Differential Shedding of Transmembrane Neuregulin Isoforms by the Tumor Necrosis Factor-α-Converting Enzyme

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Molecular and Cellular Neuroscience 16, 631–648 (2000)

doi:10.1006/mcne.2000.0896, available online at http://www.idealibrary.com on MCN

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Differential Shedding of TransmembraneNeuregulin Isoforms by the Tumor NecrosisFactor-a-Converting Enzyme

Juan Carlos Montero,1 Laura Yuste,1 Elena Dıaz-Rodrıguez,zucena Esparıs-Ogando, and Atanasio Pandiella2

Instituto de Microbiologıa Bioquımica and the Centro de Investigacion del Cancer, ConsejoSuperior de Investigaciones Cientıficas–Universidad de Salamanca, 37007 Salamanca, Spain

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The neuregulins (NRGs) are a family of EGF-like factorsthat activate receptor tyrosine kinases of the ErbB/HERtype. Some NRGs are membrane anchored and are re-leased upon cleavage of the ectodomain. Here we haveinvestigated the characteristics of the cleavage of differ-ent transmembrane NRG isoforms (proNRG) that divergein domains that have been implicated in the regulation ofthe cleavage of other membrane-anchored growth fac-tors. We show that cleavage of proNRGs is complex andgenerates several cell-bound truncated fragments. Com-parison of the resting generation of these truncated frag-ments between proNRG forms that diverge in the linkerregion that connects the EGF-like module to the trans-membrane domain revealed that proNRGb2a was rela-tively resistant to processing compared to proNRGb4a

hich was processed more efficiently than proNRGa2a.n important role for this linker in proNRG cleavage wasupported by deletion analysis of this region that pre-ented NRG solubilization. Studies aimed at the identifi-ation of the proteolytic machinery responsible forroNRG processing indicated that metalloproteases were

nvolved in proNRG processing. This was further sup-orted by the fact that cleavage of proNRGa2c was de-ective in fibroblasts derived from TACE2/2 animals thatxpress an inactive form of the metalloprotease TACE.

INTRODUCTION

The neuregulins (NRGs) are a family of epidermalgrowth factor-like (EGF-like) factors that activate recep-tor tyrosine kinases of the ErbB/HER type (Burden and

1 Equal contributors.2 To whom correspondence should be addressed at Instituto de

Microbiologıa Bioquımica, Edificio Departamental, Avenida delCampo Charro s/n, 37007-Salamanca, Spain. Fax: 134-923-224876.E-mail: [email protected].

1044-7431/00 $35.00Copyright © 2000 by Academic Press

ll rights of reproduction in any form reserved.

Yarden, 1997; Fischbach and Rosen, 1997). Studies car-ried out on cellular and animal models have supportedan essential role of NRGs and their receptors in nervoussystem physiology (Burden and Yarden, 1997). NRGshave been implicated in the induction of acetylcholinereceptors at the neuromuscular junction (Falls et al.,993; Fischbach and Rosen, 1997), the stimulation ofrogenitor neural cell proliferation (Shah et al., 1994),

and the regulation of Schwann cell survival and growth(Adlkofer and Lai, 2000; Wolpowitz et al., 2000). Genedisruption of the NRG1 gene in mice results in aberrantneural and cardiac development (Meyer and Birch-meier, 1995; Meyer et al., 1997). In these animals trabe-culation of the heart is deficient and as a consequencemouse embryos die in utero and present severe malfor-

ation of cranial and sympathetic ganglia (Britsch et al.,998; Meyer and Birchmeier, 1995; Meyer et al., 1997).s expected, some of these phenotypic abnormalitiesatch with those observed in mice deficient for the

rbB2, ErbB3, or ErbB4 receptors (Britsch et al., 1998;assmann et al., 1995; Lee et al., 1995; Riethmacher et al.,

997).Up to four distinct NRG genes have been identified

hat produce a wide variety of NRG isoforms (Busfieldt al., 1997; Carraway et al., 1997; Chang et al., 1997; Fallst al., 1993; Harari et al., 1999; Holmes et al., 1992;

archionni et al., 1993; Peles et al., 1992; Yang et al.,998; Zhang et al., 1997). Variability between isoforms ofhe same gene occurs by alternative splicing of the

RNA, giving rise to both soluble and membrane-nchored NRG forms (Marchionni et al., 1993; Wen et

al., 1994). In transmembrane NRGs derived from theNRG1 gene, differences in the intracellular domain af-fect both the length and the primary sequence (Wen etal., 1994). Variations in length give rise to short (or “c”

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type) and long (or “a” type) isoforms. In addition “b”forms, that are of intermediate length, are identical tothe “c” forms but contain a characteristic extra 39-amino-acid sequence at the C-terminal intracellular do-main. Another divergent region of membrane-anchoredNRGs is located between the fifth cysteine residue ofthe EGF-like unit and the transmembrane domain anddistinguishes between a and b NRG isoforms (Holmeset al., 1992). A region in this subdomain, the linker,extends from the sixth cysteine residue to the beginningof the transmembrane domain. The linker is also highlyvariable in both length and sequence between NRGisoforms of the same subfamily. Thus b2 isoforms con-ain the shorter linker, b4 has the larger, and b1 has anntermediate length. b3 isoforms are characterized by

the presence of a stop codon in the region correspond-ing to the linker that prevents their association to theplasma membrane and creates an isoform that is poorlyreleased to the extracellular media (Holmes et al., 1992).

The mechanisms and proteolytic components respon-sible for the solubilization of transmembrane NRGs arepoorly known. Release of NRGs has been shown to bestimulated by serum factors (Loeb et al., 1998) or byactivation of protein kinase C (PKC) (Burgess et al.,1995; Loeb et al., 1998). Studies carried out with othermembrane-anchored EGF-like growth factors indicatethat solubilization of the ectodomain of these factors iscontrolled by a class of cell surface proteolytic enzymes,generically termed secretases or sheddases, whose ac-tivity is highly regulated (Hooper et al., 1997; Massagueand Pandiella, 1993). Initial pharmacological experi-ments employing inhibitors of the different proteasefamilies pointed to metalloproteases as the enzymesresponsible for membrane protein ectodomain cleavage(Gearing et al., 1994; McGeehan et al., 1994; Mohler et al.,1994). Later, by the use of these inhibitors (Moss et al.,1997) and in vitro peptide cleavage assays (Black et al.,1997) the pro-tumor necrosis factor-a (TNFa)-convert-ing enzyme (TACE), that participates in the solubiliza-tion of the TNFa from its precursor, has been isolated.Structurally, TACE is a type I membrane protein thatcontains several domains in the extracellular region,including disintegrin and metalloprotease domains,characteristic of the ADAM subfamily of metallopro-teases (Black and White, 1998; Blobel, 1997). In additionto its role in TNFa solubilization, TACE may also par-ticipate in the cleavage of other transmembrane pro-teins. Thus, cells from animals expressing an inactiveform of TACE not only have a defect in the productionof soluble TNFa, but also fail to efficiently cleave pro-TGFa, L-selectin, the p75TNFR (Peschon et al., 1998), orb-amyloid precursor protein (bAPP) (Buxbaum et al.,998). On the other hand, it is possible that other pro-

teases may be involved in the regulation of membraneprotein ectodomain cleavage. In fact, solubilization ofthe ectodomain of the angiotensin-converting enzymeis unaffected in fibroblasts derived from TACE-inactiveanimals (Sadhukhan et al., 1999). Furthermore, screen-ings for PKC-interacting proteins have identified an-other ADAM family member, ADAM9/MDC9/mel-tring, as a PKC-regulated metalloprotease that controlsheparin-binding EGF-like (HB-EGF) growth factorcleavage (Izumi et al., 1998).

