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The N-terminal Region of Neuregulin Isoforms Determines the Accumulation of Cell Surface and Released Neuregulin Ectodomain

2001; Elsevier BV; Volume: 276; Issue: 4 Linguagem: Inglês

10.1074/jbc.m005700200

ISSN

1083-351X

Autores

Jay Y. Wang, Shyra J. Miller, Douglas L. Falls,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

Two neuregulin-1 isoforms highly expressed in the nervous system are the type III neuregulin III-β1a and the type I neuregulin I-β1a. The sequence of these two isoforms differs only in the region that is N-terminal of the bioactive epidermal growth factor-like domain. While the biosynthetic processing of the I-β1a isoform has been well characterized, the processing of III-β1a has not been reported. In this study, we compared III-β1a and I-β1a processing. Both III-β1a and I-β1a were synthesized as transmembrane proproteins that were proteolytically cleaved to produce an N-terminal fragment containing the bioactive epidermal growth factor-like domain. For I-β1a, this product was released into the medium. However, for III-β1a, this product was a transmembrane protein. In cultures of cells expressing III-β1a, the amount of neuregulin at the cell surface was much greater, and the amount in the medium was much less than in cultures expressing I-β1a. Phorbol ester treatment and truncation of the cytoplasmic tail had markedly different effects on III-β1a and I-β1a processing. These results demonstrate an important role for the N-terminal region in determining neuregulin biosynthetic processing and show that a major product of III-β1a processing is a tethered ligand that may act as a cell surface signaling molecule. Two neuregulin-1 isoforms highly expressed in the nervous system are the type III neuregulin III-β1a and the type I neuregulin I-β1a. The sequence of these two isoforms differs only in the region that is N-terminal of the bioactive epidermal growth factor-like domain. While the biosynthetic processing of the I-β1a isoform has been well characterized, the processing of III-β1a has not been reported. In this study, we compared III-β1a and I-β1a processing. Both III-β1a and I-β1a were synthesized as transmembrane proproteins that were proteolytically cleaved to produce an N-terminal fragment containing the bioactive epidermal growth factor-like domain. For I-β1a, this product was released into the medium. However, for III-β1a, this product was a transmembrane protein. In cultures of cells expressing III-β1a, the amount of neuregulin at the cell surface was much greater, and the amount in the medium was much less than in cultures expressing I-β1a. Phorbol ester treatment and truncation of the cytoplasmic tail had markedly different effects on III-β1a and I-β1a processing. These results demonstrate an important role for the N-terminal region in determining neuregulin biosynthetic processing and show that a major product of III-β1a processing is a tethered ligand that may act as a cell surface signaling molecule. neuregulin cysteine-rich domain C-terminal fragment Dulbecco's modified Eagle's medium epidermal growth factor N-terminal fragment phorbol 12-myristate 13-acetate transforming growth factor-α transmembrane hemagglutinin phosphate-buffered saline Hepes-buffered saline N-hydroxysuccinimide Neuregulin-1 gene products are cell-cell signaling proteins that are ligands for receptor tyrosine kinases of the ErbB/HER subfamily (for reviews, see Refs. 1Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 2Fischbach G.D. Rosen K.M. Annu. Rev. Neurosci. 1997; 20: 429-458Crossref PubMed Scopus (254) Google Scholar, 3Lemke G. Mol. Cell. Neurosci. 1996; 7: 247-262Crossref PubMed Scopus (219) Google Scholar, 4Adlkofer K. Lai C. Glia. 2000; 29: 104-111Crossref PubMed Scopus (158) Google Scholar). Signaling events mediated by neuregulins (NRGs)1 have been shown to be essential for normal development of the nervous system and heart (5Meyer D. Birchmeier C. Nature. 