Vol. 2, 351-357, July 1991 Cell Growth & Differentiation 351

Cyr6l, Product of a Growth Factor-inducible ImmediateEarly Gene, Is Associated with the ExtracellularMatrix and the Cell Surface’

George P. Yang and Lester F. Lau2

Department of Genetics, University of Illinois College of Medicine,Chicago, Illinois 60612

Abstrad

cyr6 1 is a specific target for adivation by platelet-derived growth fador and fibroblast growth factor andis inducible by the oncogene v-src. It is a member ofthe class of immediate early genes that includes thoseencoding protooncogene produds, transcriptionfadors, and cytokines. We have previouslycharaderized the synthesis and degradation of thecyr6l-encoded mRNA and protein. Although thededuced Cyr6l protein sequence contains an NH2-terminal secretory signal, it is not detedable in theconditioned medium of serum-stimulated cells. Weshow here that in rapidly growing cell cultures, newlysynthesized Cyr6l is secreted and is associated withboth the extracellular matrix and the cell surface. Incontrast, Cyr6l secreted in serum-stimulated quiescentcells is direded to the cell surface and is notincorporated into the extracellular matrix. Onceassociated with the extracellular matrix, Cyr6l has ahalf-life of greater than 24 h, whereas intracellular andcell surface-associated CyrGl has an apparent half-lifeof approximately 30 mm. Furthermore, Cyr6l appearsto bind heparin with high affinity. These observationssuggest similarities among Cyr6l, the fibroblast growthfadors (heparin-binding growth fadors), and theprotooncogene produd lnt-1 and are consistent withthe hypothesis that Cyr6l plays a role in cell-cellcommunication involving the interaction ofneighboring cells.

Introdudion

The proliferation of mammalian cells is controlled bypolypeptide growth factors. In mouse fibroblasts, serumgrowth factors rapidly activate the expression of a set ofcellular immediate early genes without requiring de novoprotein synthesis (1-3). Among these genes are thosethought to encode proteins that regulate the subsequentcellular responses to the growth factors. Consistent withthis hypothesis, a number of immediate early genes havebeen found to encode known or putative transcriptionfactors, including members of the Fos and Jun families

Received 2/25/91.

1 This work was supported by a grant from the National Institutes of

Health (ROl CA46565). L. F. L. is the recipient of an American CancerSociety Junior Faculty Research Award and is a Pew Scholar in theBiomedical Sciences.2 To whom requests for reprints should be addressed, at Department ofGenetics, University of Illinois College of Medicine, 808 South WoodStreet, Chicago, Illinois 60612.

(1), Myc (4), Rel (5), SRF (6), and at least four zinc fingerproteins (1, 2, 7). In addition, some murine immediateearly genes encode secretory proteins related to cyto-kines, including KC/gro, whose human homologue is amelanoma growth factor (8), and JE, whose human horn-ologue is a monocyte chemoattractant (9).

cyr6l was originally identified as a growth factor-in-ducible immediate early gene in mouse BALB/c 3T3fibroblasts (10). It is transcriptionally activated withinminutes of stimulation by serum or purified platelet-derived growth factor. Its mRNA level peaks by 60 to 90mm after stimulation and decays thereafter. The decreasein cyr6l mRNA levels is due to two effects: transcriptionalrepression and rapid mRNA degradation (ii, 12). cyr6l

is expressed during the first hour of liver regenerationfollowing partial hepatectomy (1 3), suggesting that it mayplay a role during the G0-G1 transition in the living animal.Its chicken homologue, CU-JO, is inducible by the viraloncogene v-src (14). The induction of cyr6l by such avariety of mitogenic stimuli in various cell types supportsthe contention that its expression may be an importantcomponent in the genetic program for growth.

