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• W1988629688 abstract "A disintegrin and metalloproteinase 10 (ADAM10) is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a number of proteins implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its shedding activity. Here we report the identification of an endoplasmic reticulum (ER) retention motif within the ADAM10 intracellular C-terminal tail. Sequential deletion/mutagenesis analyses showed that an arginine-rich (723RRR) sequence was responsible for the retention of ADAM10 in the ER and its inefficient surface trafficking. Mutating the second arginine to alanine was sufficient to allow ER exit and surface expression in both heterologous cells and hippocampal neurons. As synapse-associated protein 97 (SAP97) binds ADAM10 at its cytoplasmic tail and facilitates forward ADAM10 trafficking in neurons, we tested whether SAP97 could modulate ER export. However, neither expression nor Ser-39 phosphorylation of SAP97 in heterologous cells or hippocampal neurons were sufficient to allow the ER exit of ADAM10, suggesting that other signaling pathways or alternative binding partners are responsible for ADAM10 ER exit. Together, these results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10. A disintegrin and metalloproteinase 10 (ADAM10) is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a number of proteins implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its shedding activity. Here we report the identification of an endoplasmic reticulum (ER) retention motif within the ADAM10 intracellular C-terminal tail. Sequential deletion/mutagenesis analyses showed that an arginine-rich (723RRR) sequence was responsible for the retention of ADAM10 in the ER and its inefficient surface trafficking. Mutating the second arginine to alanine was sufficient to allow ER exit and surface expression in both heterologous cells and hippocampal neurons. As synapse-associated protein 97 (SAP97) binds ADAM10 at its cytoplasmic tail and facilitates forward ADAM10 trafficking in neurons, we tested whether SAP97 could modulate ER export. However, neither expression nor Ser-39 phosphorylation of SAP97 in heterologous cells or hippocampal neurons were sufficient to allow the ER exit of ADAM10, suggesting that other signaling pathways or alternative binding partners are responsible for ADAM10 ER exit. Together, these results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10. A disintegrin and metalloproteinase 10 (ADAM10) 2The abbreviations used are: ADAMa disintegrin and metalloproteinaseADAlzheimer DiseaseFITCfluorescein isothiocyanateERendoplasmic reticulumGFPgreen fluorescent proteinPCproprotein convertase. belongs to a large family of membrane-anchored metalloproteases, which are known as the ADAM protein family. ADAMs mediate the proteolytic cleavage of transmembrane proteins in their juxtamembrane region, causing their shedding, i.e. the release of their extracellular domain in a soluble form. In addition, through the intracellularly retained stubs, ADAMs can initiate the activation of intracellular signaling cascades. Because of their metalloprotease, integrin binding, cell adhesion, and signaling functions, ADAMs are well positioned to coordinate cellular processes that are required for neural development, plasticity, and repair (1.Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (922) Google Scholar, 2.Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (892) Google Scholar, 3.Pruessmeyer J. Ludwig A. Semin Cell Dev. Biol. 2009; 20: 164-174Crossref PubMed Scopus (182) Google Scholar). a disintegrin and metalloproteinase Alzheimer Disease fluorescein isothiocyanate endoplasmic reticulum green fluorescent protein proprotein convertase. ADAM10 works as a sheddase for a large number of transmembrane proteins involved in a variety of biological functions, and has been implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease (AD) (4.Kheradmand F. Werb Z. Bioessays. 2002; 24: 8-12Crossref PubMed Scopus (110) Google Scholar, 5.Moss M.L. Bartsch J.W. Biochemistry. 