1995; 378: 386-390Crossref PubMed Scopus (1049) Google Scholar, 6Kramer R. Bucay N. Kane D.J. Martin L.E. Tarpley J.E. Theill L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4833-4838Crossref PubMed Scopus (190) Google Scholar, 7Liu X. Hwang H. Cao L. Buckland M. Cunningham A. Chen J. Chien K.R. Graham R.M. Zhou M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13024-13029Crossref PubMed Scopus (82) Google Scholar, 8Wolpowitz D. Mason T.B. Dietrich P. Mendelsohn M. Talmage D.A. Role L.W. Neuron. 2000; 25: 79-91Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 9Sandrock A.W. Dryer S.E. Rosen K.M. Gozani S.N. Kramer R. Theill L.E. Fischbach G.D. Science. 1997; 276: 599-603Crossref PubMed Scopus (245) Google Scholar). Roles for NRG-1 proteins in the development of other organs and in the adult have also been suggested. Gene transcripts encoding at least 14 different NRG isoforms have been identified (2Fischbach G.D. Rosen K.M. Annu. Rev. Neurosci. 1997; 20: 429-458Crossref PubMed Scopus (254) Google Scholar). Based on the structure of their N-terminal region, NRG-1 isoforms can be divided into three types (Refs. 1Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar and 10Meyer D. Yamaai T. Garratt A. Riethmacher-Sonnenberg E. Kane D. Theill L.E. Birchmeier C. Development. 1997; 124: 3575-3586Crossref PubMed Google Scholar; see "Neuregulin Isoforms and Nomenclature" and Fig. 1 A). The NRG isoforms originally called neu differentiation factor (11 and 12) and heregulin (13Holmes 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 (926) Google Scholar) have a type I N terminus. The N terminus of chick ARIA (14Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (553) Google Scholar) is most similar to the mammalian type I N terminus. The isoforms originally called glial growth factor (15Goodearl A. Davis J.B. Mistry K. Minghetti L. Otsu M. Waterfield M.D. Stroobant P. J. Biol. Chem. 1993; 268: 18095-18102Abstract Full Text PDF PubMed Google Scholar, 16Marchionni 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 (682) Google Scholar) that include a "kringle domain" have a type II N terminus. The isoforms originally called SMDF, nARIA, or CRD-NRG (17Ho 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, 18Yang X. Kuo Y. Devay P., Yu, C. Role L. Neuron. 1998; 20: 255-270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Bermingham-McDonogh O. Xu Y.T. Marchionni M.A. Scherer S.S. Mol. Cell. Neurosci. 1997; 10: 184-195Crossref PubMed Scopus (60) Google Scholar, 20Carroll S.L. Miller M.L. Frohnert P.W. Kim S.S. Corbett J.A. J. Neurosci. 1997; 17: 1642-1659Crossref PubMed Google Scholar, 21Rosenbaum C. Karyala S. Marchionni M.A. Kim H.A. Krasnoselsky A.L. Happel B. Isaacs I. Brackenbury R. Ratner N. Exp. Neurol. 1997; 148: 604-615Crossref PubMed Scopus (70) Google Scholar) have a type III N terminus. The type I and type II isoforms contain an Ig-like domain in the N-terminal region (Ig-NRGs), whereas the type III isoform contain a cysteine-rich domain (CRD-NRGs). Studies of the temporal and spatial expression patterns of NRG isoforms suggest that type I/II and type III NRGs serve distinct functions (10Meyer D. Yamaai T. Garratt A. Riethmacher-Sonnenberg E. Kane D. Theill L.E. Birchmeier C. Development. 1997; 124: 3575-3586Crossref PubMed Google Scholar, 18Yang X. Kuo Y. Devay P., Yu, C. Role L. Neuron. 1998; 20: 255-270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). This inference has been confirmed by "knockouts" that have specifically deleted the Ig-NRGs (6Kramer R. Bucay N. Kane D.J. Martin L.E. Tarpley J.E. Theill L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4833-4838Crossref PubMed Scopus (190) Google Scholar) or the CRD-NRGs (Ref. 8Wolpowitz D. Mason T.B. Dietrich P. Mendelsohn M. Talmage D.A. Role L.W. Neuron. 2000; 25: 79-91Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar; see also Ref. 10Meyer D. Yamaai T. Garratt A. Riethmacher-Sonnenberg E. Kane D. Theill L.E. Birchmeier C. Development. 