To understand the role of cyr6l in cell growth regula-tion, we have studied its gene product. cyr6l encodes a379-amino acid protein containing 38 cysteine residueswith no sequence homology to any protein of knownfunction. The deduced sequence of Cyr6i contains aputative NH2-terminal signal peptide, suggesting that it isa secreted protein. Synthesis of Cyr6i in vitro in a rabbitreticulocyte lysate in the presence of microsomesshowed evidence of processing, consistent with secre-tion (12). Nevertheless, we were unable to detect it inthe conditioned medium of serum-stimulated BALB/c3T3 cells by immunoprecipitation (12). We show herethat Cyr6i is secreted and that the secreted protein isassociated with the ECM3 and the cell surface. Further-more, it has an apparent affinity for heparin in vitro.These data point to similarities among Cyr6i, the pro-tooncogene product lnt-i (Wnt-i), and the FGF familyof proteins. We hypothesize that Cyr6i may function incell-cell communication among neighboring cells.

Results

Cyr6l Is Associated with the Extracellular Matrix. Al-though the Cyr6i protein contains an NH2-terminal se-cretory signal, it is not accumulated in the culture me-dium (12). One interpretation of this is that Cyr6i issecreted but is sequestered by the ECM. To test thispossibility, we separated BALB/c 3T3 cell cultures intocellular, medium, and ECM fractions for analysis. The

3 The abbreviations used are: ECM, extracellular matrix; FGF, fibroblastgrowth factor; PBS, phosphate-buffered saline; kD, kilodalton(s); MEM,

minimal essential medium; FCS, fetal calf serum.

I 234 5 6 #{149}78

A

130.75

50

�

39

27

Competitor : TrpE- Cyr6l TrpE

B

Cell ECM‘I 23 4’

fig. 1. Cyr6l in the ECM. A, imniunoblot of Cyr6l. Quiescent (Lanes 1

and 3) and serum-stimulated (Lanes 2 and 4-8) confluent cell cultureswere fractionated into cellular (Lanes 1, 2, 5, and 7) and ECM (Lanes 3,4. 6, and 8) fractions. Immunoblot was probed with affinity-purified anti-

Cyr6l antibodies either without competitor or with 10 �g of TrpE-Cyr6lfusion protein (Lanes 5 and 6) or TrpE protein (Lanes 7 and 8) ascompetitor. B, trypsin sensitivity. Cellular fractions (Lanes 1 and 2) fromserum-stimulated subconfluent cells were collected by lysis in RIPA buffer

(Lanes I and 3) or by trypsinization (Lanes 2 and 4), and the ECM fractionswere collected as described. Each lane represents the cellular or ECMfraction from 9 X 10� cells. In A and B, molecular weights of marker

proteins are given at the left; arrows point to the Cyr6l band.

352 (�r6 1 Is Asso iate’d with ECM ai�1 (elI Surface

medium fraction was collected directly, and cells on thedish were washed with PBS. The cellular fraction wascollected by lysis with RIPA buffer at 4CC; this fraction

includes all intracellular components, cell membranes,and cell surface-associated proteins not removed by thePBS wash. After removal of the cellular fraction, the ECMwas collected by incubation in RIPA buffer at 85CC for 1h followed by scraping of the dish as described (15).

The cellular and ECM fractions were first examined byirnrnunoblotting, using as a probe affinity-purified anti-

Cyr6l antibodies raised against TrpE-Cyr6l fusion pro-tein. We detected a band in the ECM fraction of M,41,000, as expected for Cyr6l (Fig. 1A). Detection of this

protein was specifically blocked by incubation of theirnrnunoblot with the TrpE-Cyr6i fusion protein, but notwith the TrpE protein (Fig. 1A, Lanes 5-8). As expected,Cyr6l was not detectable in the cellular fraction fromquiescent cells but was detected in cells stimulated withserum for 1 h. Somewhat unexpectedly, Cyr6l was alsodetected in the ECM of quiescent cells (Fig. 1A, Lane 3).This result argues against the possibility that the presenceof Cyr6l in the ECM is due to leakage out of lysing cells,since there is clearly no cellular source for the protein inthis instance.

To confirm the extracellular location of Cyr6i, we

tested its susceptibility to trypsin digestion without priorlysis of the cells. BALB/c 3T3 cells were serum stimulatedfor 1 h and metabolically labeled for another 1 5 mm. Thecellular fraction was collected either by trypsinization orby lysis with RIPA buffer, followed by collection of theECM. Cyr6l in the ECM fraction was almost completelydegraded by trypsinization but not by RIPA lysis, whereasin the cellular fraction, it was protected in both isolationprocedures (Fig. 1B).