2004; 43: 7227-7235Crossref PubMed Scopus (119) Google Scholar). Because of these links, much effort is currently directed toward developing tools which modulate ADAM10 activity and can be used to target these pathologies. ADAM10 is a multidomain transmembrane glycoprotein which is expressed ubiquitously (6.Prinzen C. Muller U. Endres K. Fahrenholz F. Postina R. Faseb. J. 2005; 19: 522-1524Crossref Scopus (116) Google Scholar). It contains an N-terminal signal sequence followed by a prodomain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, an EGF-like repeat, a transmembrane domain and a SH3-binding cytoplasmic tail (7.Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3922-3927Crossref PubMed Scopus (985) Google Scholar). ADAM10 is synthesized in an inactive form that carries a proprotein convertase (PC) recognition sequence between the prodomain and the catalytic domain. Both PC7 and furin can cleave ADAM10 at the predicted PC cleavage motif to yield a mature, active form (8.Anders A. Gilbert S. Garten W. Postina R. Fahrenholz F. Faseb. J. 2001; 15: 1837-1839Crossref PubMed Scopus (187) Google Scholar). ADAM10 cleavage and maturation occur in the trans-Golgi network, in vesicles of the secretory pathway and at the cell surface (9.Schäfer W. Stroh A. Berghöfer S. Seiler J. Vey M. Kruse M.L. Kern H.F. Klenk H.D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (220) Google Scholar, 10.Wouters S. Leruth M. Decroly E. Vandenbranden M. Creemers J.W. van de Loo J.W. Ruysschaert J.M. Courtoy P.J. Biochem. J. 1998; 336: 311-316Crossref PubMed Scopus (33) Google Scholar); indeed, it has been suggested that the active form of ADAM10 can exert its catalytic activity both along the secretory system and at the plasma membrane (7.Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3922-3927Crossref PubMed Scopus (985) Google Scholar). Yet, despite the importance of forward secretory trafficking for ADAM10 activity, the mechanisms regulating its trafficking and localization are largely unknown. Multiple signaling pathways could regulate ADAM10 trafficking and, consequently, modulate its activity in different cellular systems. For instance, calcium influx stimulates ADAM10 activity in fibroblasts, an effect that requires the intracellular C terminus (11.Horiuchi K. Le Gall S. Schulte M. Yamaguchi T. Reiss K. Murphy G. Toyama Y. Hartmann D. Saftig P. Blobel C.P. Mol. Biol. Cell. 2007; 18: 176-188Crossref PubMed Scopus (182) Google Scholar). In neurons, the interaction between ADAM10 and synapse-associated protein-97 (SAP97), a protein involved in the dynamic trafficking of proteins to excitatory synapses, regulates the localization of ADAM10 at the postsynaptic membrane (12.Marcello E. Gardoni F. Mauceri D. Romorini S. Jeromin A. Epis R. Borroni B. Cattabeni F. Sala C. Padovani A. Di Luca M. J. Neurosci. 2007; 27: 1682-1691Crossref PubMed Scopus (154) Google Scholar). However, before reaching the plasma membrane and inserting into synapses, newly synthesized ADAM10 must be transported through the endoplasmic reticulum (ER)-Golgi secretory pathway via a set of processes that remain poorly understood. ER retention is a common mechanism used by many cell types to control the forward trafficking and surface expression of integral membrane proteins. Specific ER retention/retrieval signals and ER export signals have been identified in the intracellular domains of various channels and receptors, and these signals govern their surface density (13.Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 14.Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15.Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar). ER exit has also been reported to be a rate-limiting step for the surface trafficking of ADAM12 and ADAM22, two members of the ADAMs family. This process is regulated by ER retention signals in their cytoplasmic tails, and binding to 14-3-3 proteins allows ER release and membrane targeting of ADAM22 by masking the retention signals (16.Hougaard S. Loechel F. Xu X. Tajima R. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 275: 261-267Crossref PubMed Scopus (52) Google Scholar, 17.Cao Y. Kang Q. Zhao Z. Zolkiewska A. J. Biol. Chem. 2002; 277: 26403-26411Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 18.Gödde N.J. D'Abaco G.M. Paradiso L. Novak U. J. Cell Sci. 2006; 119: 3296-3305Crossref PubMed Scopus (20) Google Scholar). We have now investigated the trafficking and surface expression of ADAM10, and report the identification of a novel arginine-rich ER retention motif within its intracellular C terminus. Our results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10. The following antibodies were used: mouse anti-SAP97 (NeuroMab, Davis, CA), rabbit anti-calnexin (Stressgen Biotechnologies, Victoria, Canada), rabbit anti-calreticulin (Affinity Bioreagents, Golden, CO), rat anti-ADAM10 (R&D, Minneapolis, MN), chicken anti-GFP (Millipore, Billenica, MA), goat anti-Tac antibody (Sigma Aldrich, St. Louis, MO), rabbit anti-giantin (Covance, Princeton, NJ); mouse anti-Tac antibody (clone 7G7) was kindly provided by Dr. Bonifacino. Primary neuronal cultures were prepared from E18-E19 rat hippocampi as described (19.Gardoni F. Bellone C. Viviani B. Marinovich M. Meli E. Pellegrini-Giampietro D.E. Cattabeni F. Di Luca M. Eur. J. Neurosci. 2002; 16: 777-786Crossref PubMed Scopus (35) Google Scholar). Neurons were transfected using the calcium phosphate precipitation method at 10 days in vitro. COS7 cells were grown in DMEM supplemented with 10% bovine serum, 1 mm sodium pyruvate, and 50 units/ml penicillin/streptomycin. COS7 cells were transiently transfected with cDNA expression constructs using Superfect® Transfection Reagent (Qiagen, Hilden, Germany) and were grown for 24 h before fixation for immunocytochemistry or lysis for Western blot analysis. Chimeras of the extracellular domain of the human interleukin-2 receptor (Tac) with the intracellular C-terminal domain of mouse ADAM10 were generated by amplifying the ADAM10 C-terminal domain with the following set of primers: forward 5′-TGCCCAAGCTTCCGGATTTATCAAGATTTGCAGTG-3′ and reverse 5′-GCTCTAGATTAGCGTCGCATGTGTCCC-3′. Deletion mutants Tac737Δ, Tac734Δ, and Tac721Δ were generated by PCR amplification using the same forward primer and different reverse primers: 5′-GCTCTAGATTACCTCTGACGCGGGGGCTG-3′ for Tac737Δ, 5′-GCTCTAGATTACGGGGGCTGCTGAATGGG-3′ for Tac734Δ, 5′-GCTCTAGATTATAAAGTGCCTGGAAGTGGTTT-3′ for Tac721Δ. After digestion with HindIII and XbaI, PCR fragments were ligated into the linearized Tac pCDM8 expression vector. TacADAM10, full-length mouse ADAM10 (flADAM10) point mutations, and the stop codon resulting in the flADAM10 721Δ construct were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), following the manufacturer's instructions. All constructs were verified by sequencing. To evaluate surface and total staining, transfected COS7 cells or neurons were fixed with 4% paraformaldehyde, 4% sucrose in phosphate-buffered saline pH 7.4, and then incubated with either anti-Tac 7G7 antibody (for Tac constructs) or anti-ADAM10 antibody. To visualize surface expression, cells were then blocked with 4% normal serum, followed by a Cy3-conjugated secondary antibody. Afterward, cells were permeabilized with 0.1% Triton X-100 for 10 min and intracellular expression was determined by incubating cells with the appropriate antibody and labeling the total receptor fraction with a FITC-conjugated secondary antibody. For colocalization experiments, transfected COS7 cells were permeabilized with 0.2% saponin before incubation with anti-Tac 7G7 antibody and anti-calnexin/calreticulin (ER marker) or giantin antibodies (Golgi marker) followed by corresponding secondary antibodies. Wide-field fluorescence images were acquired in a Zeiss Axiovert 200M epifluorescence microscope with a Zeiss ×40 or 25 objective and a CoolSnap CCD camera. Images were analyzed using Metamorph Imaging software (Molecular Devices, Sunnyvale, CA). For quantification of surface and total expression intensities, images were acquired using the same settings and exposure