse transcription (Superscript II; Invitrogen) using oligo(dT) or Pcdhγ constant region-specific priming of mouse brain total RNA. Full-length PcdhγA3 and PcdhγC3 constructs were generated from PCR amplifications with Pcdh-specific primers on cDNA (constant region) and on BAC-DNA spanning the Pcdhγ cluster (11Wu Q. Zhang T. Cheng J.F. Kim Y. Grimwood J. Schmutz J. Dickson M. Noonan J.P. Zhang M.Q. Myers R.M. Maniatis T. Genome Res. 2001; 11: 389-404Crossref PubMed Scopus (199) Google Scholar). These PCR products were cloned into the reading frame of the pEGFPN1 expression vector (Clontech) to give COOH-terminal fusions of the Pcdhs with the enhanced green fluorescent protein (eGFP). For expression of untagged Pcdhs γ, constructs were cloned into the pcDNA3.1.zeo expression vector (Invitrogen, Germany). For the NH2-terminal fusion with GFP, the signal sequence of PcdhγC3 in pcDNA3.1.zeo was replaced in frame by the signal sequence of Igα followed by a FLAG tag and eGFP taken from a construct kindly provided by Dr. Stefan Kuppig (Max-Planck Institute of Immunobiology, Freiburg, Germany). This same sequence (without GFP) was used for cloning of the extracellularly truncated version of Pcdhs to produce FLAG-γA3 and FLAG-γC3 COOH-terminal GFP fusions. Table I lists all of the proteins analyzed in this study.Table IProtocadherin γ-molecules analyzed in this study Open table in a new tab Anti-PcdhγC3 antibodies were raised against an extracellular epitope (γC3a, amino acids 71–86) and an intracellular epitope (γC3b, amino acids 792–809) of PcdhγC3 by boosting rabbits with these peptides coupled to keyhole limpet hemocyanin at 30-day intervals (Pineda Antibody Service, Berlin, Germany). The antisera were tested on 293 cells transiently transfected with PcdhγC3-eGFP. The rabbit antibodies to the Pcdhγ constant domain have been described previously (20Phillips G.R. Tanaka H. Frank M. Elste A. Fidler L. Benson D.L. Colman D.R. J. Neurosci. 2003; 23: 5096-5104Crossref PubMed Google Scholar).Metabolic Labeling and Immunoprecipitations—Cells were cultured in 6-well plates and starved for 1–2 h at 37 °C in 1 ml/well of Dulbecco's modified Eagle's medium lacking methionine and cysteine, supplemented with 10% dialyzed fetal calf serum, prior to biosynthetic labeling with 80 μCi of [35S]methionine and [35S]cysteine (Promix™; Amersham Biosciences). Cell culture supernatants and washed cells were collected after overnight incubation. For pulse-chase experiments, cells were labeled for 30 min or 1 h, and the chase was initiated by washing the cells and replacing the labeling medium with complete medium. After the times indicated, washed cells were scraped from the plates and solubilized in 300 μl of NET buffer (21Kessler S.W. J. Immunol. 1975; 115: 1617-1624PubMed Google Scholar). Immunoprecipitations were performed using 1–2 μg of antibody and 25 μl of postnuclear supernatant/sample, essentially as described (22Kaloff C.R. Haas I.G. Immunity. 1995; 2: 629-637Abstract Full Text PDF PubMed Scopus (67) Google Scholar).SDS-PAGE, Western Blotting, and Detection of Radiolabeled Proteins—Lysate or immunoprecipitated proteins were separated by SDS-PAGE on 10 or 8–16% gradient gels (Fermentas, St. Leon-Rot, Germany). Gels were stained with Coomassie Blue and incubated in Amplify (Amersham Biosciences) before fluorography (Eastman Kodak Co.) or used for protein transfer onto nitrocellulose membranes (Schleicher & Schuell). Reactive bands were detected using the following primary antibodies: rabbit anti-GFP (1:500; Clontech), rabbit anti-FLAG (1:400; Sigma), rabbit anti-C3a (1:2,000), rabbit anti-C3b (1: 20,000), and rabbit antibodies to the Pcdhγ constant domain (1:2,000). After incubation with peroxidase-coupled anti-rabbit antibodies (Jackson/Dianova, Heidelberg, Germany), the blots were developed with chemiluminiscent reagents (Pierce).Fluorescence Microscopy and Imaging—Analysis of eGFP fluorescence in living cells grown on glass bottom dishes was done by confocal laser scanning microscopy on a Leica DMIre2 inverted microscope setup using appropriate filter settings for eGFP detection. In addition, eGFP fluorescence was analyzed in cells grown on coverslips in 6-well plates. After fixation with paraformaldehyde, the coverslips were mounted on slides with Fluoromount-G (Southern Biotechnology, Birmingham, AL). Films of fluorographs, gels, and