To test whether newly synthesized Cyr6l was incor-porated into the ECM, we carried out pulse-chase ex-periments. Confluent, quiescent BALB/c 3T3 cells wereserum stimulated and pulse labeled, as described above,

and further incubated in unlabeled medium for variousdurations. In these confluent cells, newly synthesizedCyr6l was found only in the cellular fraction. Althoughmost of the protein in the cellular fraction had decayedor was undetectable within 1 h, no Cyr6l was found ineither the ECM or the medium (Fig. 2A). However, sincewe have shown by imrnunoblotting that it is present in

the ECM in confluent quiescent cells (Fig. 1A), we rea-soned that it must be incorporated into the ECM as the

cells grow to confluence. This interpretation is consistentwith the observation that cyr6l is constitutively ex-pressed in all phases of the cell cycle in logarithmicallygrowing cells (12). We therefore examined the fate of

Cyr6l in subconfluent, rapidly growing BALB/c 3T3 cellsusing the same pulse-chase protocol (Fig. 2B). In this

case, the level of Cyr6i decreased in the cellular fractionas it increased in the ECM fraction, indicating that newlysynthesized Cyr6i was incorporated into the ECM.

These results show that subconfluent, logarithmicallygrowing 3T3 cells secrete Cyr6i , and the secreted proteinis accumulated in the ECM. Once sequestered in the

ECM, Cyr6l appears to be stable and is thus detectablein the ECM of confluent quiescent cells even thoughthese cells do not synthesize it. To test this interpretation,

we determined the half-life of Cyr6l in the ECM directly,

using pulse-chase labeling, and found it to be greater

. -

98

68 .-.

29

than 24 h. In contrast, Cyr6l in the cellular fraction has

a half-life of approximately 30 mm (Ref. 1 2; Fig. 3).

Cyr6l Has a High Affinity for Heparin. Heparan sul-

fate is attached to the core proteins of proteoglycans

found in the ECM of many cell types. A number of growth

factors and protooncogene products are thought to as-

sociate with the ECM due to their affinity for heparan

sulfate or related glycosaminoglycans (15, 16). We there-

fore tested whether Cyr6l would bind to heparin in vitro.

Cell Growth ,5 I)ifterentiation 353

4 G. P. Yang and I.. F. Lao, unpublished data.

ACell ECM

I 1 I -U

01234 QI234

Medium

01234

B

Cell ECM MediumI 1 I U I

1234 234 234

68

43

29

a a

68 C

43 !!!.-“ �

29

#{163} #{163}

Fig. 2. Incorporation of newly synthesized Cyr6l into the ECM. A, confluent BALB/c 3T3 cell cultures fractionated into (ellular, [CM, or mediumfractions. Cells were quiescent (Q) or serum stimulated for 1 h prior to pulse labeling followed by � hase in unlabeled medium for 0 (L,ines I (, 1 5 (Lanes

2), 30 (Lanes :1), or 60 (Lanes 4) mm. Each lane represents immunoprecipitates from 9 x 1O� cells. B, identical experiment to that des rilwd in .-\, exceptthat subconfluent cell cultures were used. In A and B, molecular weights of marker proteins are given at the left.

Total cell lysate from metabolically labeled, serum-stirn-

ulated 3T3 cells was incubated with heparin-agarosebeads. Proteins bound to the beads were eluted withincreasing salt concentrations in RIPA buffer and irnrnu-noprecipitated with anti-Cyr6i antibodies. Cyr6i was

eluted in the range of 0.6 to 1.0 M NaCI, with peakelution at 0.8 M NaCI (Fig. 4). This result suggests thatCyr6i binds heparin with high affinity, although we can-not rule out the possibility that it may bind to heparin

via interactions with another molecule.Cyr6l Is Secreted in Both Confluent and Subconfluent