times. The average intensity of surface fluorescence staining (Cy3, red) was determined after cell tracing and normalized to the total intensity (FITC, green) to correct for differences in expression. Surface ratios were obtained by dividing the background subtracted Cy3 and FITC fluorescence intensities. Statistical differences were analyzed by ANOVA followed by the Bonferroni test. Aliquots of transfected COS7 lysates were treated with or without 2 units of N-glycosidase F for 2 h at 37 °C in the buffer recommended by the supplier (Roche Diagnostics, Manheim, Germany). Endoglycosidase H (EndoH; 5 milliunits, Roche Diagnostics) was added to aliquots of transfected COS7 proteins and incubated in 40 mm sodium acetate, pH 5.4 for 17 h at 37 °C. Denaturing buffer was added to digested samples, which were loaded on SDS-PAGE and probed by immunoblotting using goat anti-Tac antibody. A control digestion with no enzyme added demonstrated that samples did not undergo spontaneous degradation during incubations. Previous studies suggested that ADAM10 traffics inefficiently to the plasma membrane in recombinant systems and is retained in intracellular compartments, with only a small fraction reaching the cell surface (7.Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3922-3927Crossref PubMed Scopus (985) Google Scholar). Because we had previously reported that the C terminus of ADAM10 is responsible for its intracellular trafficking (12.Marcello E. Gardoni F. Mauceri D. Romorini S. Jeromin A. Epis R. Borroni B. Cattabeni F. Sala C. Padovani A. Di Luca M. J. Neurosci. 2007; 27: 1682-1691Crossref PubMed Scopus (154) Google Scholar), we constructed chimeras of the ADAM10 C-terminal tail (695–749 amino acids of mouse ADAM10) with the surface reporter protein Tac (human interleukin-2 receptor α-subunit) (20.Bonifacino J.S. Cosson P. Klausner R.D. Cell. 1990; 63: 503-513Abstract Full Text PDF PubMed Scopus (197) Google Scholar). Tac is normally transported to the plasma membrane, where it accumulates at steady state and has been used extensively as a tool to define signals involved in secretory and endocytic membrane trafficking by attaching candidate sequences to its C terminus (15.Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 21.Jansen E.J. Holthuis J.C. McGrouther C. Burbach J.P. Martens G.J. J. Cell Sci. 1998; 111: 2999-3006Crossref PubMed Google Scholar, 22.Ghosh R.N. Mallet W.G. Soe T.T. McGraw T.E. Maxfield F.R. J. Cell Biol. 1998; 142: 923-936Crossref PubMed Scopus (213) Google Scholar). Either Tac alone or TacADAM10 were transiently transfected into COS7 cells, and their surface expression was evaluated using an immunofluorescence-based antibody uptake assay. Consistent with previous reports, Tac displayed strong plasma membrane expression (Fig. 1A). Remarkably, addition of the C-terminal tail of ADAM10 completely prevented surface localization, despite intense intracellular labeling (Fig. 1, A and B, Tac surface/total expression = 1.99 ± 0.27; TacADAM10 surface/total expression = 0.02 ± 0.003; p < 0.001). The intracellular staining patterns of Tac and TacADAM10 also differed; whereas Tac was present in intracellular vesicles, intracellular TacADAM10 exhibited a reticular appearance (see insets in Fig. 1A) and accumulated in a perinuclear compartment. To identify the intracellular compartments where TacADAM10 was retained, we analyzed the subcellular localization of Tac and TacADAM10 using fluorescence microscopy. Tac was present along the secretory pathway, as showed by its partial colocalization with the ER marker calreticulin (Fig. 2A, top) and the Golgi-resident protein giantin (Fig. 2B, top), and its localization in vesicular structures. In contrast, TacADAM10 largely colocalized with two different ER markers, calreticulin and calnexin (Fig. 2A), and was excluded from Golgi structures (Fig. 2B, bottom). Further evidence of ER retention of TacADAM10 was obtained by Western blot analysis. Whereas a single band was present in homogenates of COS7 cells transfected with TacADAM10, two different bands appeared in cells transfected with Tac (Fig. 2C). The result is consistent with previous observations (15.Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar) that Tac is expressed as both mature high and immature low molecular weight species, and suggests that TacADAM10 is present only as an ER-retained immature species. This was confirmed by deglycosylation experiments where we compared the glycosylation state of Tac and TacADAM10 by assessing their sensitivity to two different glycosidases: endoglycosidase H (EndoH), an enzyme that hydrolyzes the high mannose N-glycans present on immature secretory proteins in the ER (23.Trimble R.B. Maley F. Anal. Biochem. 1984; 141: 515-522Crossref PubMed Scopus (200) Google Scholar) but not complex forms of N-linked oligosaccharide, and N-glycosidase F (PNGase F), an enzyme that cleaves all N-linked oligosaccharides (24.Tarentino A.L. Gómez C.M. Plummer Jr., T.H. Biochemistry. 1985; 24: 4665-4671Crossref PubMed Scopus (917) Google Scholar). Because conversion of high mannose glycans into complex oligosaccharides occurs in the Golgi, resistance to EndoH indicates that a glycoprotein has reached this compartment. On the contrary, EndoH sensitivity is considered an indicator of immaturity. Treatment with EndoH demonstrated that the single TacADAM10 band was EndoH-sensitive and confirmed that it was retained in the ER as an immature protein (Fig. 2D, lower panels). In contrast, Tac appeared as both a mature, high molecular weight, EndoH-resistant species and a immature EndoH-sensitive species that migrated at a lower molecular weight (Fig. 2D, top panels). All Tac and TacADAM10 species exhibited increased electrophoretic mobility after PNGase F treatment, demonstrating PNGase F sensitivity (Fig. 2D). This combination of biochemical and immunocytochemical experiments provides strong evidence of selective ER retention mediated by the C-terminal domain of ADAM10. Next, we analyzed the amino acid sequence of the ADAM10 cytoplasmic tail to search for putative ER retention motifs. Based upon homology with known ER retention/retrieval consensus sequences in KATP channels and the NMDA receptor subunit NR1 (13.Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 14.Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15.Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar), we noted three arginine-based motifs within the C-terminal tail that could be responsible for ER retention (for review see Refs. 25.Teasdale R.D. Jackson M.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 27-54Crossref PubMed Scopus (447) Google Scholar, 26.Ma D. Jan L.Y. Curr. Opin. Neurobiol. 2002; 12: 287-292Crossref PubMed Scopus (152) Google Scholar and Fig. 3A). Then we made a series of C-terminal truncations in TacADAM10 to evaluate the role of the motifs 723RRR, 734RQR, and 748RR in ER retention and surface expression. Deleting the last 12 amino acids of ADAM10 (Tac737Δ) did not allow surface expression. Similar results were obtained with the Tac734Δ mutant (Fig. 3B), indicating that the 748RR and 734RQR sequences do not play a major role in ER exit. In contrast, removal of the last 28 amino acids yielded robust surface staining of the Tac721Δ mutant when compared with TacADAM10 (TacADAM10 surface/total expression = 0.04 ± 0.001; Tac721Δ surface/total expression = 2.38 ± 0.74, p < 0.01, Fig. 3B). We then assessed the intracellular localization of the deletion mutants by colocalization with calnexin. Tac737Δ and Tac734Δ mutants showed, as TacADAM10, strong perinuclear staining which colocalized with calnexin, suggesting that they were confined to the ER (supplemental Fig. S1). The surface-expressed Tac721Δ mutant displayed a more diffuse intracellular pattern and did not colocalize with calnexin (Fig. 3C, top). In a small percentage of cells, Tac721Δ accumulated intracellularly in giantin-positive structures (Fig. 3C, bottom), indicating that it was released from the ER and reached the Golgi apparatus. However, most cells exhibited a diffuse staining reflecting an efficient forward