Indirect evidence suggests that cleavage of NRGsmay be functionally important. In fact, transmembraneNRGs are expressed by the endocardial layer of theheart (Erickson et al., 1997; Meyer and Birchmeier, 1994,1995; Meyer et al., 1997), but their receptors are locatedat a significant distance (in the myocardium (Gassmannet al., 1995) or the heart valve mesenchyma (Erickson etal., 1997; Riethmacher et al., 1997)) from the site of NRGbiosynthesis, indicating that cleavage of NRGs may beessential for their function. In addition, animals ex-pressing uncleavable mutant NRGs that lack the cyto-solic domain display a phenotype analogous to NRG-null mice (Liu et al., 1998a,b). Here we have investigatedseveral properties related to the cleavage of membrane-bound NRGs. We show that cleavage of membrane-anchored NRGs generates a complex pattern of cell-bound truncated fragments and that this effect can beneutralized by hydroxamic acid-based metalloproteaseinhibitors. Studies on extracellular determinants indi-cate that cleavage is more efficient in NRG isoformscontaining a long linker region and also suggest thatother determinants may also be involved in proNRGcleavage. We also report that cleavage of NRGs is im-paired in cells derived from animals that express aninactive form of the metalloprotease TACE, indicating arole of this enzyme in NRG solubilization.

RESULTS

Since variability between proNRG isoforms affectsregions that have been implicated in the regulation ofthe cleavage of other membrane-anchored growthfactors (Massague and Pandiella, 1993), we reasonedthat such a variability could confer distinct solubili-zation properties to different proNRG isoforms. Thelong proNRGa2a, proNRGb2a, and proNRGb4a areidentical in their transmembrane and cytosolic do-mains, but diverge in their extracellular juxtamem-brane linker region (Fig. 1A) (Wen et al., 1994). Com-parison of the shedding of these proNRG isoformscould thus inform about a possible role of the linkerlength and sequence in NRG solubilization. The

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proNRGa2c isoform shares identity in the extracellu-lar and transmembrane regions with proNRGa2a, butontains only 157 of the 374 amino acids of the intra-ellular domain of proNRGa2a. Thus any possible

differences in the shedding of NRGa2a versusNRGa2c could be attributed to sequences locatedexclusively in the intracellular domain.

FIG. 1. Expression of transmembrane NRGs in 293 cells. (A) SchemaroNRGs. The divergent linker region sequences of the forms analyze

in 293 cells. Cells transfected with the cDNAs coding for proNRGa2aanti-ectodomain (anti-HA) or anti-endodomain (anti-endo311) antibodwere probed with these antibodies. Where indicated, 10 mg of the peptgG, heavy IgG chain. The M r (in kDa) of the markers is shown at thells was collected over 24 h and immunoprecipitated with polyclonaample buffer and then subjected to 10% SDS–PAGE and Western blohe top of the gel. Where indicated, 10 mg of the HA peptide usmmunoprecipitated sample. (D) Effect of conditioned medium from 2

to confluence and then serum starved overnight. Five milliliters of confor 10 min to MCF7 monolayers. Lysates were prepared and p185HER2 twith the 4D5 antibody, followed by anti-phosphotyrosine Western.

Cleavage of ProNRG Generates SeveralCell-Bound Truncated Fragments

We used an approach successfully employed to ana-lyze the cleavage of other membrane-anchored growthfactors of the EGF family, such as proTGFa (Pandielland Massague, 1991). A polyclonal antibody (Ab311)

presentation of domains common to several different transmembranethis work are shown. (B) Expression of proNRGa2a and proNRGa2cproNRGa2c were lysed and samples were immunoprecipitated withmmunoprecipitates were resolved in 8% SDS–PAGE gels, and blotssed for immunization was included during the immunoprecipitation.t. (C) Identification of released NRG. Culture medium from 293-a2c

-HA antibodies. Samples were boiled in nonreducing electrophoresiswith the monoclonal anti-HA antibody. Unreduced IgG migrated atgenerate the polyclonal anti-HA antibodies was included in the

c cells on p185HER2 tyrosine phosphorylation. MCF7 cells were growned medium (CM) or 10 nM recombinant NRGa (rNRGa) were added

ne phosphorylation analyzed by immunoprecipitation of the receptor

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that recognized an epitope located in the intracellulardomain was raised, and in addition HA tags were in-serted at the ectodomain between the Ig- and the EGF-like domains (Fig. 1A). The different NRG isoformswere transfected into HEK293 or CHO cells, and se-lected clones were tested for expression by Western blotanalyses using combinations of the anti-ectodomainand anti-endodomain antibodies (Fig. 1B).

Immunoprecipitation and Western blotting of 293 cellstransfected with the cDNA coding for the long proNRGisoform a2a (293-a2a cells) with the anti-endodomain an-tibody 311 (Fig. 1B, top left panel) identified three bands.The most abundant and slowest migrating band corre-sponded to mature proNRGa2a; and the intermediatecorresponded to immature proNRGa2a. The faster mi-grating band was poorly reactive with the anti-HA anti-body (Fig. 1B, top right panel), indicating that this formprobably corresponded to a truncated form lacking theIg-like domain. This was also supported by the failure ofthe 7D5 anti-ectodomain mAb to recognize such band(data not shown). The specificity of the anti-endodomain311 and anti-HA antibodies to transfected proNRG wasconfirmed by preincubation of the immunoprecipitateswith the peptide against which each antibody was raised(Fig. 1B) and by comparing the immunoprecipitatedbands detected in cells transfected with the differentproNRG isoforms with those from untransfected 293 cells(data not shown). Soluble NRG derived from these trans-fectants could be detected in the culture medium afterimmunoprecipitation with the anti-HA antibody (Fig. 1C).The presence of the HA tag did not apparently affect thebioactivity of HA-NRG, as indicated by the ability ofconditioned medium from HA-tagged proNRGa2a-ex-

ressing cells to induce tyrosine phosphorylation of185HER2 in MCF7 cells (Fig. 1D).The expression profile of the short proNRGa2c pre-

cursor isoform paralleled that of proNRGa2a, but thebands recognized had a lower M r than the proNRGa2a-derived forms as expected from the significantly shortercytosolic tail of the proNRGa2c isoform (Fig. 1B). Thetruncated form of proNRGa2c migrated close to the Igheavy chain and could only easily be distinguishedfrom the latter in higher resolution SDS–PAGE gels(e.g., Fig. 2). An analogous pattern of expression ofproNRGa2a and proNRGa2c was observed in CHO celltransfectants; however, their resting release of NRGwas found to be higher than in 293 transfectants (datanot shown).

Immunoprecipitation and Western blot analyses withthe 311 antibody of 293-a2a cells treated with the PKCactivator PMA showed a substantial decrease in theamount of the mature and truncated proNRGa2a forms(Fig. 2A). The cleavage of the ectodomain of
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proNRGa2a was accompanied by increased release ofNRG to the culture medium with respect to restingrelease (Fig. 2A), together with the generation of severaltruncated cell-associated fragments, whose M r rangedfrom 50 to 65 kDa (Fig. 2A). Higher autoradiographicexposure times revealed that a small amount of tailfragments was present in untreated proNRGa2a cellsdata not shown, see also Fig. 5C), indicating that cleav-ge occurred under steady-state conditions, but coulde accelerated upon PKC activation. An analogous pat-ern of truncated fragments was also observed in anti-ndodomain immunoprecipitates from proNRGa2c

(Fig. 2B), in which up to four different proNRG-derivedtruncated fragments were identified in cells treatedwith PMA (Figs. 2B and 2C). The acute action of PMAwas prevented by a long-term incubation with thephorbol ester (Fig. 2D) and by the PKC inhibitor bisin-dolylmaleimide (BIM) (Fig. 2E), supporting that theeffect of PMA on proNRG cleavage was due to PKCactivation. The cleavage of proNRGa2a andproNRGa2c was observed both in extracellularly HA-tagged forms and in untagged versions of the samemolecule indicating that the tag did not affect PMA-regulated proNRG cleavage (data not shown).