Cells. The apparent affinity ofCyr6l for heparin providedus with an assay for its secretion. The addition of soluble

heparin to the culture medium could act to adsorb se-

Ce II

hrs 0 I 3 6 1218

98

68

43

29

18

ECM

24 0 I 3 6 121824

t

fig. 3. Stability of Cyr6l in the [CM. 3T3 cell cultures were serum

stimulated for 1 h, pulse labeled for 15 mm, and chased in unlabeledmedium; lane numbers indicate the chase period in hours. Cultures wereseparated into cellular and ECM fractions; each lane represents immu-

noprecipitates from 4 x i0� cells. Molecular weights of marker proteins

are given at the left.

creted Cyr6i, making it detectable in the culture me-diurn. Since we had shown that Cyr6l synthesized byserum-stimulated confluent cells was found in neither

the ECM nor the conditioned medium, we used thisheparin-binding assay to determine whether Cyr6i pro-duced by these cells was secreted or remained intracel-lular. Confluent cells were stimulated with serum for 1 hand pulse labeled for 15 mm, and then chased in unla-beled medium with added soluble heparin. Under theseconditions, Cyr6i was chased from the cell into theconditioned medium within 1 h, indicating that it wassecreted and was bound by the soluble heparin (Fig. 5A).The same result was observed in subconfluent cells, but

Cyr6l was not incorporated into the ECM as seen pre-viously (compare Fig. 2B and Fig. 5B). The simplest

interpretation of this result is that in the subconfluentcells, secreted Cyr6i may be quantitatively adsorbed bythe soluble heparin, making it unavailable for associationwith the ECM.

These observations indicate that Cyr6l is secreted inboth confluent and subconfluent cells and is thereforefound in the cell medium in the presence of solubleheparin. One explanation for the absence of Cyr6i in

cell medium without added heparin is that secretedCyr6i not bound to the ECM is susceptible to serumproteases. To test this possibility, we partially purifiedCyr6l by affinity chromatography using heparin-agarosebeads (0.6 to 1.0 M NaCl elution) and incubated it inconditioned medium at 37#{176}Cfor 30 mm in the presenceor absence of heparin. No proteolytic activity that de-graded Cyr6l was detected in either case:i

As judged by the heparin-binding assay, a substantialfraction of Cyr6l was secreted in both confluent and

subconfluent cells, whereas a small fraction was incor-porated into the ECM in subconfluent cells. The lack ofdetectable Cyr6l in the medium without added heparin

98

68 �

43

-�

29 �

NaCI 0.15 0.4 0.6 0.8 1.0 1.2 1.5 2.0 M

fig. 4. Heparin binding. Labeled IT I cell lysates we’re incubated withheparin-agarose beads followed by (‘lotion at the indi ated NaCI concen-Irations. Arrow, Cyr6 1 band. Mole’ ular weight markers are shown at left.

Cell ECM Medium Cell ECM MediumI 1 1

01234�i

01234I

01234�-i I

1234I I

1234I U

1234

354 Cyr6l Is Asso jated with ECM and Cell Surface

I 2345678

and the disparity between the amount of secreted Cyr6i

detected by the heparin-binding assay and the amount

seen in the ECM (compare Figs. 28 and 5B) suggest that

a portion of secreted Cyr6l might be bound to a structureother than the ECM, most likely the cell surface.

Cyr6l Is Associated with the Cell Surface. To testdirectly whether secreted Cyr6l is associated with the

cell surface, we used a lactoperoxidase-mediated ra-

dioiodination procedure to label cell surface proteins(i7). Monolayers of intact BALB/c 3T3 cells were radioi-

odinated, and the cellular fraction containing the cell

membrane but not the ECM was harvested. Cell lysateswere imrnunoprecipitated with antibodies specific for

Cyr6i and Nur77. Nur77 is a 68 kD protein encoded byan immediate early gene which is detected by [US]-methionine metabolic labeling under the same condi-

tions as Cyr6l and is localized to the cytoplasm andnucleus (18). Labeled Cyr6l was detected in serum-stimulated confluent and subconfluent cells, but not inunstimulated quiescent cells (Fig. 6). Nur77 was notlabeled under any of these conditions, indicating that nointracellular proteins were labeled. These results showthat Cyr6l is associated with the cell surface.

