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• W2050479911 abstract "The β-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the β-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other β-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor. The β-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the β-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other β-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor. epidermal growth factor sensory and motor neuron-derived factor polymerase chain reaction Chinese hamster ovary Dulbecco's modified Eagle's medium. Proliferation of Schwann cells during embryonic and early postnatal development is necessary to provide sufficient numbers of Schwann cells to ensheath and myelinate peripheral axons. A series of studies carried out with purified cultures of Schwann cells and sensory nerve cells established that the mitogenic signal that drives Schwann cell proliferation is associated with the axons of these nerve cells (1Wood P. Bunge R.P. Nature. 1975; 256: 662-664Crossref PubMed Scopus (315) Google Scholar, 2Salzer J.L. Bunge R.P. J. Cell Biol. 1980; 84: 739-752Crossref PubMed Scopus (313) Google Scholar, 3Salzer J.L. Bunge R.P. Glaser L. J. Cell Biol. 1980; 84: 767-778Crossref PubMed Scopus (224) Google Scholar, 4Salzer J.L. Williams A.K. Glaser L. Bunge R.P. J. Cell Biol. 1980; 84: 753-766Crossref PubMed Scopus (198) Google Scholar). This mechanism, in which the Schwann cell mitogen is immobilized on the axonal surface, ensures proper matching of Schwann cell numbers and axons during development. Although the axon-associated mitogen has not been purified, there is evidence that it is a member of the heregulin family of polypeptide growth factors (5Morissey T.K. Levi A.D.O. Nuijens A. Sliwkowski M.X. Bunge R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1431-1435Crossref PubMed Scopus (247) Google Scholar, 6Dong Z. Brennan A. Liu N. Yarden Y. Lefkowitz G. Mirsky R. Jessen K.R. Neuron. 1995; 15: 585-596Abstract Full Text PDF PubMed Scopus (395) Google Scholar). The heregulins form a subset of the epidermal growth factor (EGF)1 family of polypeptide growth factors. At least 12 heregulin isoforms have been described, all of which appear to be generated by alternative splicing of a single gene (7Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-Mcdonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (674) Google Scholar, 8Meyer D. Yamaai T. Garratt A. Reithmacher-Sonnenberg E. Kane D. Theill L.E. Birchmeier C. Development. 1997; 124: 3575-3586Crossref PubMed Google Scholar). All heregulin isoforms contain a domain of approximately 50 amino acids that is homologous to the active domain of EGF and is responsible for receptor binding (9Barbacci E.G. Guarino B.C. Stroh J.G. Singleton D.H. Rosnack K.J. Moyer J.D. Andrews G.C. J. Biol. Chem. 1995; 270: 9585-9589Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Heregulins exert their biological activities by binding and activating receptor tyrosine kinases, called ErbB2, ErbB3, and ErbB4, that bear structural similarity to the EGF receptor (10Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (922) Google Scholar, 11Reise D.J. van Raaij T.M. Plowman G.D. Andrews G.C. Stern D.F. Mol. Cell. Biol. 1995; 15: 5770-5776Crossref PubMed Scopus (346) Google Scholar, 12Pinkas-Kramarski R. Shelly M. Glathe S. Ratzkin B.J. Yarden Y. J. Biol. Chem. 1996; 271: 19029-19032Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). ErbB receptors undergo tyrosine autophosphorylation upon ligand-dependent activation. This leads to activation of cytoplasmic signaling pathways, such as the mitogen-activated protein kinase pathway, which accounts for their mitogenic activity. Results of in situ hybridization studies suggest that the principal form of β-heregulin expressed by sensory nerve cells is sensory and motor neuron-derived factor (SMDF) (13Ho W.-H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Analyses of mouse embryos with targeted heregulin gene mutations provide indirect evidence that SMDF is important for early Schwann cell development (8Meyer D. Yamaai T. Garratt A. Reithmacher-Sonnenberg E. Kane D. Theill L.E. Birchmeier C. Development. 