The effect of PMA on the generation of the truncatedfragments and the cleavage of the mature form wasdose dependent, with a half maximal effect at concen-trations of 50–100 nM (Fig. 3A, top and right panels).Generation of the truncated fragments correlated withsolubilization of bioactive NRG, as indicated by theability of conditioned medium from 293-a2c cellstreated with different concentrations of PMA to stimu-late tyrosine phosphorylation of p185HER2 in MCF7 cells(Fig. 3A, bottom and right panels).

To analyze whether such a complex pattern of cleav-age could be reproduced in cells that autoctonouslycontain NRG1, we searched for cell lines that expressedtransmembrane NRG1 forms. RT-PCR analysis of sev-eral human cell lines with oligonucleotide pairs thatincluded extracellular and cytosolic sequences (Fig. 3B)showed that the pancreatic adenocarcinoma cell lineNP18 contained transmembrane NRGs (Fig. 3C). TheSKNBE neuroblastoma cell line also expressed NRGsbut to a much lesser extent than NP18 cells (Fig. 3C).The most abundant NRG band split into two on high-resolution acrylamide gels, indicating that NP18 cellsexpressed at least two different NRG1 isoforms (Fig.3D, filled arrows). In cell lysates from resting NP18cells, the anti-endodomain 311 antibody immunopre-cipitated two major bands with a M r between 110 and0 kDa (Fig. 3E, arrows). Treatment of NP18 cells withMA induced disappearance of the proNRG isoformsith generation of several cell-bound tail fragments of

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M r between 50 and 65 kDa (Fig. 3E), indicating that thecleavage of NRG in this cell line paralleled that ob-tained in the transfected 293 cells.

All the Tail Fragments Derived from ProNRG AreMembrane Bound

The heterogeneous pattern of the truncated cell-bound tail fragments derived from the differentproNRG isoforms raised a question about their mecha-nism of generation. In the case of notch, cleavage at theectodomain precedes a secondary cleavage that occurswithin the intracellular domain (Brou et al., 2000). If ananalogous mechanism operated to generate the differ-ent proNRG-derived cell-bound fragments was investi-gated by two experimental approaches. First, an HA tagwas inserted at the transmembrane–juxtamembrane

FIG. 2. PKC activation generates several cell-bound truncated fragmeells were treated with PMA (1 mM) for 20 min and then cell lysatescells) or anti-HA (media) antibodies. Samples were resolved in 8%nti-HA (media) antibodies. (B) Effect of PMA on proNRGa2c cleavag

and Western blot procedures were as above, except that the SDS–PAGgel. (C) Magnification of the region where the proNRGa2c fragmentsthe cleavage of proNRGa2c. (D) Depletion of PKC by prolonged treaWhere indicated (chronic PMA) 293-a2c cells were incubated with PImmunoprecipitation and Western blot with the anti-endodomainPMA-induced proNRGa2c cleavage. Where indicated 293-a2c cells waddition. Ig, immunoglobulin heavy chain.

border in the intracellular domain of proNRGa2c (Fig.4A). Western blot analysis of the extracellularly andintracellularly HA-tagged proNRGa2c indicated thatthe mature forms of both constructs were of similar M r

(Fig. 4B). However, the tail fragments were of signifi-cantly higher M r in the case of proNRGa2c-HAintra,when compared to the ectodomain HA-tagged counter-part (Fig. 4B), suggesting that cleavage occurred N-terminal to the tag. Second, in cell fractionation exper-iments all the truncated fragments derived fromproNRGa2c cleavage partitioned with the microsomalpellet (Fig. 4C), indicating that they preserved a mem-brane anchor.

To analyze if the different M r of the proNRG-derivedcell-bound fragments were due to PKC-induced phos-phorylation of the cytosolic tail of proNRGa2c, immu-noprecipitates from PMA-treated cells were incubated

(A) Effect of the PKC activator PMA on proNRGa2a cleavage. 293-a2adium samples were immunoprecipitated with anti-endodomain 311

PAGE gels, and Western blots were analyzed with the 311 (cells) or-a2c cells were treated with PMA as above. The immunoprecipitationl used for the separation of the distinct 311-reactive bands was a 12%ated. The arrows indicate up to four distinct fragments derived fromt with phorbol esters inhibited PMA-induced proNRGa2c cleavage.for 24 h, and then PMA was added again to the samples indicated.antibody were as above. (E) Effect of the PKC inhibitor BIM onreated with the PKC inhibitor BIM (10 mM) for 15 min before PMA

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with calf intestinal phosphatase before Western blotanalysis. This treatment, that efficiently dephosphory-lated the TrkA receptor tyrosine kinase (data notshown), did not substantially affect the mobility of thePMA-induced proNRGa2c tail fragments (Fig. 4D).

FIG. 3. Cleavage of proNRG isoforms in cells that autoctonouslyproNRGa2c and effect of conditioned medium on p185ErbB2 tyrosine phoncentrations of PMA, and cell lysates were subjected to immunonditioned medium from the same experiment was concentratedhosphorylation of p185ErbB2 was analyzed as described under Expe

maximal p185ErbB2 tyrosine phosphorylation. Quantitation of the doseragments generated, and p185ErbB2 tyrosine phosphorylation is graph

(arrows) with respect to the different proNRG domains. (C) RT-PCRarrow indicates a major product with the expected bp content. Two o(D) Acrylamide gel electrophoresis of the PCR-amplified fragments frimmunoreactive with the anti-endodomain 311 antibody, and effect ocipitated with the 311 antibody and probed in Western blot with the0 min, and then lysates were immunoprecipitated with the 311 antroNRG isoforms (arrows) disappear upon PMA treatment and tail

Differential Shedding of ProNRG Isoforms

The kinetics of PMA-induced cleavage of differentproNRG isoforms was next investigated (Fig. 5). Upon

ess proNRG. (A) Dose–response effect of PMA on the cleavage oforylation. 293-a2c cells were incubated for 20 min with the indicated

cipitation and Western blot analysis with the 311 antibody (top).added to MCF7 cells in the presence of 10 mM BIM, and tyrosinental Procedures (bottom). rNRG (10 nM) was used as a control foronse data with respect to the maximal amount of mature proNRG,shown at the right. (B) Schematic representation of the primer sites

lysis of proNRG expression in different human cell lines. The filledands (hollow arrowheads) were also amplified in the NP18 cell line.P18 cells. (E) Expression in NP18 cells of different proNRG isoformsA on proNRG cleavage in NP18 cells. Cell lysates were immunopre-e antibody. Where indicated, cells were treated with 1 mM PMA for

followed by Western blot with the same antibody. The two majorents are generated.

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PMA treatment a decrease in the mature proNRG pre-

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cursor was observed in all NRG isoforms analyzed (Fig.5A). This effect was dose dependent (data not shown)and reached a maximum at times above 20 min oftreatment with the phorbol ester. The levels of the trun-cated proNRG form decreased following a similar timecourse with somewhat faster and more efficient cleav-age than mature proNRG. In contrast, the immatureform was largely resistant to PMA treatment at earlytime points, and its amount was up-regulated after20–30 min of treatment.