Western blots were scanned with a flatbed scanner, and digital images were imported and processed in Photoshop software (Adobe).RESULTSMatrix Metalloproteinase-mediated Cleavage of Pcdhγ into an Amino-terminal Fragment Shed from the Membrane and a Membrane-bound Carboxyl-terminal Fragment—According to sequence homologies, γ-protocadherins fall into three subclasses, Pcdhγ A, B, and C. We used 293 cells expressing two different Pcdhγ proteins fused carboxyl-terminal to GFP (i.e. PcdhγC3-GFP and PcdhγA3-GFP). Cellular extracts were analyzed by Western blotting using an antiserum directed against the constant domain of Pcdhγ proteins (Fig. 1A, lanes 1–3) as well as anti-GFP antibodies (Fig. 1A, lanes 4–6). Both antibodies detected bands corresponding to the full-length Pcdhγ-GFP fusion proteins, which migrated at the expected position of ∼150 kDa. In addition, smaller proteins were detected, with apparent molecular masses of ∼50 kDa in the case of PcdhγC3-GFP and ∼65 kDa in the case of PcdhγA3-GFP (Fig. 1A). Analysis of a second member of the PcdhγA subclass, PcdhγA1-GFP, also showed a fragment of 65 kDa (data not shown).Fig. 1γ-Protocadherins are cleaved by MMP into a COOH-terminal fragment associated with the cell membrane and an NH2-terminal fragment shed from the cell surface.A, lysates of transfected 293 cells stably expressing PcdhγC3-GFP (lanes 1, 4, 7, and 10) or PcdhγA3-GFP (lanes 2, 5, 8, and 11) or of untransfected 293 cells (lanes 3, 6, 9, and 12) were probed with antibodies to the Pcdhγ constant domain (anti-γconst., lanes 1–3), GFP (lanes 4–6), PcdhγC3a (lanes 7–9), or PcdhγC3b (lanes 10–12). Anti-C3a and -C3b recognize epitopes situated at the NH2 and COOH terminus of the PcdhγC3 variable domain, respectively. Note that the GFP moiety is at the COOH terminus of the fusion proteins. B, 293 cells stably expressing PcdhγA3- or PcdhγC3-GFP fusion proteins were biosynthetically labeled for 30 min. Postnuclear supernatants were prepared at the chase times indicated and used for immunoprecipitation of GFP fusion proteins. CTF is generated from full-length protein as evidenced by the time-dependent appearance of the labeled fragments (arrowheads). C, 293 cells stably expressing GFP-PcdhγC3 fusion protein or untransfected controls were biosynthetically labeled for 12 h in the absence or presence of GM6001 (4 μg/ml), as indicated. Cell culture supernatants or cell lysates were used for immunoprecipitation of GFP-containing proteins. Note that here the GFP moiety is at the NH2 terminus of PcdhγC3. The NH2-terminal fragment (arrowhead) immunoprecipitated from the cell culture supernatant (sup) is smaller than the full-length protein present in the cellular lysate (lys).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further characterize these fragments, antibodies reacting with two different epitopes of PcdhγC3 were used. Anti-C3a antibodies recognize a peptide epitope present in the first extracellular domain and detected full-length PcdhγC3-GFP but not the lower molecular weight fragment (Fig. 1A, lane 7). In contrast, anti-C3b antibodies, raised against a peptide at the very end of the PcdhγC3 variable domain, recognized both the full-length PcdhγC3-GFP protein and the fragment (Fig. 1A, lane 10), whereas no protein from untransfected or PcdhγA3-GFP-expressing cells was labeled. From these results, we conclude that the Pcdhγ fragments are composed of the GFP moiety, the Pcdhγ constant domain, and a carboxyl-terminal portion of the variable domain.To investigate whether this carboxyl-terminal fragment (CTF) is derived from a processing event, we performed pulse-chase experiments with stably transfected 293 cells that express PcdhγA3-GFP or PcdhγC3-GFP and immunoprecipitated the proteins carrying the GFP moiety. Directly after the pulse, only full-length PcdhγA3- or PcdhγC3-GFP fusion proteins were detected (Fig. 1B). Increasing amounts of CTF appeared in a time-dependent fashion in both cell lines, thus demonstrating that the fragments result from endoproteolytic processing of full-length PcdhγA3-GFP and PcdhγC3-GFP, respectively. In all experiments, PcdhγA3 appeared to be more rapidly processed into CTF than PcdhγC3.Carbonate extraction of membrane fractions obtained by differential centrifugation of mechanically broken cells indicated that CTF is an integral membrane protein (data not shown). These findings suggest that the site of endoproteolytic cleavage is located in the Pcdhγ extracellular domain, possibly mediated by an MMP. If so, the generation of CTF should be accompanied by the shedding off of the membrane of a corresponding soluble NH2-terminal fragment (NTF). The release of such a fragment should be inhibited by GM6001, a synthetic broad range metalloproteinase inhibitor (23Galardy R.E. Cassabonne M.E. Giese C. Gilbert J.H. Lapierre F. Lopez H. Schaefer M.E. Stack R. Sullivan M. Summers B. Ann. N. Y. Acad. Sci. 1994; 732: 315-323Crossref PubMed Scopus (153) Google Scholar).