1997; 124: 3575-3586Crossref PubMed Google Scholar). With respect to the type III NRGs, analysis of the knockout animals indicates that type III NRGs play a critical role in the interactions of peripheral nerve axons with muscle and with Schwann cells. Defects in animals lacking type III NRGs include retraction of nerve terminals from newly formed synapses, absence of Schwann cells from peripheral nerves, and loss of motor and sensory neurons (8Wolpowitz D. Mason T.B. Dietrich P. Mendelsohn M. Talmage D.A. Role L.W. Neuron. 2000; 25: 79-91Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The cellular processing of type I NRGs has been the subject of several investigations (22Burgess T.L. Ross S.L. Qian Y.X. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 23Lu H.S. Hara S. Wong L. Jones M.D. Katta V. Trail G. Zou A.H. Brankow D. Cole S. Hu S. Wen D.Z. J. Biol. Chem. 1995; 270: 4775-4783Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 24Loeb J.A. Susanto E.T. Fischbach G.D. Mol. Cell. Neurosci. 1998; 11: 77-91Crossref PubMed Scopus (59) Google Scholar, 25Han B. Fischbach G.D. J. Biol. Chem. 1999; 274: 26407-26415Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Most type I NRG isoforms include a hydrophobic stretch of amino acids C-terminal of the epidermal growth factor (EGF)-like) domain that serves as a transmembrane (TM) domain. These type I isoforms are synthesized as transmembrane "proproteins" from which a paracrine signal can be produced by proteolytic cleavage in the stalk region (Fig. 1 B). The release of the ectodomain into the extracellular space may require transport of the uncleaved proprotein to the cell surface, followed by stalk cleavage and "shedding" of the ectodomain from the surface. Alternatively, after arriving at the surface, the proprotein may be internalized into endocytotic pathway compartments in which proteolytic processing occurs with subsequent secretion of the ectodomain fragment (24Loeb J.A. Susanto E.T. Fischbach G.D. Mol. Cell. Neurosci. 1998; 11: 77-91Crossref PubMed Scopus (59) Google Scholar). Release of the type I NRG ectodomain into culture medium is accelerated by activation of protein kinase C (12Wen D.Z. Suggs S.V. Karunagaran D. Liu N.L. Cupples R.L. Luo Y. Janssen A.M. Benbaruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W.N. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (234) Google Scholar, 22Burgess T.L. Ross S.L. Qian Y.X. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Loeb J.A. Susanto E.T. Fischbach G.D. Mol. Cell. Neurosci. 1998; 11: 77-91Crossref PubMed Scopus (59) Google Scholar) and blocked by shortening the cytoplasmic tail to fewer than ∼90 amino acids (7Liu X. Hwang H. Cao L. Buckland M. Cunningham A. Chen J. Chien K.R. Graham R.M. Zhou M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13024-13029Crossref PubMed Scopus (82) Google Scholar, 26Liu X. Hwang H. Cao L. Wen D. Liu N. Graham R.M. Zhou M. J. Biol. Chem. 1998; 273: 34335-34340Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or by deletion of specific segments within this 90-amino acid stretch (26Liu X. Hwang H. Cao L. Wen D. Liu N. Graham R.M. Zhou M. J. Biol. Chem. 1998; 273: 34335-34340Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). NRG III-β3 was the first reported NRG isoform with a type III N-terminal region (17Ho 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). This isoform, originally referred to as SMDF, has a β3 type of EGF-like domain, and thus lacks the transmembrane domain C-terminal of the EGF-like domain (Fig. 1 A). Subsequent to the isolation of the III-β3 isoform, a III-β1a isoform (Fig. 1 A) was discovered (Refs. 18Yang X. Kuo Y. Devay P., Yu, C. Role L. Neuron. 1998; 20: 255-270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Bermingham-McDonogh O. Xu Y.T. Marchionni M.A. Scherer S.S. Mol. Cell. Neurosci. 1997; 10: 184-195Crossref PubMed Scopus (60) Google Scholar, 20Carroll S.L. Miller M.L. Frohnert P.W. Kim S.S. Corbett J.A. J. Neurosci. 1997; 17: 1642-1659Crossref PubMed Google Scholar; see also Ref. 27Yang J.F. Zhou H. Choi R.C. Ip N.Y. Peng H.B. Tsim K.W. Mol. Cell. Neurosci. 1999; 13: 415-429Crossref PubMed Scopus (11) Google Scholar). Based on RNA studies (10Meyer D. Yamaai T. Garratt A. Riethmacher-Sonnenberg E. Kane D. Theill L.E. Birchmeier C. Development. 