1997; 124: 3575-3586Crossref PubMed Google Scholar). SMDF contains a β-heregulin EGF homology domain near the carboxyl-terminal end of the protein (13Ho W.-H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). With the exception of this domain, however, the rest of the SMDF sequence appears to be unique to this heregulin isoform. The structural variation among isoforms produces multiple molecular forms of heregulins. Some heregulins contain amino-terminal signal peptides that can mediate secretion or membrane insertion of the polypeptides (7Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-Mcdonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (674) Google Scholar). Other isoforms lack such sequences (10Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (922) Google Scholar, 14Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Levy R.B. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (521) Google Scholar). There is evidence that both structural varieties can associate with membranes, however, and/or be released in soluble form into the medium of cultured cells expressing these proteins. These properties could result from the utilization of an internal hydrophobic sequence, present in most heregulins, to mediate membrane insertion and/or function as a membrane-spanning domain. The available data on membrane-associated forms of heregulins suggest these proteins are oriented with the amino-terminal ends exposed to the extracellular space and the carboxyl-terminal ends in the cytoplasm (14Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Levy R.B. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 15Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). This orientation places the EGF homology domain of these heregulins on the external side of the plasma membrane. SMDF lacks an apparent amino-terminal signal peptide, but contains an internal hydrophobic sequence that could serve as a membrane insertion site (13Ho W.-H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The structure of SMDF differs from most other heregulin isoforms, however, in that it contains a cysteine-rich domain in place of the Ig domain. In SMDF, as well as some other heregulin isoforms, the EGF homology domain of SMDF is positioned near the carboxyl-terminal end of the polypeptide. Thus, the nature of the association of SMDF with the plasma membrane would have important consequences for its biological activity. Several heregulins have also been shown to bind to heparin and heparan sulfate (10Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (922) Google Scholar, 16Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Levy R.B. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (474) Google Scholar, 17Loeb J.A. Fischbach G.D. J. Cell Biol. 1995; 130: 127-135Crossref PubMed Scopus (114) Google Scholar, 18Sudhalter J. Whitehouse L. Rusche J.R. Marchionni M.A. Mahanthappa N.K. Glia. 1996; 17: 28-38Crossref PubMed Scopus (33) Google Scholar). Binding of some growth factors, such as basic fibroblast growth factor, to cell surface heparan sulfate molecules has been shown to have important consequences for the activities of the factors (19Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-846Abstract Full Text PDF PubMed Scopus (2070) Google Scholar, 20Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1284) Google Scholar). There is evidence that heparan sulfate-dependent interactions are involved in Schwann cell mitogenesis (18Sudhalter J. Whitehouse L. Rusche J.R. Marchionni M.A. Mahanthappa N.K. Glia. 1996; 17: 28-38Crossref PubMed Scopus (33) Google Scholar, 21Ratner N. Bunge R.P. Glaser L. J. Cell Biol. 1985; 101: 744-754Crossref PubMed Scopus (127) Google Scholar, 22Ratner N. Hong D. Lieberman M.A. Bunge R.P. Glaser L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6992-6996Crossref PubMed Scopus (113) Google Scholar). The ability of glial growth factor-2 to activate ErbB receptors and stimulate Schwann cell proliferation is inhibited by exogenous heparin or heparan sulfate or by incubation of the Schwann cells in medium containing an inhibitor of proteoglycan synthesis (18Sudhalter J. Whitehouse L. Rusche