γ-Protocadherins have an apparent molecular mass of ∼120 kDa (20Phillips G.R. Tanaka H. Frank M. Elste A. Fidler L. Benson D.L. Colman D.R. J. Neurosci. 2003; 23: 5096-5104Crossref PubMed Google Scholar). Calculating a molecular mass of 20–25 kDa for the CTF of unmodified PcdhγC3 (see Fig. 3B), the NH2-terminal fragment should have a mass of ∼100 kDa. To detect NTF, we analyzed transfected 293 cells producing a PcdhγC3 protein with a GFP tag at the NH2 terminus, as pictured in Table I. Biosynthetic labeling was performed in the absence or presence of GM6001, and cell lysates as well as cell culture supernatants were analyzed for GFP-tagged proteins by immunoprecipitation. In the lysate of transfected cells, only full-length GFP-PcdhγC3 protein with a molecular mass of ∼150 kDa was detected (Fig. 1C). A GFP-containing NTF of the expected size (100 kDa + GFP = 130 kDa) was detected exclusively in the cell culture supernatant of transfected cells (Fig. 1C, arrowhead). When the labeling was performed in the presence of GM6001, only marginal amounts of NTF were detected, supporting our hypothesis that the cleavage is MMP-dependent. Processing of γ-protocadherins seems to be a general phenomenon, also detected with endogenous Pcdhγ protein in several cell lines. For example, we found low steady state levels of PcdhγC3 and CTF in mouse embryonic fibroblasts (MEF; see results below), but not when the MEF were cultured in the presence of GM6001. PcdhγC3-CTF was also detected in P19 embryonic carcinoma cells, which have been widely used as a model for neuronal differentiation (24Finley M.F. Kulkarni N. Huettner J.E. J. Neurosci. 1996; 16: 1056-1065Crossref PubMed Google Scholar, 25Kotani M. Osanai T. Tajima Y. Kato H. Imada M. Kaneda H. Kubo H. Sakuraba H. J. Neurosci. Res. 2002; 67: 595-606Crossref PubMed Scopus (29) Google Scholar). Interestingly, the low levels of endogenous PcdhγC3 expressed by P19 cells increased upon differentiation induced by retinoic acid. Accordingly, PcdhγC3-CTF was readily detected, providing suggestive evidence for efficient proteolytic processing in those cells (data not shown).Fig. 3Inhibition of presenilin function leads to accumulation of Pcdhγ-CTF.A, pulse-chase experiment with 293 cells stably expressing PcdhγA3 fusion protein. Cells were biosynthetically labeled for 1 h and chased for 5 h, all in the absence or presence of the specific presenilin inhibitor L685,458. Anti-GFP antibodies were used for immunoprecipitation of PcdhγA3-GFP and CTF. The inhibitor leads to strong accumulation of CTF (arrowhead). B, presenilin-dependent processing of both recombinant untagged and endogenous PcdhγC3. 293-PcdhγC3 cells (left panel) or WT or mutant (PS1–/–, PS2–/–) MEF (right panel) were cultured in the absence or presence of L685,458, as indicated. Western blots were probed with anti-C3b antibodies. Note that CTF produced from both untagged and endogenous PcdhγC3 has an apparent molecular mass of ∼25 kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Taken together, our results provide strong evidence for an extracellular cleavage of Pcdhγ mediated by a matrix metalloproteinase to produce soluble Pcdhγ-NTF and membrane-bound Pcdhγ-CTF.MMP Cleavage in Pcdhγ Ectodomains Occurs Close to the Membrane—To narrow down the MMP cleavage sites, we designed truncated PcdhγA3 and PcdhγC3 molecules, lacking almost the complete extracellular domain (see Table I). A signal sequence followed by a FLAG tag was introduced 18 amino acids NH2-terminal to the respective transmembrane segment of PcdhγA3 and PcdhγC3 (Fig. 2A). 