1997; 124: 3575-3586Crossref PubMed Google Scholar, 12Wen D.Z. Suggs S.V. Karunagaran D. Liu N.L. Cupples R.L. Luo Y. Janssen A.M. Benbaruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W.N. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (234) Google Scholar, 18Yang X. Kuo Y. Devay P., Yu, C. Role L. Neuron. 1998; 20: 255-270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Bermingham-McDonogh O. Xu Y.T. Marchionni M.A. Scherer S.S. Mol. Cell. Neurosci. 1997; 10: 184-195Crossref PubMed Scopus (60) Google Scholar, 20Carroll S.L. Miller M.L. Frohnert P.W. Kim S.S. Corbett J.A. J. Neurosci. 1997; 17: 1642-1659Crossref PubMed Google Scholar, 28Meyer D. Birchmeier C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1064-1068Crossref PubMed Scopus (165) Google Scholar, 29Chen M.S. Bermingham-McDonogh O. Danehy F.T. Nolan C. Scherer S.S. Lucas J. Gwynne D. Marchionni M.A. J. Comp. Neurol. 1994; 349: 389-400Crossref PubMed Scopus (109) Google Scholar, 30Corfas G. Rosen K.M. Aratake H. Krauss R. Fischbach G.D. Neuron. 1995; 14: 103-115Abstract Full Text PDF PubMed Scopus (210) Google Scholar), it is likely that the III-β1a isoform is one of the most abundant NRG isoforms in the late embryonic and postnatal nervous system and that the III-β3 isoform is relatively rare. The III-β1a and I-β1a isoforms differ in their N-terminal regions but are identical in their EGF-like, "stalk," transmembrane, and cytoplasmic regions (Fig. 1 A). Based on the similarity in structure of the III-β1a and I-β1a isoforms, schematic diagrams (4Adlkofer K. Lai C. Glia. 2000; 29: 104-111Crossref PubMed Scopus (158) Google Scholar,8Wolpowitz D. Mason T.B. Dietrich P. Mendelsohn M. Talmage D.A. Role L.W. Neuron. 2000; 25: 79-91Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) have represented the topology of III-β1a as similar to I-β1a (Fig. 1, B and C). This model of III-β1a topology suggests that III-β1a, like I-β1a, will be cleaved in the stalk region and that the consequence of this cleavage will be release of the entire N-terminal fragment into the extracellular fluid. To test this model and to determine whether the cellular mechanisms that regulate I-β1a topology and biosynthetic processing also govern III-β1a, we have compared III-β1a processing to I-β1a processing using biochemical and immunocytochemical techniques. The isoform designation "III-β 1a" refers to a NRG with a "type III" N-terminal region, a "β " type EGF-like domain, a "1" type sequence at the carboxyl terminus of the EGF-like domain, and an "a" type cytoplasmic tail (see Fig. 1 A for illustration). NRG isoforms can differ in the sequence of their N-terminal region (type I, II, and III), EGF-like domain (α or β), the C-terminal end of the EGF-like domain (1, 2, 3, or 4), and cytoplasmic tail (a, b, c, or none). In NRG isoforms with β1, β2, or β4 EGF-like domains, there is a stalk region, transmembrane domain, and cytoplasmic tail C-terminal of the EGF-like domain. (The stalk is the sequence between the EGF-like domain and the transmembrane domain.) These features are not found in NRG isoforms with a β3 EGF-like domain. Most NRGs in the nervous system have a β-type EGF-like domain and a-type tail (12Wen D.Z. Suggs S.V. Karunagaran D. Liu N.L. Cupples R.L. Luo Y. Janssen A.M. Benbaruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W.N. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (234) Google Scholar, 28Meyer D. Birchmeier C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1064-1068Crossref PubMed Scopus (165) Google Scholar). The amino acid sequence of rat NRG I-β1a can be found in Ref. 12Wen D.Z. Suggs S.V. Karunagaran D. Liu N.L. Cupples R.L. Luo Y. Janssen A.M. Benbaruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W.N. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (234) Google Scholar, and the amino acid sequence of the rat type III N terminus can be found in Ref. 19Bermingham-McDonogh O. Xu Y.T. Marchionni M.A. Scherer S.S. Mol. Cell. Neurosci. 