J.R. Marchionni M.A. Mahanthappa N.K. Glia. 1996; 17: 28-38Crossref PubMed Scopus (33) Google Scholar). The present study was carried out to address several specific questions related to SMDF structure and function. The data presented demonstrate that SMDF is a cell surface transmembrane protein that is oriented in the plasma membrane with the carboxyl-terminal EGF homology domain exposed to the extracellular compartment. Membrane-associated SMDF activates ErbB receptors on Schwann cells. In contrast with some heregulin isoforms, SMDF does not bind heparin, and its activity is not dependent on Schwann cell heparan sulfate molecules. Human SMDF cDNA was cloned by nested PCR amplification from a human brain stem cDNA library (LMG2, American Type Culture Collection, 37432). The primers were based on the published human SMDF cDNA sequence (13Ho W.-H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and are shown in Table I. Conditions for PCR reactions were 94 °C for 1 min (denaturation), 60 °C for 2 min (annealing) and 72 °C for 10 min (extension) for 30 cycles. The resulting PCR product was cloned into pCRII (Invitrogen). The insert was excised from this vector by digestion with EcoRI and subcloned into the plasmid pCMVneo. For the addition of FLAG epitope tags, the insert was reamplified by PCR using primers that overlapped the ends of the protein coding sequence and were extended on their 5′-ends by sequence coding for the FLAG epitope sequence. These products were cloned into the T/A expression plasmid pCR3.1 (Invitrogen). Sequences of expression plasmids were verified by DNA sequence analysis.Table IPrimers used for PCR amplification of human SMDFPrimerSequenceFirst round SenseGCC TCT GCG TGG TAA TGG AC AntisenseAAT GTT CTC ATG CGA CAG GCSecond round SenseCTT CTG GAG GTG AGC CGA TG AntisenseAAG CAG CAC CAA CTG AGC AT Open table in a new tab Chinese hamster ovary (CHO) and human embryonal kidney (293) cells were transfected with these plasmids using LipofectAMINE (Life Technologies, Inc.). The cells were cultured in DME medium/10% fetal calf serum on uncoated polystyrene tissue culture dishes. To isolate stably transfected lines, 3 days after transfection the cells were switched to growth medium containing G418 (400 μg/ml). Aliquots of cell lysates or membrane fractions were subjected to SDS gel electrophoresis on 7.5% or 5–15% polyacrylamide gels. The resolved proteins were blotted onto Immobilon membranes, blocked with 5% nonfat milk in 0.1 m NaCl, 0.01m Tris-HCl, pH 7.5, and stained with mouse monoclonal anti-FLAG or rabbit polyclonal anti-β-heregulin EGF homology domain antibodies. Bound antibodies were detected by enhanced chemiluminescence. To digest SMDF exposed on the cell surface, the cells were incubated in trypsin (2.5 mg/ml) for 2 min. The trypsin solution was removed, the residual trypsin activity was blocked by the addition of soybean trypsin inhibitor (2.5 mg/ml), and the cells were lysed for immunoblot analysis. Biotinylation of cell surface proteins was carried out as follows. Transfected cells were rinsed twice with HBS buffer (20 mmHEPES, pH 7.5, 150 mm NaCl, 2 mmCaCl2) and then incubated in HBS containing 0.2 mg/ml NHS-LC biotin (Pierce) for 1 h on ice. The medium was removed, and the cells were rinsed with 0.8 m glycine in HBS, followed by HBS. The cells were scraped into immunoprecipitation buffer (0.5% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mmTris-HCl, pH 7.5). The lysates were clarified by centrifugation for 2 min at 10,000 × g, and anti-FLAG M2 monoclonal antibody (Sigma) was added (1:1000 final dilution). The samples were incubated for 1 h at 4 °C, and then the antibodies were precipitated by the addition of protein A-Sepharose. The bound proteins were eluted by incubation of the Sepharose beads in SDS gel sample buffer, separated on 7.5% SDS-polyacrylamide gels, and stained with anti-FLAG antibodies. To isolate membranes from transfected cells, the cells were lysed in hypotonic HME buffer (20 mm HEPES, 2 mmMgCl2, 1 mm EDTA, pH 7.4). Cell lysates were centrifuged at 6000 × g for 10 min to prepare a low speed pellet containing plasma membranes. The resulting supernatant fraction was centrifuged at 100,000 × g for 1 h to prepare a high speed pellet, containing the remaining membranes. In some experiments, the membranes in the low speed pellet were purified further. The pellet was resuspended in HME buffer and layered over a solution of 40% sucrose. The tubes were centrifuged at 25,000 rpm for 1 h in an SW41 rotor (Beckman). The purified membranes were recovered from the solution interphase. Cell surface proteins were selectively immunoprecipitated as follows. Transfected cells were rinsed twice with DME medium and then incubated in medium containing anti-FLAG antibody (1:1000 final dilution in DME medium) for 30 min on ice. The antibody solution was removed, and the cells were rinsed twice with DME medium. The cells were subjected to hypotonic lysis in HME buffer as described above, and a crude membrane fraction was prepared by centrifugation at 85,000 rpm for 25 min in a TLA 100.3 rotor (Beckman). The supernatant solution was removed, and the membrane pellet was resuspended in HME buffer and layered over 40% sucrose. The tubes were centrifuged at 25,000 rpm for 1 h. The purified membranes were recovered from the solution interphase, diluted in HME buffer, and centrifuged in the TLA 100.3 rotor as described above. The membranes were dissolved in immunoprecipitation buffer, and the anti-FLAG antibodies were collected by adsorption to protein A-Sepharose. The immunoadsorbed proteins were solubilized in SDS gel sample buffer and subjected to immunoblot analysis with anti-FLAG antibodies. As a control for surface immunoprecipitation, membranes were isolated from transfected cultures, incubated with anti-FLAG antibodies, dissolved in immunoprecipitation buffer, and then treated as described above. Triton X-100 extracts of membranes prepared from SMDF-expressing cells were subjected to heparin affinity chromatography. The extracts were diluted with 0.025 m NaCl, 0.01 m Tris-HCl, pH 7.5, mixed overnight at 4 °C with heparin-Sepharose equilibrated in the same buffer, and then packed into a column. The column flow-through was collected, the beads were washed with equilibration buffer, and the bound proteins were eluted by a NaCl step gradient. Aliquots of the flow-through, wash, and elution fractions were subjected to immunoblot analysis. A 52-amino acid peptide corresponding to the EGF homology domain of β-heregulin (9Barbacci E.G. Guarino B.C. Stroh J.G. Singleton D.H. Rosnack K.J. Moyer J.D. Andrews G.C. J. Biol. Chem. 1995; 270: 9585-9589Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) was synthesized in the Weis Center for Research Core Molecular Biology Laboratory and purified by reverse phase high pressure liquid chromatography. The peptide was used to immunize rabbits using a synthetic adjuvant system (Ribi Immunochemicals). Anti- β-heregulin antibodies were purified by affinity chromatography using the synthetic peptide covalently coupled to CNBr-activated Sepharose. Cells to be used for immunofluorescence microscopy were grown on glass slide chambers. For surface staining, the cells were incubated with primary antibody solution before fixation and without permeabilization. For intracellular staining, the cells were fixed with 3% paraformaldehyde, 0.15 m NaCl, 50 mm sodium phosphate, pH 7.4, and then permeabilized by incubation for 2 min in 0.05% Triton X-100 before incubation with the primary antibody. Blocking and washing steps were carried out as described previously (23Carey D.J. Stahl R.C. J. Cell Biol. 1990; 111: 2053-2062Crossref PubMed Scopus (58) Google Scholar). Bound antibodies were detected by incubation with Texas Red-conjugated secondary antibodies and visualized using a Nikon Optiphot microscope equipped for epifluorescence. Schwann cells were prepared from neonatal rat sciatic nerves and cultured as described previously (23Carey D.J. Stahl R.C. J. Cell Biol. 1990; 111: 2053-2062Crossref PubMed Scopus (58) Google Scholar). Schwann cells were exposed to membrane fractions or conditioned medium obtained from SMDF-transfected cells for 15 min in serum-free medium (Ham's F-12, DME medium 1:1, 0.1 μg/ml insulin, 50 μg/ml transferrin) and then lysed in immunoprecipitation buffer. Aliquots were incubated with anti-ErbB3 