trafficking of this construct. In Western blot analyses, the Tac721Δ mutant was present as both high and low molecular weight species, whereas TacADAM10 and Tac734Δ mutants appeared as single bands. The Tac737Δ mutant was expressed as a doublet (Fig. 3D). Both the single Tac734Δ band and the Tac737Δ doublet were, as TacADAM10, EndoH-sensitive (Fig. 3E), confirming that these constructs do not exit the ER and suggesting that the Tac737Δ doublet reflects multiple glycosylation sites rather than states of maturation. In contrast, the prominent higher molecular weight Tac721Δ band was EndoH-resistant, indicating transport to the late Golgi. As a forward trafficking index, we measured the optical densities of the mature and immature species, and compared the mature/immature ratio of the Tac721Δ mutant with that of Tac alone. The Tac721Δ mature/immature ratio was 2.26, higher than the ratio of 1 estimated for Tac. This result is consistent with a trend toward higher surface expression of the Tac721Δ mutant when compared with Tac (Fig. 3B) and indicates that removal of the distal part of the ADAM10 cytoplasmic tail, along with deleting the ER retention motif, could unmask forward trafficking signals. Taken together, these experiments demonstrate that a 12 amino acid fragment (721–734) in ADAM10 plays an inhibitory role on its trafficking to post-ER compartments and toward the cell surface. To map the motif responsible for ER retention, we carried out alanine substitutions in the arginine-rich putative ER retention sequence (723RRR) located within the 12 amino acid fragment and analyzed the behavior of the TacADAM10 mutants by immunofluorescence and deglycosylation experiments. Disruption of the RRR sequence by replacing all three arginine residues with alanine yielded strong surface expression (RRR to AAA = 5.53 ± 0.22-fold increase in the surface/total ratio compared with TacADAM10, p < 0.001, Fig. 4, A and B). Substituting the first or third arginines with alanine had no effect, but mutation of the second arginine was sufficient to allow surface expression (RRR to RAR = 3.64 ± 0.22-fold increase in the surface/total ratio compared with TacADAM10, p < 0.001, Fig. 4, A and B). Nevertheless, the effect on surface expression of this single substitution was significantly lower than that of the triple alanine mutant (p < 0.001, RRR to AAA versus RRR to RAR, Fig. 4B). To evaluate the importance of the sequence context, we generated double arginine mutants and observed a significantly increased surface expression of the RRR to AAR mutant when compared with the RRR to RAR mutant (p < 0.001, Fig. 4B). Indeed, the surface/total expression ratio of the RRR to AAR mutant was virtually identical to the triple mutant. Western blot and deglycosylation analyses revealed that the RRR to RAR mutant was present as both a mature, high molecular weight, EndoH-resistant species and a smaller, immature EndoH-sensitive species, as previously seen for Tac721Δ (Fig. 4, C and D). Notably, the RRR to AAA and RRR to AAR mutants were detected mainly as a high molecular weight mature species (Fig. 4C) not sensitive to EndoH treatment (Fig. 4D). Single RRR to ARR and RRR to RRA mutants were detected as a single EndoH-sensitive band, further confirming their ER retention (Fig. 4, C and D). Therefore, and despite a major role of the second arginine within the 723RRR motif in TacADAM10 ER retention, the amino acid context also influences TacADAM10 maturation and surface expression. To rule out the possibility that ER retention of TacADAM10 reflects, rather than an endogenous mechanism to limit surface expression, a lack of trafficking factors in COS7 cells which would be available in a native environment for ADAM10 expression, we monitored the surface expression of TacADAM10 in neurons. Cultured hippocampal neurons were transfected with either Tac or TacADAM10 constructs, and their localization was analyzed by immunofluorescence. Whereas Tac was expressed at the surface and displayed punctate intracellular labeling along dendrites, TacADAM10 