Discussion

The induction of cell proliferation by growth factors isthought to involve the activation of a complex geneticprogram (1). Immediate early genes are the first to beactivated, and in many instances, they encode regulatorymolecules that appear to control the subsequent cellularresponses to growth factors. Recent characterization ofimmediate early genes has already yielded a wealth ofnew information on potential regulators of cell growth.The immediate early gene Cyr6l is a specific target forinduction by platelet-derived growth factor, FGF, phor-bol esters, and the oncogene v-src, and it is also activatedduring liver regeneration (1 1-14). To understand its func-tion and role in the mitogenic response, we have exam-ned the biochemical properties of the cyr6l-encodedprotein.

Cyr6l is a secreted protein but is not found in theculture medium. In rapidly growing cells, newly synthe-sized Cyr6i is secreted and is associated with both theECM and the cell surface. ECM-associated Cyr6l is sta-ble, and thus it is found in both growing and quiescentcell cultures. In serum-stimulated confluent cells, newly

synthesized Cyr6l is preferentially associated with thecell surface. The simplest explanation for the differentsecretory targets is that, in quiescent confluent cells, mostbinding sites in the ECM are occupied, and new matrixformation is limited. Alternatively, in confluent cells,there may be more binding sites or sites of higher affinityon the cell surface. Another possibility is that Cyr6lsynthesized under varying growth conditions is differen-tially modified, and the modifications may target the

A

98

68

43 1

B

98

68

43 0

29 * 29

fig. 5. Detection of Cyr6l in culture medium in the presence of added soluble heparin. A. confluent quiescent (Q) 3T3 cell cultures were serum

stimulated, labeled, and frationated into cellular, ECM, and media fractions as in Fig. 2, except for the addition of 50 big/mI of soluble heparin in themedium. Cells were pulse labeled and chased for F) (Lanes 1), 15 (Lanes 2), 30 (Lanes fl, or 60 (Lanes 4) mm. Each lane represents immunoprecipitates

from 6 X 1O� cells. B, identical experiment to that described in A, except that subconfluent cell cultures were’ used. Molecular weights of marker proteinsare given at left.

Cyr6l Nur77U �1 I

QCSQCS

68

43

29

,-..�

,�s,t4.t ,

,-,

Fig. 6. Detection at the cell surface. Quiescent confluent (Q), serum-

stimulated confluent (C), and serum-stimulated subconfluent (5) cellcultures were washed with PBS, and extracellular proteins were labeled

by enzymatic radioiodination. Cell lysates were collected and immuno-precipitated with affinity-purified anti-Cyr6l or anti-Nur77 antibody. Each

lane represents immunoprecipitates from 4 X 10� cells or 1 x iO� cells in

confluent and subconfluent cell cultures, respectively. Arrow, Cyr6l

band. Molecular weights of marker proteins are given at left.

Cell Growth & Differentiation 355

protein to different structures. The association of Cyr6lwith the matrix and the cell surface suggests that itsaction is likely to be localized rather than systemic.

The half-life of Cyr6l in the cellular fraction is approx-imately 30 mm. Since this fraction includes cell surface-associated proteins, we infer that the cell surface-asso-ciated Cyr6i must also have a short half-life. Consistentwith this interpretation, Cyr6l was not detected on thecell surface of quiescent cells by radioiodination. In con-trast, [CM-associated Cyr6i has a half-life of greater than24 h and is detected in the [CM of quiescent cells. Theseobservations clearly indicate that extracellular Cyr6i iscompartmentalized into an unstable and a stable corn-ponent. Our fractionation data indicate that the stablefraction is ECM associated and the unstable fraction iscell surface associated. One likely interpretation is thatthere is rapid reuptake and degradation of Cyr6l by thecell, as evidenced by the short half-life of the cellularfraction that includes the secreted, cell surface-associ-ated Cyr6l.