Analysis of the truncated tail fragments revealed dif-ferences in their time course of generation. In general,the fragments with a higher M r were the first to begenerated, being detectable in proNRGa2a at 2.5 min ofPMA treatment. The presence of the two upper frag-ments was maintained for 30 min, decreasing thereaf-ter. The lower M r fragments increased their presencewith a significant delay, being clearly detectable be-tween 10 and 20 min of treatment and remaining forlonger times than the upper fragments.

The time course of generation of the fragments de-rived from the a isoforms was analogous, indicatingthat differences in the length of the intracellular domainare not apparently reflected in changes in their solubi-

FIG. 4. PMA-induced cleavage of proNRG occurs at the ectodomain(B) Cleavage of proNRGa2c tagged with HA at the ectodomainImmunoprecipitations and Western blots were performed with the anfrom proNRGa2c to the microsomal fraction of 293-a2c cells. Cells we

nder Experimental Procedures. (D) Effect of calf intestinal phosphaated with the anti-endodomain 311 antibody and, where indicateDS–PAGE and Western blot with the 311 antibody was as above.

lization properties in response to PMA; however, dif-ferences were found when the rate of generation ofa-derived fragments was compared to the b2a-derivedtail fragments (Figs. 5A and 5B). A larger lag withrespect to the a-derived fragments was detectable whenthe generation of b2a-derived fragments was analyzedin PMA-treated 293-b2a cells. In the latter, cleaved frag-ments were clearly detectable only after 5 min of PMAtreatment, compared to the detection of truncated frag-ments at 2.5 min of PMA treatment in proNRGa formsFigs. 5A and 5B).

b4a-expressing 293 cells contained a significantamount of high M r truncated fragments under restingconditions, and addition of PMA decreased its presenceto generate truncated fragments of lower M r (Fig. 5A).

The Linker Region Is Determinant in ProNRGCleavage

Comparison of the basal presence of the truncatedfragments between different proNRG isoforms indi-cated a correlation between the length of the linkerregion and the resting cleavage of proNRG (Fig. 5C).ProNRGb4a-expressing cells accumulated a substantial

Schematic representation of intracellularly HA-tagged proNRGa2c.2c-HA; see also Fig. 1B) or at the endodomain (293a2c-HAintra).odomain 311 antibody. (C) Association of the tail fragments derived

ctionated and analyzed for proNRG by Western blotting as describedn proNRGa2c fragment mobility. Cell lysates were immunoprecipi-e immunoprecipitates were incubated with phosphatase for 1 h.

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638 Montero et al.

amount of truncated fragments, while cells transfectedwith the shorter b2a form contained little amounts oftruncated fragments. The levels of truncated fragmentsin 293-a2a cells were higher than those present in 293-b2a, but in both cases lower than those of b4a-express-ing cells. Resting accumulation of soluble bioactiveNRG was more substantial in 293-a2a culture medium,as indicated by the quantitatively higher tyrosine phos-phorylation of p185HER2 in MCF7 cells treated with con-ditioned medium from 293-a2a cells compared to theeffect of medium from 293-b2a cells (Fig. 5D).

The above fact, together with the ample spectrum ofRG-derived cell-associated fragments, led us to inves-

igate the possible role that determinants located in theinker region may have on proNRG cleavage. For thisurpose, deletion mutants that affected distinct regionsf the linker were created. Initially, a large deletion (20mino acids) that brought the sixth cysteine residue of

FIG. 5. Cleavage of different proNRG isoforms. (A) 293 cells expresimes and then subjected to 8% SDS–PAGE gels, except for the lower peveloped with the 311 antibody. (B) Quantitative analysis of the different proNRG isoforms at different times (as indicated) of PMA

obtained in every single experiment. (C) Accumulation of truncattransfected with different proNRG isoforms were lysed and immunopare shown. (D) Effect of conditioned medium on p185HER2 tyrosine phoCM a2a) or proNRGb2a (CM b2a) was added to MCF7 monolayers fo

content of the receptor was analyzed. Recombinant NRGa (rNRGa,

he EGF-like domain to one amino acid from the start of

the transmembrane domain was generated. This con-struct, termed D223–242, was highly resistant to PMA-induced cleavage, as indicated by the strong inhibitionof PMA-induced generation of cell-bound truncatedfragments (Fig. 6A). In addition, conditioned mediumfrom this mutant failed to stimulate phosphorylation ofp185HER2 (data not shown) or the downstream signalingmolecules Erk1/2 (Fig. 6G). A marginal amount of atruncated fragment could in some experiments be de-tected in 293-D223–242 cells treated with PMA (Fig. 6A,arrow). To analyze if the failure to cleave the D223–242

mutant was due to missorting of the mutant protein,protease protection experiments and immunofluores-cence analyses were carried out. In protease protectionexperiments, the mature and truncated forms of wild-type and the D223–242 mutant were sensitive to proteinaseK treatment (Fig. 6B), indicating that they were exposedat the plasma membrane. However, the immature form

different proNRG isoforms were treated with PMA for the indicatedf the bottom panel that was ran on 12% gels. The Western blots were

rom three independent experiments to measure the cleavage of thement. The data (mean 6 SD) are referred to the maximal cleavageagments in 293 cells expressing different proNRG isoforms. Cellsitated with the 311 antibody. Two different exposures of the same blotrylation. Medium conditioned for 2 h by cells expressing proNRGa2amin and then p185HER2 immunoprecipitated and the phosphotyrosine) was used as a control.

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FIG. 6. Analysis of mutants of proNRGa2c with deletions in the linker region. (A) Effect of the D223–242 deletion on cleavage. Immunoprecipi-ation and Western blotting with the 311 antibody was as described under Experimental Procedures. The arrow indicates a small amount of aruncated fragment generated upon treatment of the mutant-expressing cells with PMA. (B) Protease protection experiments of wild-type andhe D223–242 mutant. Cells were treated with proteinase K as described under Experimental Procedures and then lysed and subjected tommunoprecipitation and Western blotting with the 311 antibody. (C) Immunofluorescence analysis of the subcellular distribution of wild-typend D223–242 mutant proNRGa2c. Cells were plated on coverslips, allowed to attach for at least 2 days, and then permeabilized and incubated withhe 311 antibody. Images were captured using a Zeiss LSM510 confocal microscope. Bar, 15 mm. (D) Description of the sequences deleted in the

different mutants of the linker region of proNRGa2c. (E) Cleavage of the different deletion mutants shown. Where indicated, PMA (1 mM) wasdded for 20 min and then cell lysates were prepared for immunoprecipitation with the 311 anti-endodomain antibody. The Western blot was

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640 Montero et al.

form was located in a cellular compartment not acces-sible to the exogenously added protease. These experi-ments also revealed increased amounts of the immatureform of the mutant D223–242 with respect to the wild type.mmunofluorescence experiments with the anti-ndodomain antibody 311 indicated that wild-typeroNRGa2c and the D223–242 mutant were localized at the

same subcellular sites, i.e., the cell surface, and intracel-lular perinuclear sites (Fig. 6C). This intracellular stain-ing partially colocalized with the Golgi marker lentillectin (data not shown). In agreement with the proteaseprotection experiments, staining in intracellular com-partments was more pronounced in D223–242 mutant thanin wild-type proNRGa2c-expressing cells. Taken to-gether, the above data suggested that deficient cleavageof D223–242 was not due to missorting of the mutantprotein to the cell surface.