antibodies (Santa Cruz Biotechnology, Inc.), and immune complexes were precipitated by the addition of protein A-Sepharose beads coated with anti-rabbit IgG. The immunoprecipitates were solubilized in buffer containing 2% SDS, and the proteins were subjected to SDS gel electrophoresis and transferred to Immobilon membranes. The membranes were stained with anti-phosphotyrosine antibodies. Schwann cells were incubated in growth medium supplemented with 1 mm4-methylumbelliferyl-β-d-xyloside (Sigma) or 30 mm sodium chlorate (Sigma) for 24 h. The media were removed, and the cells were stimulated with SMDF as described above. Human SMDF cDNA was cloned as described under “Experimental Procedures.” The sequence of this cDNA matched the published human SMDF sequence reported previously (13Ho W.-H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and contained coding information for a polypeptide with a predicted M r of approximately 32,000. The SMDF cDNA was used to generate several full-length expression constructs, including forms with FLAG epitope tags at the amino-terminal (NT-SMDF) or carboxyl-terminal (CT-SMDF) end of the protein coding sequence (Fig. 1,top). These were used for transient and stable transfection of CHO cells and human embryonic kidney (293) cells. Similar results were obtained in all cases. Transfected cells expressed an 83-kDa polypeptide that was not seen in untransfected cells or cells transfected with vector lacking the SMDF cDNA insert. An apparently identical 83-kDa product was seen in cells transfected with NT-SMDF or CT-SMDF (detected on immunoblots with anti-FLAG antibody) or SMDF lacking an epitope tag (detected with anti-EGF homology domain antibodies). In most experiments, an additional band of 43 kDa was also seen in SMDF-transfected cells. Anti-FLAG immunoreactive polypeptides of identical sizes were seen in cells transfected with NT-SMDF or CT-SMDF. This suggests that these polypeptides contain the full-length SMDF sequence and that SMDF undergoes some type of processing or modification to produce the 83-kDa product. An alternative possibility that cannot be excluded is that the 43-kDa bands seen in NT-SMDF- and CT-SMDF-transfected cells are proteolytic fragments of SMDF that result from cleavage at a site that produces amino-terminal and carboxyl-terminal products of identical size. Since the 83-kDa product is the cell surface form of SMDF (see below) the 43-kDa band was not characterized further. The deduced amino acid sequence of human SMDF lacks an apparent amino-terminal signal peptide to mediate either secretion or membrane insertion of the polypeptide. There is, however, an internal hydrophobic sequence that could function as an uncleaved membrane insertion signal. To determine whether SMDF was expressed on the cell surface, transfected cells were treated with trypsin. As shown in Fig. 2 A, trypsin removed completely the slow migrating form of SMDF, but it had no effect on the fast migrating form. These results suggest that the 83-kDa form of SMDF is expressed on the cell surface and that the 43-kDa form is intracellular. This conclusion was further supported by results of cell surface labeling of SMDF-expressing cells. Surface proteins were covalently labeled with biotin by incubation of intact cells with a membrane-impermeant protein-modifying reagent. Immunoprecipitation of lysates from these cells with anti-FLAG antibodies and detection of biotin-labeled polypeptides with an avidin-conjugated probe revealed mainly the 83-kDa form of SMDF (Fig. 2 B). To provide additional evidence for membrane association of SMDF, transfected cells were subjected to subcellular fractionation. When CHO cells that were stably transfected with NT-SMDF were lysed in the absence of detergent and subjected to differential centrifugation, the bulk of SMDF was present in the low speed pellet that was enriched in plasma membranes (Fig. 3 A). Only small amounts of SMDF were present in the high speed pellet, composed mainly of membranes of intracellular organelles. SMDF was not detected in the soluble fraction of the cells (Fig. 3 A). SMDF was also associated a purified membrane fraction obtained by sucrose density gradient