293 cells were transiently transfected with the FLAG-tagged constructs, and Western blots were performed using the anti-FLAG antibodies. Remarkably, the two proteins migrated with a considerably different mobility (Fig. 2B), although they have a similar molecular mass (Fig. 2A). This difference in mobility resembled that of the respective CTFs in Fig. 1A and could be due to the different charge properties of the respective proteins. When anti-GFP antibodies were used, faint bands below the FLAG-tagged proteins became detectable, indicating that both FLAG-γA3-GFP and FLAG-γC3-GFP were still slowly processed into CTF. However, the cleavage efficiency is apparently higher with the nontruncated proteins (compare Figs. 2B and 1A).Fig. 2MMP cleavage in both PcdhγA3 and -C3 proteins occurs in close vicinity to the membrane.A, schematic representation of the FLAG-tagged PcdhγA3-GFP (FLAG-γA3-GFP) and the FLAG-tagged PcdhγC3-GFP (FLAG-γC3-GFP). The signal sequence preceding the FLAG tag is not indicated. The various domains of the proteins are indicated by boxes: FLAG tag (striped); transmembrane (TM) segment (black); carboxyl-terminal end of the variable domain (white); constant domain of Pcdhγ (γ-const., light gray); and GFP moiety (dark gray). The outlined Pcdhγ-specific sequences of 18 amino acids separate the FLAG tag from the respective Pcdhγ-transmembrane segment. B, cell surface expression of FLAG-γ-C3-GFP. 293 cells were stably transfected to express FLAG-γ-C3-GFP, and fixed cells were examined by confocal microscopy. C, 293 cells were transfected to express the proteins shown in A. Lysates were analyzed by Western blotting (WB) using anti-FLAG or anti-GFP antibodies, as indicated. Note that CTF (arrowheads) is detected only by anti-GFP antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)From these results, we conclude that the MMP cleavage in both PcdhγA3 and -C3 proteins occurs in close vicinity to the membrane, giving rise to CTF proteins of similar molecular weight.Inhibition of Presenilin Action Leads to the Accumulation of Pcdhγ-CTF—The MMP-dependent processing of γ-protocadherins into CTF and NTF is reminiscent of the cleavage preceding the presenilin-mediated processing of classical cadherins (7Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Baki L. Wen P. Efthimiopoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (616) Google Scholar, 8Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). To investigate a possible involvement of presenilin, we cultured PcdhγA3-GFP-expressing 293 cells in the presence of the aspartyl protease transition state mimic L685,458, a highly specific presenilin-1 inhibitor (26Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (364) Google Scholar). We found that inhibition of presenilin-1 by L685,458 resulted in a time-dependent accumulation of CTF (data not shown), which would be expected when presenilin-dependent downstream processing of CTF is blocked. Consistent with this scenario, the presence of L685,458 led to the accumulation of PcdhγA3-GFP-CTF in a pulse chase (Fig. 3A). Treatment with the inhibitor also led to strong enrichment of a protein of the molecular mass expected for unmodified CTF (∼25 kDa) when 293 cells expressed recombinant PcdhγC3 without the GFP tag (Fig. 3B, left panel). In addition, a slightly larger band of unknown origin was detected, possibly reflecting a second MMP cleavage site in PcdhγC3. If presenilins are indeed involved in the downstream processing of Pcdhγ-CTF, one would expect an accumulation of Pcdhγ-CTF in cells lacking γ-secretase activity. To address this possibility, we used MEF established from wild type (WT) or double mutant embryos (PS1–/–; PS2–/–) lacking both presenilin genes (27Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar). Remarkably, these fibroblasts express PcdhγC3 endogenously, and full-length PcdhγC3 (∼120 kDa) was detected in both wild type and mutant MEF with anti-C3b antibodies (Fig. 3B, right panel). An additional fragment corresponding to PcdhγC3-CTF was detected in the mutant but not in WT MEF. Importantly, treatment of WT MEF with the presenilin i" @default.
• W1971775650 created "2016-06-24" @default.
• W1971775650 creator A5024449849 @default.
• W1971775650 creator A5043535434 @default.
• W1971775650 creator A5051460980 @default.
• W1971775650 creator A5052103880 @default.
• W1971775650 date "2005-03-01" @default.
• W1971775650 modified "2023-10-02" @default.
• W1971775650 title "Presenilin-dependent Processing and Nuclear Function of γ-Protocadherins" @default.
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