Intermediate deletions (13 amino acids) of the linkerregion close to the transmembrane (D228–240) or to theEGF-like module (D223–235) also prevented regulatedcleavage (Figs. 6D and 6E) and accumulation of bioac-tive NRG in the conditioned medium (Fig. 6G); as didshorter deletions of seven to eight amino acids per-formed on different sites of the linker region (D224–230

and D234–241, Figs. 6D and 6E). Even though the D228–240

mutant was highly resistant to PMA-induced cleavage,a small amount of truncated fragments could be de-tected in cells treated with the phorbol ester (Fig. 6E,arrows). Finally, since rat a NRGs have been shown tobe processed at the MKV subdomain within the linkerunder resting conditions (Lu et al., 1995), a deletionmutant consisting in the elimination of these residueswas created (DMKV). As shown in Fig. 6F this shortdeletion mutant also failed to be cleaved in response toPMA, and cells transfected with the DMKV were unableto produce bioactive NRG (Fig. 6G).

Resting and Regulated ProNRG Cleavage IsSensitive to Hydroxamic Acid-DerivedMetalloprotease Inhibitors

Resting and regulated release of several membrane-bound proteins has been shown to be inhibited byhydroxamic acid-derived metalloprotease inhibitors(Black and White, 1998; Mullberg et al., 1997). One of

probed with the same antibody. The arrows indicate two fragmentsdeletion on proNRGa2c cleavage. (G) Effect of conditioned mediumphosphorylation of the downstream molecules Erk1 and Erk2. MCFmedium from 293 cells transfected with wild-type proNRGa2c or thelysates by Western blotting with an anti-p-Erk antibody.

these compounds, BB3103, was able to reduce the rest-ing accumulation of soluble NRG in the culture me-dium, as indicated by the profound inhibition ofp185HER2 phosphorylation upon treatment of MCF7 cellswith media from 293-a2c cells cultured in the presenceof the protease inhibitor (Fig. 7A).

To analyze whether BB3103 also affected regulatedproNRG cleavage, cells were incubated with this com-pound for 30 min and then treated with PMA. BB3103prevented both the regulated release of NRG to theculture medium (Fig. 7B) and the phorbol ester-inducedgeneration of fragments derived from proNRGa2c (Fig.7C) or proNRGa2a (Fig. 7D). Other metalloproteaseinhibitors such as 1,10-phenanthroline (Fig. 7E) orEDTA (data not shown) did not significantly affectproNRG-regulated release. Treatment with 1,10-phenanthroline (2–10 mM) for long periods was foundto affect cell viability thus preventing us to correctlyevaluate its potential inhibitory effect on resting NRGrelease (data not shown).

Deficient Cleavage of ProNRG in FibroblastsExpressing an Inactive Form of TACE

By several approaches, including the use of theabove-mentioned hydroxamic acid-derived compounds(Black et al., 1997; Moss et al., 1997), the metalloproteaseTACE, that processes certain membrane proteins (Bux-baum et al., 1998; Peschon et al., 1998), has been isolated.Thus, the possible role of TACE in the regulated cleav-age of proNRGa2c was next investigated. For this weused a fibroblast cell line (herewith referred asTACE-DZn/DZn) derived from mice homozygous for aform of TACE in which the metal binding pocket of theprotease had been deleted (Peschon et al., 1998). As aconsequence, cells derived from these animals ex-pressed a TACE form with a M r below that of TACEfrom cells obtained from wild type animals (Fig. 8A). Inthese cells transfection with the cDNA coding forproNRGa2c (TACE-DZn/DZn-a2c cells) resulted in the ex-pression of proNRGa2c forms analogous to those foundin 293-a2c cells (Fig. 8B). However, in contrast to thesubstantial sensitivity of proNRGa2c to PMA-inducedcleavage in 293-a2c cells, treatment of TACE-DZn/DZn-a2ccells with the phorbol ester did not induce a decrease in

ere detectable in D228–240 and D234–241 mutants. (F) Effect of the MKVm 293 cells transfected with different proNRGa2c mutants on thels were treated with recombinant NRG (rNRG) or the conditionedted mutants. Active forms of Erk1 and Erk2 were identified in MCF7

that wfro

7 celindica

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the amount of the mature proNRGa2c or the generationof truncated fragments (Fig. 8B, top panel). In addition,PMA failed to induce accumulation of bioactive NRG inTACE-DZn/DZn-a2c cells (Fig. 8B, bottom panel). In con-trast, transfection of proNRGa2c in TACE1/1 fibroblastsresulted in efficient cleavage of proNRGa2c even underesting conditions (Fig. 8C). To investigate if the failuref PMA to induce processing of proNRGa2c in

TACE-DZn/DZn cells was due to a general defect in thePKC pathway, another independent action of PKC, i.e.,the regulation of the MAPK upstream activating ki-nases, was analyzed. Western blot analysis of Erk1/2 bythe use of antibodies that recognize the dually phos-phorylated active form of these kinases showed thatPKC activation resulted in efficient phosphorylation ofErk1/2 (Fig. 8D), thus supporting that other actions ofPKC in TACE-DZn/DZn cells were preserved.

DISCUSSION

Studies on the expression and function of NRGs haveindicated that cleavage of transmembrane NRGs maybe an essential step in the physiological actions of thesefactors. In fact, transmembrane NRGs are expressed bythe endocardial layer of the heart (Erickson et al., 1997;

FIG. 7. Effect of metalloprotease inhibitors on proNRG cleavage. (A93-a2c, or 293-a2c cells treated with the protease inhibitor BB3103 (xperimental Procedures. (B) Effect of BB3103 on PMA-induced gene

or 20 min before addition of PMA (1 mM). Culture media were harve) Effect of BB3103 (20 mM) on the cleavage of proNRGa2a and

,10-phenanthroline (5 mM) on PMA-induced cleavage of proNRGa2

eyer and Birchmeier, 1994, 1995; Meyer et al., 1997),ut their receptors are located at a significant distancein the myocardium (Gassmann et al., 1995) or the heartalve mesenchyma (Erickson et al., 1997; Riethmacher etl., 1997)) from the site of NRG biosynthesis. In addi-ion, animals expressing uncleavable mutant NRGs dis-lay a phenotype analogous to NRG-null mice (Liu etl., 1998a), also indicating that cleavage of NRGs may beritical for their function. This, together with the facthat certain naturally occurring NRG isoforms divergen the region where cleavage of the membrane-boundrecursor is expected to occur, led us to investigate theechanisms, molecular determinants, and proteolytic

ntities that participate in NRG solubilization.An interesting conclusion of the present study was

he complex nature of expression and cleavage of theifferent NRG isoforms. Western blotting analysis ofroNRGs expressed in 293 and CHO cells indicated thatt least three different forms could be detected. The twolower migrating bands corresponded to the maturend immature forms previously reported in CHO cellsBurgess et al., 1995). The third, faster migrating form,orresponded to a truncated version of NRG. An anal-gous lower M r form has also been described in COS

cells expressing proARIAb1a (Han and Fischbach,1999). In the latter, the lower M was attributed to a

F7 monolayers were incubated with conditioned medium from 293,M), and p185HER2 phosphorylation was analyzed as described under

of soluble NRG. 293-a2c cells were incubated with BB3103 (20 mM)nd soluble NRG detected as described in the legend to Fig. 1. (C andNRGa2c in 293 cells. (E) Effect of a 30-min preincubation with293 cells.