1997; 10: 184-195Crossref PubMed Scopus (60) Google Scholar. Based on the results presented in this study, we propose that the III-β1a proprotein has a cytoplasmic tail at both its N terminus and C terminus. However, to be consistent with the published literature, the term "cytoplasmic tail" used without qualification refers to the cytoplasmic region C-terminal to the EGF-like domain. For example, III-β1a has the "a-tail" type of cytoplasmic tail. We refer to neuregulin proteins with both an ectodomain epitope and cytoplasmic tail epitope as "proproteins," in recognition of their potential to be biosynthetic precursors to cleaved bioactive products. However, this does not exclude the possibility that these "proproteins" are themselves bioactive signaling proteins. Throughout this paper, "neuregulin" and the abbreviation "NRG" refer only to the proteins encoded by the nrg-1 gene. Three related genes (nrg-2, nrg-3, andnrg-4) have now been identified, but the proteins encoded by these genes are not discussed. All neuregulin proteins analyzed in this study included an HA epitope tag in the ectodomain (Fig. 1 D). The sole exception is untagged I-β1a (labeled "native") used in the experiment illustrated in Fig. 2. Standard recombinant DNA techniques (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory, NY1989Google Scholar, 32Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1995Google Scholar) were used to create expression vectors for NRG proteins with an HA epitope tag in the ectodomain, just N-terminal of the EGF-like domain. The sequences of I-β1a and III-β1a differ at their N-terminal ends but are identical from a serine residue located 14 amino acids N-terminal of the first cysteine residue of the EGF-like domain through their C termini (Fig.1 A). The PCR-based strategy used to insert the HA tag changes the sequence between this serine and cysteine from STSTSTTGTSHLIKC to STSTSTTGTSIDYPYDVPDYASLHLIKC (underlined residues represent HA epitope tag). All HA-tagged constructs used contained this identical sequence within the ectodomain. A similar polymerase chain reaction-based strategy was used to introduce a FLAG epitope tag at the N terminus of an HA-tagged III-β1a construct for examination of the membrane orientation of the III-β1a N-terminal fragment. In this FLAG-III-β1a construct, the N-terminal sequence was changed from MEIYSP … toMDYKDDDKEFGGMEIYSP … (underlined sequence represents FLAG tag). The fidelity of all sequences amplified by polymerase chain reaction was verified by DNA sequencing. The vector backbone for all constructs was pcDNA 3.1 (Invitrogen). The tail deletion constructs and NRG I-β1c construct included a C-terminal Myc epitope tag. The Myc tag was not utilized in this study. Details regarding the primers used and the specific procedures employed to create these constructs are available upon request. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. PC 12 cells (N21 strain; Ref. 33Burry R.W. Perrone-Bizzozero N.I. J. Neurosci. Res. 1993; 36: 241-251Crossref PubMed Scopus (32) Google Scholar) were a gift from Richard Burry (Ohio State University). PC12 cells were cultured in DMEM containing 10% horse serum and 5% FetalClone (Hyclone) with 50 ng/ml NGF (Life Technologies, Inc.). Schwann cells were dissociated and purified from neonatal rat sciatic nerves (56Brockes J.P. Fields K.L. Raff M.C. Brain Res. 1979; 165: 105-118Crossref PubMed Scopus (915) Google Scholar). Purified Schwann cells were cultured on poly-l-lysine-coated slides in DMEM supplemented with 10% fetal calf serum, 2 μm forskolin (Calbiochem), and 5 ng/ml glial growth factor (a gift from Mark Marchionni (Cambridge Neuroscience)). PC12 and COS-7 cells were maintained in 5% CO2 at 37 °C. Schwann cells were maintained in 7.5% CO2 at 37 °C. For immunocytochemistry, cells were plated and transfected in eight-well Permanox chamber slides (Nunc, Naperville, IL). For biochemical experiments, cells were plated in 100-mm culture dishes. Plasmids were prepared for transfection using Qiagen MaxiPrep kits. Transfection of