Cyr6i appears to bind heparin with high affinity, sug-gesting that it may bind to the ECM via association withheparan sulfate or related glycosaminoglycans found inthe [CM. The high NaCI concentration (0.8 M) requiredto dissociate Cyr6i from heparin-agarose suggests thatthis interaction may be physiologically significant. Cyr6imay potentially bind to heparan sulfate proteoglycans onthe cell surface (19), raising the possibility that it may actas a cell adhesion molecule by mediating interactionsbetween the cell surface and the ECM. Since Cyr6i doesnot have the RGD sequence, it is unlikely that it binds tothe integrin family of proteins (20). Alternatively, Cyr6imight bind to a specific cell surface receptor. The appar-ent rapid rate of Cyr6i turnover on the cell surface isconsistent with the hypothesis that it binds to a specificcell surface receptor, and then the receptor-ligand corn-plex is rapidly internalized and degraded. The possible

existence of a specific cell surface receptor may imply adirect role for Cyr6l in cell growth control, possibly asan autocrine or paracrine factor.

Our findings suggest similarities between Cyr6l andthe protein product of the protooncogene mt-i (wnt-1),a cysteine-rich, 370-amino acid protein with a secretorysignal (21). Initial studies failed to detect Int-1 in condi-tioned media, although it was found in the secretorypathway (22-24). Recently, lnt-1 was found associatedwith the [CM (15) and the cell surface (17). Int-1 alsobinds heparin-agarose and is eluted optimally at 0.6 M

NaCI (15). Some properties shared by Cyr6i and Int-1are also shared by the FGFs. FGFs are associated withthe ECM (16, 25) and have high affinity for heparin,dissociating at greater than 1.0 M NaCI (26, 27). Theheparin-binding properties of FGFs and related proteinshave functional significance, since heparin enhances themitogenic effect of basic FGF (28). Accordingly, FGFsand related proteins have been referred to as the heparin-binding growth factor family of proteins (16). Further-more, granulocyte/macrophage colony-stimulating factorand interleukin 3 will support hernopoiesis only whenbound to heparan sulfate in the ECM (29). FGFs havebeen isolated from a variety of tissues, including brainand muscle, and are thought to play roles in angiogenesisand development (16). lnt-i can cause partial transfor-mation of mammary epithelial cell lines (30) and is alsoimplicated in neural development (31, 32). Althoughthere is no sequence homology among Int-1, FGF, andCyr6i, their similar biochemical properties raise the in-triguing possibility that they may be functionally relatedas regulatory molecules important in growth anddevelopment.

We have begun to assess the functional role of Cyr6l.Experiments comparing the growth of BALB/c 313 cellsin the presence of anti-Cyr6i immune and preimmunesera show a decline in the growth rate of cells exposed

to anti-Cyr6l immune serum.4 Although these results aresubject to alternative interpretations, taken together withevidence that Cyr6l is a mitogen-induced protein thatbinds heparin, associates with the ECM and cell surface,and has a short half-life when cell surface associated, wehypothesize that Cyr6i plays a role in cell-cell cornrnu-nication among neighboring cells. Purification and analy-sis of the Cyr6l protein will allow this hypothesis to betested.

Materials and Methods

Antibody Preparation and Affinity Purification. Poly-

clonal rabbit antisera were prepared against a TrpE-Cyr6lfusion protein expressed in bacteria as described (12).To affinity-purify the antibodies, antisera were firstpassed through a column comprised of purified TrpEprotein coupled to Affi-Gel 10 (Bio-Rad), followed bypassage over a similar column with TrpE-Cyr6l fusionprotein. Anti-Cyr6i antibodies were eluted with 0.1 M

glycine, pH 2.5, and collected in tubes containing neu-tralizing Tris-HCI and bovine serum albumin (0.2 mg/mIfinal concentration).

Immunoblotting. One-tenth ofthe cellular fraction andone-fifth of the ECM fraction from a 100-mm dish wereobtained as described below without radiolabeling. Sam-pies were separated on sodium dodecyl sulfate-10%

polyacrylamide gels, electroblotted to nitrocellulose fil-

356 Cyr6l Is Associated with ECM and Cell Surface

ters (Schleicher and Schuell), and probed with the affin-ity-purified anti-Cyr6i antibodies (1 :50 dilution), bio-tinylated goat anti-rabbit antibody [1 :200 dilution (VectorLaboratories)], and avidin-conjugated alkaline phospha-tase [1 :200 dilution (Vector Laboratories)] in that order.Protein bands were detected by incubation in 0.03%nitroblue tetrazolium, 0.02% bromochloro-indolyl phos-phate in alkaline phosphatase buffer (100 mtvi NaCI-5 m�iMgCI2-100 mM Tris, pH 9.5).