) MC20 mrationsted a

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642 Montero et al.

truncation in the intracellular domain of proARIA (Hanand Fischbach, 1999). It is unlikely that this may explainthe truncated form we identified. Thus, inclusion of anHA tag in the area linking the Ig- and the EGF-likeregions suggested that the lower M r form correspondedto a truncation between these two domains, as indicatedby the poor reactivity of the truncated form with theanti-HA antibody. In addition, in proNRGa2c an intra-cellular truncation similar to that described forproARIA should result in the removal of the epitoperecognized by the anti-endodomain 311 antibody, thuspreventing its identification with this reagent. How-ever, the truncated form also appeared in proNRGa2c-expressing transfectants immunoprecipitated by suchantibody. These data, based mainly on epitope map-ping, indicate that the truncated form must be gener-ated by removal of the N-terminal extension, including

FIG. 8. Participation of TACE in the cleavage of proNRGa2c. (A) Exfibroblasts derived from wild-type (TACE1/1) and mutant (TACE-DZn/

and the Western blot was probed with the same antibody (top panbioactive NRG from 293-a2c and TACE-DZn/DZn-a2c cells, as measured bof proNRGa2c in 293 or TACE-DZn/DZn cells were treated with PMA wdentified by Western blotting as described. (C) Same experiment

TACE-DZn/DZn. (D) Action of PMA on the phosphorylation of the MAP kwith PMA and cell lysates prepared to analyze Erk1/2 levels (anti-Erkprobe).

the Ig-like domain. An analogous N-terminal cleavagehas been reported in the maturation of the precursor forproTGFa (Massague and Pandiella, 1993). This proteinis biosynthesized as an 18-kDa form that matures intoheterogenously glycosylated 20- to 22-kDa forms (Pan-diella and Massague, 1991). Removal of the N-terminalextension of proTGFa that contains the glycosylationsites results in the generation of a mature precursor of17 kDa that accumulates at the cell surface (Teixido etl., 1987). Whether the truncated proNRG form arisesy an analogous proteolytic cleavage or results fromeading of an open frame in the region between the Ig-nd the EGF-like domains is under study.Cleavage of several membrane proteins has been

hown to be stimulated by agents that activate intracel-ular signaling cascades such as the PKC pathwayHooper et al., 1997; Massague and Pandiella, 1993).

ion of TACE and the truncated inactive form of TACE (TACE-DZn) inice. Lysates were immunoprecipitated with the anti-TACE antibody,

he bottom panel shows the effect of PMA on the release of soluble85HER2 tyrosine phosphorylation in MCF7 cells. (B) Stable transfectants

indicated, and proNRGa2c and the cell-associated fragments wereB, but using wild-type TACE1/1-transfected cells instead of the

s Erk1/2. Where indicated, TACE-DZn/DZn or 293-a2c cells were treatede) or the phosphorylated and active forms of these kinases (anti-p-Erk

pressDZn) mel). Ty p1hereas ininaseprob

l

RIsa(


This pathway has also been shown to control the cleav-age of proNRG (Burgess et al., 1995; Loeb et al., 1998)suggesting that processing of proNRG could be con-trolled by mechanisms analogous to those operating forother membrane molecules that undergo regulatedshedding. Interesting was the heterogeneous nature ofthe cell-bound fragments generated upon PKC-medi-ated proNRG cleavage. Activation of PKC provoked thegeneration of at least four different cell-associated frag-ments. Analogous multiple cleavages also occur inother membrane proteins that undergo regulated cleav-age of their ectodomains, such as the bAPP (Zhong etal., 1994) or notch (Brou et al., 2000). Different mecha-nisms may be responsible for the generation of multiplecell-associated fragments of these transmembrane pro-teins. bAPP may be cleaved at its ectodomain at severalsites generating multiple cell-associated fragments(Zhong et al., 1994). In addition, cleavage of the endodo-main of this molecule also generates truncated frag-ments that lack the transmembrane anchor and are thussoluble in the cytosol (Gervais et al., 1999). In the case ofnotch, cleavage at the ectodomain precedes a secondarycleavage that occurs at the endodomain (Brou et al.,2000). Apparently, all the different truncated fragmentsderived from proNRG occurred by cleavage outside theendodomain, i.e., at the ectodomain or within the mem-brane. This was supported by the data with intracellu-larly HA-tagged proNRGa2c, as well as by cell fraction-ation experiments. The latter demonstrated that all ofthe cell-bound fragments derived from proNRG weremembrane-associated, supporting that the heterogene-ity in the fragments generated probably resulted fromcleavage of the proNRG precursors at different siteswithin their ectodomains or in a transmembrane regionthat would preserve at least part of their membraneanchor. Interestingly, the tail fragments generated uponproNRG cleavage span over a range of '7 kDa. Theinker region in proNRGa contains 26 amino acids,

corresponding to '3 kDa. Apparently, therefore, cleav-age at different sites within the linker may not com-pletely explain the ample range of fragments generated.Possibilities such as cleavage at regions outside thelinker (in the EGF-like or the transmembrane domain)or posttranslational modifications may explain this ap-parent discrepancy. It is highly unlikely that posttrans-lational modifications such as glycosylation or phos-phorylation may account for the different mobility ofthe fragments detected. In fact, the linker region doesnot contain any common consensus glycosylation site(Wen et al., 1994); and in vitro phosphatase treatment ofthe fragments did not substantially affect their mobility.In addition, in the presence of agents that preventproNRG cleavage, such as BB3103, we failed to detect

heterogeneity in the precursor forms upon PMA treat-ment. However, other modifications such as acylation(that occurs in the cytoplasmic tail of the related EGFfamily growth factor proTGFa (Bringman et al., 1987))or intracellular proteolysis of C-terminal residues (asreported in proARIA (Han and Fischbach, 1999)) maycontribute to the heterogeneous nature of the cell-bound fragments. It should be pointed out that whilethe 311 antiserum recognizes a C-terminal epitope ofproNRGa2c, deletion by proteolysis of a short sequenceat the C-terminus of proNRGa2c could result in a trun-cated form still recognized by antibodies present in theantiserum that interact with an epitope located at theN-terminal end of the peptide sequence used in thegeneration of the antiserum.

Alternative splicing of the mRNA coding for se-quences where proNRG cleavage occurs gives rise todifferent proNRG isoforms (Fischbach and Rosen,1997). The comparative analysis of the shedding ofsome of these isoforms from their respective precur-sors indicated differences between both resting andregulated release. Alternative splicing of the mRNAin analogous regions of other membrane-anchoredgrowth factors has been reported (Flanagan et al.,1991; Hill and Sternberg, 1992; Huang et al., 1992;

ettenmier and Roussel, 1988; Rettenmier et al., 1987).n the stem cell factor the region affected by theplicing event contains the major cleavage site; andn isoform lacking this region is released inefficientlyFlanagan et al., 1991; Huang et al., 1992). In the case

of the precursors for the colony-stimulating factor-1,cells expressing the short isoform release the factor tothe medium, but very slowly compared to cells ex-pressing the large isoform (Rettenmier and Roussel,1988; Rettenmier et al., 1987). It has also been pro-posed that the length of the linker region may beimportant in proARIA cleavage, independently of theprimary sequence (Han and Fischbach, 1999). Thelatter hypothesis cannot, however, fully explain thedata we obtained by the deletion analysis of thelinker region of proNRGa2c. The length of the linkerregion in proARIA (from the sixth cysteine residue ofthe EGF-like module to the transmembrane domain)is of 26 amino acids, i.e., 5 amino acids longer thanproNRGa2c. Deletion of up to 9 amino acid residuesin proARIA did not affect the efficiency of release ofsoluble ARIA to the culture medium of COS cellstransfected with the mutant precursor (Han and Fisch-bach, 1999). However, deletion of 11 amino acidsdecreased proARIA solubilization, indicating that aminimum length of 16 amino acids was required forefficient processing of proARIA (Han and Fischbach,1999). The data we obtained by the deletion analysis