COS-7 cells using DEAE-dextran was performed as described previously (34Wang J.Y. Frenzel K.E. Wen D. Falls D.L. J. Biol. Chem. 1998; 273: 20525-20534Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) using 0.2 μg of DNA/well and 10 μg of DNA/100-mm dish. Transfection of PC 12 cells was performed using LipofectAMINE 2000 (Life Technologies) according to the manufacturer's instructions. PC12 cells were transfected 1 day after plating (∼90% confluent); 1 μg of DNA was used per well. Schwann cell cultures were transfected when 50–80% confluent. Transfection of Schwann cells was performed with FuGene reagent (Roche Molecular Biochemicals) according to the manufacturer's directions. For each well, 0.75 μl of FuGene was mixed with 25 μl of serum-free DMEM and then incubated with 0.5 μg of DNA for 15 min at room temperature prior to adding to the media of the cultured cells. COS-7 and PC 12 cells were processed 72 h after transfection. Schwann cells were analyzed 48 h after transfection. COS-7 cells transfected with the III-β1a construct were washed twice with PBS (0.1 mphosphate buffer, pH 7.4, 150 mm NaCl). One ml of hypotonic lysis buffer (20 mm Hepes, 2 mmMgCl2, pH 7.4) was added per 100-mm dish of transfected cells. The cells were collected in this buffer using a rubber policeman and passed through a 21-gauge needle 30 times. Nuclei and unbroken cells were removed by centrifugation at 200 × g for 10 min at 4 °C. The supernatant from this low speed spin (Input in Fig. 5 A) was centrifuged at 125,000 × g for 1 h at 4 °C using a Beckman TLA 100.3 rotor. To analyze the proportion of cellular III-β1a proprotein and its products bound to membranes, the supernatant (S2) and membrane pellet (P2) from this high speed spin were prepared in SDS sample buffer. For membrane extraction experiments, the membrane pellet was extracted on ice for 30 min in hypotonic lysis buffer with 1 m KCl or with 50 mm Na2CO3, pH 12. Following extraction, the samples were centrifuged at 125,000 ×g for 1 h at 4 °C to separate the soluble (S; supernatant) and insoluble (I; pellet) fractions. An equal percentage of the starting material for supernatant and pellet fractions was analyzed by Western blotting. Triton X-114 partitioning experiments were performed using a modification of Bordier's original protocol (35Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar). The membrane pellet (see above) was resuspended in 960 μl of hypotonic lysis buffer and mixed with 240 μl of 8% (v/v) Triton X-114 in 10 mmTris, pH 7.5, 150 mm NaCl (final volume 1.2 ml; final Triton X-114 concentration of 1.6%). This mixture was centrifuged at 125,000 × g for 1 h at 4 °C using a Beckman TLA 100.3 rotor to produce a soluble and an insoluble (pellet) fraction. To induce phase separation, 200 μl of the soluble fraction was warmed to 28 °C for 3 min. The warmed mixture was then layered onto a 6% sucrose cushion (300 μl) in a microcentrifuge tube and centrifuged at 200 × g for 5 min. This resulted in a three-phase solution: aqueous, top; sucrose cushion, middle; detergent, bottom. The aqueous phase was mixed with an equal volume of 2× sample buffer. The detergent phase was adjusted to 200 μl with lysis buffer and then mixed with an equal volume of 2× sample buffer. To prepare lysates for Western blot analysis, cells in 100-mm dishes were rinsed twice with HBS (20 mm Hepes, pH 7.4, 150 mmNaCl, 2 mm CaCl2) and lysed by scraping in 400 μl of lysis buffer (1% SDS, 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl). The lysate was passed through a 27-gauge needle four times to shear DNA and cleared of insoluble material by centrifugation at 15,000 × g for 20 min at 4 °C. The samples analyzed in Fig. 2 were total cell lysates, not cleared lysates. These samples were prepared by harvesting the cells directly in 400 μl of 2× SDS sample buffer/dish. Western blot analysis was performed as described elsewhere (34Wang J.Y. Frenzel K.E. Wen D. Falls D.L. J. Biol. Chem. 1998; 273: 20525-20534Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Samples were heated for 5 min at 