Cell Labeling and Fradionation. BALB/c 313 cells weremaintained in Eagle’s MEM (GIBCO) with 10% FCS (Biol-ogos) at 37#{176}Cand made quiescent by growth to conflu-ence and shift to MEM-0.5% FCS for 2 days. Quiescentcells were preincubated in MEM devoid of FCS andcysteine (GIBCO) for 1 h prior to the addition of [355]�

cysteine (100 .tCi/100-mm dish; ICN) for a 15-mm label-ing. Serum-stimulated confluent and subconfluent cellswere incubated in MEM-10% FCS without cysteine for 1h prior to the addition of [35S]cysteine for a 15-mmlabeling. For pulse-chase analysis, cells were washedthree times with PBS after labeling and incubated inunlabeled MEM-iO% FCS for the indicated times.

Conditioned medium was collected directly; phenyl-methylsulfonyl fluoride (Sigma) was added to 1 mt�’i, andthe sample was centrifuged at 1,500 x g to removedebris. Cells were washed with ice-cold PBS, and thecellular fraction was collected by adding 0.5 ml of RIPAbuffer (150 mM NaCI-1.0% Nonidet P-40-0.5% deoxy-cholate-0.1% sodium dodecyl sulfate-SO m� Tris, pH8.0-1 mM phenylmethylsulfonyl fluoride) to each 100-mm dish and pipetting off the cell lysate. Cell lysateswere incubated on ice for 1 h and centrifuged at 16,000x g to remove debris. Following removal of cell lysate,

dishes were washed with PBS. ECM fractions were col-lected by adding 0.5 ml RIPA buffer to each dish, fol-lowed by incubation at 85#{176}Cfor 1 h, and scraping asdescribed (iS). Immunoprecipitations were carried outas described (12). Soluble heparin (Sigma) was added toa final concentration of SO �g/ml where indicated.

Trypsin Sensitivity. Cells were labeled as describedabove for 1 h and washed with PBS. The cellular fractionwas collected with RIPA as described above or withtrypsin as follows. To each dish, 1.0 ml of 10 mg/mItrypsin was added for S mm at room temperature. Cellswere pipetted off, and 1.0 ml MEM-1O% FCS was addedto inhibit trypsin activity. Cells were spun down, washedonce with PBS with 5% FCS, resuspended in 0.5 ml RIPAbuffer, and lysed on ice for 1 h, followed by centrifuga-tion to remove debris. In both cases, dishes were washedwith PBS following collection of the cellular fraction, andthe [CM fraction was collected as described above.

Heparin-Agarose Binding and Elution. [35S]Cystemne-labeled cell lysate (1.0 ml) prepared as described abovewas added to 125 zI of a 70% slurry of heparmn-agarosebeads (Sigma) and incubated at 4#{176}Cfor at least 1 h. Thebeads were eluted with RIPA at various concentrationsof NaCI. At each NaCI concentration, the beads wereeluted with 40 �l three times. Elutions were pooled andbrought to a final volume of 1.2 ml with distilled H2Oprior to immunoprecipitation.

Radioiodination. Confluent and subconfluent cellswere grown on 100-mm dishes, as described above, andwashed with PBS. To each dish, 1 .0 ml of PBS was addedwith 2.5 mCi Nat25l (ICN), 125 �l Enzymobead reagent(Bio-Rad), 120 �tl 2% glucose, and the reaction was al-

lowed to proceed for 1 5 mm at room temperature (17).Labeling mixture was removed, and cells were washedwith PBS with 10 mt�’i Nal. Cellular fractions, which con-tam cell surface proteins, were collected as describedabove.

Acknowledgments

We thank A. M. C. Brown for communicating results prior to publication,T. P. O’Brien for help with antibody preparation; T. G. Hazel for anti-Nur77 antibody; T. P. O’Brien, T. G. Hazel, G. T. Williams, and A. S.Abler for critical readings of the manuscript; and members of the labo-ratory for helpful discussions.

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