was mainly found intracellularly in the cell body (Fig. 5). Surface staining of TacADAM10 represented a very faint signal at the soma, matching our results in COS7 cells (Fig. 5). In contrast, Tac721Δ and TacADAM10 mutants lacking the retention motif (RRR to AAA and RRR to RAR) were located at the neuronal surface and in intracellular vesicles along dendrites (Fig. 5), suggesting that removal of the ER retention signal favors the dendritic targeting and surface expression of TacADAM10 in hippocampal neurons and indicating that ER retention is a mechanism controlling ADAM10 localization in neurons. We then asked whether ER retention plays a role in the context of the full-length ADAM10 protein, as it is possible that folding or other factors permit ER release. Indeed, it has been previously reported that ADAM10 is localized in the Golgi, albeit it trafficks inefficiently to the surface (7.Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3922-3927Crossref PubMed Scopus (985) Google Scholar). COS7 cells were transfected with full-length ADAM10 (flADAM10), a deletion mutant lacking the last 28 amino acids (flADAM10 721Δ) or a mutant lacking the second arginine of the ER retention motif, and their surface expression was analyzed by immunofluorescence. flADAM10 showed a strong perinuclear staining and was weakly expressed at the surface, whereas the mutants displayed a more diffuse intracellular pattern and stronger surface staining (Fig. 6A). Quantification revealed significant increases in surface/total expression ratios when the RRR motif was removed (Fig. 6B, p < 0.001 versus wild-type flADAM10). Recent studies showed that PDZ (PSD95/DLG/ZO-1) domain-containing scaffolding proteins that associate with receptors and channels early in the secretory pathway can facilitate or inhibit their ER to Golgi transport (14.Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15.Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 27.Roche K.W. Tu J.C. Petralia R.S. Xiao B. Wenthold R.J. Worley P.F. J. Biol. Chem. 1999; 274: 25953-25957Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). ADAM10 binds the PDZ domain protein SAP97, interaction which facilitates its trafficking to postsynaptic compartments (12.Marcello E. Gardoni F. Mauceri D. Romorini S. Jeromin A. Epis R. Borroni B. Cattabeni F. Sala C. Padovani A. Di Luca M. J. Neurosci. 2007; 27: 1682-1691Crossref PubMed Scopus (154) Google Scholar). To test whether SAP97 could favor ADAM10 ER exit, TacADAM10 and SAP97 were cotransfected in COS7 cells and surface expression was determined. However, SAP97 did not allow TacADAM10 surface expression (Fig. 7A, center panels). The result was confirmed by deglycosylation analysis, which showed that SAP97 coexpression did not modify TacADAM10 maturation, because only the EndoH-sensitive state was attained (Fig. 7B). Alternatively, the activation of specific signaling pathways might be required for ADAM10 transport along the secretory pathway. For instance, SAP97-Ser-39 phosphorylation by CaMKII favors the release of a SAP97/NR2A complex from the ER (28.Mauceri D. Gardoni F. Marcello E. Di Luca M. J. Neurochem. 2007; 100: 1032-1046Crossref PubMed Scopus (62) Google Scholar). In order to evaluate the putative role of SAP97 phosphorylation in modulating the ER release of ADAM10, we cotransfected COS7 cells with TacADAM10 and SAP97 mutants that prevent (GFPSAP97-S39A) or mimic (GFPSAP97-S39D) CaMKII-dependent phosphorylation. Neither SAP97-S39D nor SAP97-S39A cotransfection allowed TacADAM10 surface expression in COS7 cells (Fig. 7C). Given that SAP97 can" @default.
• W1988629688 created "2016-06-24" @default.
• W1988629688 creator A5001945408 @default.
• W1988629688 creator A5006467212 @default.
• W1988629688 creator A5029825330 @default.
• W1988629688 creator A5041197792 @default.
• W1988629688 date "2010-04-01" @default.
• W1988629688 modified "2023-09-27" @default.
• W1988629688 title "An Arginine Stretch Limits ADAM10 Exit from the Endoplasmic Reticulum" @default.
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