95 °C prior to being loaded on a gel for analysis. The antibody dilutions used were as follows: α-HA monoclonal antibody 16B12 (raw ascites fluid; Berkeley Antibody Company, Richmond, CA), 1:3000; α-NRG a-tail rabbit polyclonal antibody SC348 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 0.1 μg/ml; horseradish peroxidase-conjugated goat anti-rabbit (Pierce), 1:50,000; horseradish peroxidase-conjugated donkey anti-mouse (Jackson ImmunoResearch Laboratories), 1:50,000. Blots were developed using Renaissance chemiluminescence substrate (PerkinElmer Life Sciences). Prestained standards used on Western blots (BenchMark protein ladder; Life Technologies) were calibrated against unstained molecular weight markers (Bio-Rad). A standard curve (distance migratedversus log M r) was created based on the migration of the marker proteins, and molecular weights were assigned to each band of interest according to the position of the center of the band. Experiments illustrated in the figures were repeated three times (Figs.Figure 2, Figure 3, Figure 4, 6, and 7) or twice (Figs. 5 and 8). Each repetition gave results similar to those illustrated. For quantitation of cell surface or released NRG, samples to be compared were analyzed on a single blot along with multiple dilutions of a standard sample. Films were scanned, and band "volume" (summed pixel density) was measured using the NIH Image software. A standard curve was created by plotting the pixel density for each dilution of the standard sample as a function of the relative amount loaded. The relative amount of NRG in each test sample was calculated based on this standard curve.Figure 4The 76-kDa NTF of III-β1a accumulates in the plasma membrane. A, COS-7 cells were transfected with III-β1a or I-β1a expression vectors or mock-transfected. Cell surface proteins were biotinylated (+), and the biotinylated proteins were isolated by precipitation with streptavidin agarose and analyzed by Western blot with α-HA. A strong 76-kDa band is present in the biotinylated III-β1a sample (lane 2). With heavier loadings and long exposures, a weak 140-kDa band was also detected, and this band was also detected with α-a-tail (not shown). 110- and 45-kDa bands are present in the biotinylated I-β1a sample (lane 4). These I-β1a bands are much less intense than the III-β1a NTF band. Control samples (lanes 3 and 5) were prepared identically except that the biotinylation reagent was omitted (−). The absence of signal in these no biotin controls demonstrates that NRG precipitation by streptavidin was dependent on protein biotinylation. An identically prepared sample of biotinylated proteins from mock-transfected cells was loaded in lane 1. Each lane was loaded with 10% of the fraction material derived from a 100-mm dish of transfected COS-7 cells. B, Western blot analysis with α-HA of lysates from which biotinylated proteins (A) were isolated. The band intensities are similar for the lysates prepared from dishes subjected to biotinylation (+) and to mock biotinylation (−). Each lane was loaded with 1% of the total cell lysate prepared from each dish.C, relative amounts of biotinylated NRG proteins. Bands were quantitated by densitometry as described under "Experimental Procedures." The amount of the III-β1a NTF (76 kDa) was assigned a value of 1. Mean ± S.E. (n = 3) is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6The concentration of NRG in medium conditioned by COS-7 cells expressing III-β1a is low relative to the level in medium conditioned by COS-7 cells expressing I-β1a. A, Western analysis of medium conditioned for 30 min by COS-7 cells expressing III-β1a or I-β1a and either untreated (−) or treated (+) with 100 nm PMA for 30 min. Two separate exposures of the same blot are shown, because the amount of NRG in medium conditioned by III-β1a-expressing cells is much less than in medium conditioned by I-β1a-expressing cells. Treatment with PMA dramatically increased the amount of NRG released by I-β1a-expressing cells but had l

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