mmlsivlt

s

sn

644 Montero et al.

of the proNRGa2c linker differ from those obtainedin proARIA. Thus, all of our deletion mutants, in-cluding the MKV deletion mutant, were strongly re-sistant to PMA-induced proNRG cleavage. Not onlyregulated cleavage was prevented by these trunca-tions, but also soluble NRG failed to accumulate inthe culture medium of the mutants, as indicated bythe failure of conditioned medium to stimulatep185HER2 tyrosine phosphorylation. The resultinglength of the linker region in the MKV deletion mu-tant of proNRGa2c is 18 amino acids, i.e., 2 aminoacids larger than the proARIA mutant that efficientlyreleased ARIA (Han and Fischbach, 1999). Our con-clusion is thus that the length of the linker region isnot the sole factor that restricts proNRG cleavage. Insupport of the latter is also the fact that the naturallyoccurring proNRGb2a, whose length of the linkerregion is identical to the truncated proNRGa2c-MKV

utant, is released in both a resting and a regulatedanner. Thus other determinants located in the

inker may be important for proNRG cleavage. Ithould be pointed out, however, that different exper-mental conditions (analysis of resting ARIA releaseersus PMA-stimulated cleavage of proNRG) or cel-

ular models used may explain the differences be-ween our results and those reported using proARIA.

Previous studies with other EGF-like growth factorsuch as proTGFa, HB-EGF, and other membrane-bound

proteins have indicated that metalloproteases highlysensitive to hydroxamic acid-derived compounds areresponsible for the resting and regulated cleavage ofthese proteins (Black and White, 1998; Mullberg et al.,1997). In concordance with this was the finding that onesuch compound, BB3103, inhibited both resting andPMA-induced shedding of proNRG. Thus, even thoughthe target substrates are quite different, the signalingpathways and the final components of the cleavagemachinery appear to share certain properties, such asactivation mechanisms or protease family implicated inthe cleavage. By different types of approaches, pro-teases of the ADAM family have been identified asstrong candidates for the control of the regulated cleav-age of membrane proteins (Black et al., 1997; Izumi et al.,1998; Moss et al., 1997). One of them, TACE, initiallyisolated as a proTNFa processing enzyme, has alsobeen shown to participate in the release of the ectodo-mains of several transmembrane proteins (Peschon etal., 1998). It is possible that TACE may also have arole in proNRG processing. Thus, transfection ofproNRGa2c in cells derived from animals expressing aninactive form of TACE resulted in the correct ex-pression of the proNRGa2c precursor. However,proNRGa2c was completely resistant to PMA-induced

cleavage, even though PMA-induced Erk1/2 activationremained intact, suggesting that the PKC pathway isfunctional in these cells. In contrast, expression ofproNRGa2c in cells derived from wild-type animalsresulted in cleavage and generation of the differentcell-bound truncated fragments analogous to thoseobtained in CHO or 293 cells transfected withproNRGa2c. Thus TACE may be an essential compo-nent of the machinery that cleaves proNRGs. WhetherTACE itself is directly responsible for the cleavage ofproNRGs or acts as a component of a more complexprotease cascade whose activation results in cleavage ofproNRGs at multiple sites is still unknown.

Clues for an important role of membrane-anchoredgrowth factor processing in animal physiology havebeen obtained by studies of fly (Perrimon and Per-kins, 1997) and mouse development (Luetteke et al.,1993). In fly early developmental stages the EGFRappear to control the polarization of the embryo byinteraction with several EGF-like ligands (Howes etal., 1998; Wasserman and Freeman, 1998). One ofthese ligands, spitz, is produced as a transmembraneprotein but is only active after proteolytic processingto produce the secreted protein (Golembo et al., 1996).Another indication of a role of membrane proteinectodomain cleavage in animal development is sug-gested by data obtained from the analysis of animalswith targeted mutations of the TACE locus that resultin the expression of an inactive form of TACE(Peschon et al., 1998). These animals die in utero orhortly after birth. Interestingly, some of their phe-otypic defects resemble those reported in TGFa-

deficient mice, indicating that cleavage of this mem-brane growth factor may be as important for animaldevelopment as its biosynthesis. As mentionedabove, indirect evidence also suggests that cleavageof NRGs may be essential for animal development.Final verification of the importance of NRG process-ing in development will require the elaboration ofanimal models in which proNRG processing is pre-vented. Our studies on the molecular determinantsthat play a role in the regulation of the cleavage maythus help in designing proNRG molecules that, beingbiologically active as transmembrane precursors, failto efficiently release soluble NRG.

EXPERIMENTAL PROCEDURES

Reagents

Cell culture media, sera, and G418 were purchasedfrom GIBCO BRL (Gaithersburg, MD). HRP conjugates

TaEa4trL

C

a

Shri

C

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B


of protein A were from Calbiochem Behring Corp. (SanDiego, CA) and protein A–Sepharose was from Phar-macia Fine Chemicals (Piscataway, NJ). Immobilon-Pmembranes were from Millipore Corp. (Bedford, MA).Luminol, 4-p-iodophenol, PMA, calf intestinal phospha-tase, 1,10-phenanthroline, and EDTA were purchasedfrom Sigma Chemical Co.; BIM was from Calbiochem.The inhibitor compound BB3103 was generously pro-vided by British Biotech. Other generic chemicals werepurchased from Sigma Chemical Co., Roche Biochemi-cals, or Merck.

The monoclonal anti-HA antibody was fromBABCO. The mouse monoclonal antibody 7D5, thatrecognizes a glycopeptide of the extracellular domainof rat NRG located outside the EGF-like domain(Wang et al., 1998), was obtained from Neomarkers.

he mouse monoclonal antiphosphotyrosine andnti-phospho-Erk antibodies and the polyclonal anti-rk antibody were from Santa Cruz Biotechnology,nd the monoclonal anti-HER2 ectodomain antibodyD5 was provided by Dr. M. X. Sliwkowski (Genen-ech, San Francisco, CA). HRP conjugates of anti-abbit IgG and anti-mouse IgG were from Bio-Radaboratories (Cambridge, MA).

ell Culture

All cell lines were cultured at 37°C in a humidifiedtmosphere in the presence of 5% CO2, 95% air. Cells

were grown in DMEM containing high glucose (4500mg/liter) and antibiotics (penicillin 100 U/ml, strepto-mycin 100 mg/ml) and supplemented with 5% (293,CHO, HeLa, and the fibroblast cell lines derived fromTACEDZn/DZn and TACE1/1 mice) or 10% (MCF7, BT474,SKBR3, and SKNBE) fetal bovine serum (FBS). The cellline NP18 was grown in RPMI medium containing an-tibiotics and 10% FBS.