centrifugation (Fig. 3 A). In contrast to these results, when the fractionation was carried out in buffer containing the nonionic detergent Triton X-100, all of the SMDF was recovered in the soluble fraction (Fig. 3 A). SMDF appeared to be tightly associated with cell membranes, based on the finding that extraction of the low speed pellet with 1m KCl did not solubilize the protein (Fig. 3 B). Some β-heregulins bind heparin/heparan sulfate and, thus, have the potential to associate with membranes by noncovalent binding to membrane heparan sulfate proteoglycans. Extraction of the low speed pellet with buffer containing soluble heparin did not solubilize SMDF (Fig. 3 B), indicating that association of SMDF with the membranes was not dependent on interactions with heparan sulfate. Expression of SMDF on the plasma membrane has the potential to generate growth factor molecules that can activate heregulin receptors on adjacent cells. This property would be dependent on the orientation of the polypeptide within the plasma membrane, however. The EGF homology domain of SMDF is located near the carboxyl-terminal end of the protein (Fig. 1 A). To determine the orientation of SMDF within the plasma membrane, CHO cells that were transiently transfected with either NT-SMDF or CT-SMDF were stained with anti-FLAG antibodies as either intact or permeabilized cells. As shown in Fig. 4, staining of SMDF on the surface of intact cells was observed only with CT-SMDF (A) and not with NT-SMDF (B). Staining of both forms of SMDF was observed when fixed cells were permeabilized with Triton X-100 (Fig. 4, C and D). Identical results were obtained in 293 cells (data not shown). These results demonstrate that SMDF is oriented in the plasma membrane with the carboxyl-terminal end exposed to the extracellular space. This orientation is consistent with the utilization of an internal, uncleaved signal peptide, which would direct the insertion of SMDF as a type II transmembrane protein. Furthermore, this orientation places the active EGF homology domain of SMDF on the extracellular side of the plasma membrane. Additional evidence for this membrane orientation was provided by results of immunoprecipitation experiments. Cells that were transfected with either CT-SMDF or NT-SMDF were incubated with anti-FLAG antibodies. The unbound antibodies were removed, and the cells were lysed without use of detergent and used to prepare membrane fractions. The membranes were dissolved in immunoprecipitation buffer, and the FLAG antibodies were isolated by adsorption to protein A-Sepharose. As shown in Fig. 5 A, the 83-kDa form of CT-SMDF, but not NT-SMDF, was isolated by this procedure. In contrast, when membranes were isolated from CT-SMDF- or NT-SMDF-transfected cells and then incubated with anti-FLAG antibodies and purified by adsorption to Protein A-Sepharose, both the 83- and 43-kDa forms of CT-SMDF and NT-SMDF were isolated (Fig. 5 A). These results indicate that the carboxyl-terminal, but not the amino-terminal, end of SMDF is exposed to the extracellular environment. They also demonstrate that the amino-terminal FLAG epitope is accessible to the antibody in the absence of detergent. Finally, these results provide additional evidence that the 83-kDa SMDF is the cell surface form. Essentially identical results were obtained when CT-SMDF- and NT-SMDF-transfected cells were incubated with anti-FLAG antibodies and then lysed with detergent before the addition of protein A-Sepharose (Fig. 5 B). Significant amounts of SMDF were immunoprecipitated only from cells expressing the CT-SMDF. β-heregulins exert their biological activities by binding to and activating the receptor tyrosine kinases ErbB2, ErbB3" @default.
• W2050479911 created "2016-06-24" @default.
• W2050479911 creator A5026689644 @default.
• W2050479911 creator A5074651593 @default.
• W2050479911 date "1998-11-01" @default.
• W2050479911 modified "2023-10-10" @default.
• W2050479911 title "Sensory and Motor Neuron-derived Factor Is a Transmembrane Heregulin That Is Expressed on the Plasma Membrane with the Active Domain Exposed to the Extracellular Environment" @default.
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