Generation of Antibodies

The anti-endodomain antiserum 311 was raisedagainst the sequence NH2-CETPDSYRSDPHSER-COOH, that corresponds to the 15 COOH- terminalresidues of rat NRGa2c. The polyclonal anti-HA anti-serum was obtained from rabbits that were immunizedwith the peptide: NH2-CYPYDVPDYAG-COOH. Theanti-TACE antiserum was generated by immunizationof rabbits with the peptide NH2-CKLQRQNRVD-

KETE-COOH, located in the COOH-terminal end ofuman TACE. All the antipeptide antibodies were pu-ified from the antisera by affinity chromatography us-ng peptide–Sepharose columns.
p
onstruction of Mutants and Transfections

The different isoforms of rat NRGs (a2a, a2c, b2a,b4a) used in this study were subcloned into HindIII–XbaI sites of the pCDNA3 vector (Invitrogen) and trans-fected into 293 or CHO cells by the calcium phosphatetechnique or in TACEDZn/DZn or TACE1/1 fibroblasts bylipofection. All the isoforms were mutagenized to in-troduce HA epitopes between the Ig-like and the EGF-like domains by creating EcoRI sites. To facilitate iden-tification by the anti-HA antibodies the HA-taggedversions of the proNRG isoforms included three in-frame contiguous copies (a total of 36 amino acids) ofsingle (12 amino acids) HA tags.

The different deletions in the juxtamembrane regionof proNRGa2c were generated by site-directed mu-tagenesis (MKV mutant) or PCR (rest of mutants). Mu-tants of proNRGa2c were verified by automated se-quencing.

Immunoprecipitation and Western Blotting

Cells were washed with PBS and lysed in ice-coldlysis buffer (140 mM NaCl; 10 mM EDTA; 10% glycerol;1% Nonidet P-40; 20 mM Tris, pH 8.0; pepstatin, 1 mM;aprotinin, 1 mg/ml; leupeptin, 1 mg/ml; PMSF, 1 mM;and 1 mM sodium orthovanadate). After scraping thecells from the dishes, samples were centrifuged at10,000g at 4°C for 10 min and supernatants were trans-erred to new tubes with the corresponding antibodynd protein A–Sepharose. Immunoprecipitations wereerformed at 4°C for at least 2 h, and the immuneomplexes were recovered by a short centrifugationollowed by three washes with 1 ml of cold lysis buffer.amples were then boiled in electrophoresis sampleuffer and loaded in SDS–PAGE gels. After transfer toVDF membranes, filters were blocked for 1 h in TBSTnd then incubated for 2–16 h with the correspondingntibody. After washing with TBST, filters were incu-ated with HRP-conjugated secondary antibodies for 30in, and bands were visualized by a luminol-based

etection system with p-iodophenol enhancement (Ca-rera et al., 1996).Immunoprecipitation of soluble NRG from the me-

ium was done by recovering culture supernatants fol-owed by addition of the anti-HA antibody. Incubation

ith the antibody, washes of the immunoprecipitates,nd Western blotting with the corresponding antibod-es was performed as above.

ioactivity Assay

Conditioned medium from cells transfected withroNRGs was used to stimulate MCF7 cells for 10 min.

Gd

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646 Montero et al.

The cellular MCF7 lysates were then incubated with 2mg of the 4D5 anti-HER2 antibody and proteinA–Sepharose for 2 h. The immune complexes werewashed in lysis buffer, boiled in electrophoresis samplebuffer, and resolved in 6% SDS–PAGE gels. The filterswere then incubated with anti-phosphotyrosine anti-bodies, and the bands were visualized by a luminolsystem as above. In the experiments in which the con-ditioned medium was prepared in the presence ofPMA, and to avoid the effects of the latter on theautophosphorylation properties of ErbB receptors (Pan-diella et al., 1989), preincubation of MCF7 cells with thePKC inhibitor BIM (10 mM) was carried out for 30 minbefore addition of the conditioned medium. In theseexperiments, conditioned medium from four 100-mmpetri dishes was concentrated to 500 ml with a Centricon10 device, and the concentrated medium was incubatedwith monolayers of MCF7 cells for 10 min. Cells werelysed and the phosphorylation of HER2 was analyzedas above.

Cell Fractionation

293-a2c cells from 100-mm dishes (90% confluent)were incubated with or without 1 mM PMA for 20 min,and then EDTA was added to a final concentration of 5mM. Cells were detached from the dish by pipetting,harvested, and washed by centrifugation at 3000 rpmfor 5 min at 4°C. The supernatant was eliminated andthe cellular pellet was resuspended in 10 ml of a buffercontaining 0.25 M sucrose, 10 mM EDTA, 10 mM Tris,pH 7.0, and 1 mM PMSF. After centrifugation at 3000rpm for 5 min at 4°C, the pellet was resuspended in 5 mlof buffer and manually disrupted using a glass Douncehomogenizer. Major cells debris was eliminated by cen-trifugation at 3000 rpm for 5 min at 4°C, and the super-natants were subjected to ultracentrifugation at 30,000rpm for 30 min at 4°C. The supernatant with the cyto-solic fraction was made 1% NP40, and the pellet withthe microsomal fraction was lysed in lysis buffer con-taining phosphatase (1 mM sodium orthovanadate) andprotease (1 mM pepstatin, 1 mg/ml aprotinin, 1 mg/mlleupeptin, and1 mM PMSF) inhibitors. Both fractionswere immunoprecipitated with the 311 antibody andproteins identified by Western blotting with the sameantibody.

RT-PCR Assay

Human cell lines 293, BT474, HeLa, MCF7, NP18,SKBR3, and SKNBE were analyzed for expression ofproNRG isoforms by RT-PCR. Two specific primerswere used to amplify the cDNAs (see Fig. 3B):

NRG421–435, based on the sequence 59-GCCTCAACT-AAGG-39 that codes for amino acids of the extracellularomain of human NRG; and NRG1068–1087, based on the

sequence 59-TGCTTTCAGTGTGTCCGTTG-39 that codesfor amino acids of the cytoplasmic tail of human NRG.

Protease Protection Experiments

For protease protection experiments with proteinaseK, transfected 293 cells were washed once with KRHbuffer (that contained, in mmol/liter: NaCl, 140; KCl, 5;CaCl2, 2; MgSO4, 1.2; KH2PO4, 1.2; glucose, 6; Hepes, 25,pH 7.4) and incubated in this buffer supplemented with200 mg/ml proteinase K for 90 min. After washing threetimes with PBS containing 2 mM PMSF, cells were lysedin 1 ml of immunoprecipitation buffer and analyzed fora2c fragment generation by using the 311 antiserum.

Confocal Immunofluorescence Microscopy

Cells plated on glass coverslips were washed withPBS and fixed in 2% p-formaldehyde for 30 min at roomtemperature. Monolayers were washed twice in PBST(PBS supplemented with 0,1% Triton X-100 final) andthen blocked in PBST with 5% BSA for 1 h at roomtemperature. Monolayers were then incubated with theprimary antibody in blocking solution for 2 h at roomtemperature or overnight at 4°C. After two washes of 15min each in PBST, the coverslips were incubated withcyanine-2- or cyanine-3-conjugated secondary antibod-ies for 30 min, washed 3 3 5 min each in PBST, andmounted. Samples were analyzed by confocal immuno-fluorescence microscopy using a Zeiss LSM 510 confo-cal microscope.

ACKNOWLEDGMENTS

We thank Drs. Jacques Peschon, Roy. A. Black, Mark X. Sliwkowski,and Duanzhi Wen for their generous gift of reagents. The supply ofBB3103 by British Biotech is also acknowledged. Finally, we thank thefunding from the European Community, the Fundacion Ramon

reces, and the Spanish Ministry of Education and Culture. A.E-O.as supported by a postdoctoral contract from the Spanish Ministry

f Education and Culture. E.D-R. and L.Y. were supported by aredoctoral fellowship from the same Ministry.

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Received April 19, 2000Revised June 29, 2000

Accepted July 31, 2000

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Differential Shedding of Transmembrane Neuregulin Isoforms by the Tumor Necrosis Factor-α-Converting Enzyme