Proc. Natl. Acad. Sci. USA 87 (1990) 2045

Biochemistry. In the article "Human basic fibroblast growthfactor gene encodes four polypeptides: Three initiate trans-lation from non-AUG codons" by Robert Z. Florkiewicz andAndreas Sommer, which appeared in number 11, June 1989,

ROBERT Z. FLORKIEWICZ*t AND ANDREAS SOMMERSynergen, Inc., 1885 33rd Street, Boulder, CO 80301

In addition, the footnotes in the lower right-hand corner

of Proc. Natl. Acad, Sci. USA (86, 3978-3981), the authorsrequest that the following corrections be noted: The authorand affiliation lines should read as shown below.

should read as shown below.

*Present address: The Whittier Institute for Diabetes and Endocri-nology, 9894 Genesee Avenue, La Jolla, CA 92037.tTo whom reprint requests should be addressed.

Correction

Proc. Natl. Acad. Sci. USAVol. 86, pp. 3978-3981, June 1989Biochemistry

Human basic fibroblast growth factor gene encodes fourpolypeptides: Three initiate translation from non-AUG codons

(gene expression/translational initiation/angiogenesis)

ROBERT Z. FLORKIEWICZ*t AND ANDREAS SOMMER**The Whittier Institute for Diabetes and Endocrinology, 9894 Genesee Avenue, La Jolla, CA 92037; and tSynergen, Inc., 1885 33rd Street, Boulder, CO 80301

Communicated by William B. Wood, February 17, 1989

ABSTRACT Human basic fibroblast growth factor(bFGF) is an angiogenic polypeptide mitogen present in a widevariety of mesoderm- and neuroectoderm-derived tissues.bFGF cDNA and genomic clones predict a 17.8-kDa (155-amino acid) gene product based on the presence of a singleputative translational initiator ATG codon. However, a bFGFprotein isolated from human placenta contains two additionalamino acids NH2-terminal to the predicted initiator methio-nine. We report here that the human cell line SK-HEP-1coexpresses four molecular forms (17.8, 22.5, 23.1, and 24.2kDa) of bFGF. The 17.8-kDa bFGF protein is translationallyinitiated at the previously predicted methionine (AUG) codon,whereas the 22.5-, 23.1-, and 24.2-kDa proteins initiate atunusual non-AUG codons. The higher molecular weight formsare colinear NH2-terminal extensions of the 18-kDa bFGF.

Human basic fibroblast growth factor (bFGF) is a heparin-binding polypeptide mitogen and chemoattractant for mes-enchyme-derived cells (1). It induces the synthesis of latentcollagenase and plasminogen activator in capillary endothe-lial cells (2) and is angiogenic in vivo (3). Both cDNA andgenomic clones for bFGF have been described (4, 5) and thesingle-copy gene for human bFGF (FGFB) has been localizedon chromosome 4 (6).

All characterized cDNA clones contain one putative initi-ator methionine codon (ATG) from which synthesis of a155-amino acid (17.8 kDa) bFGF protein species is thought tostart (4). However, we have shown that bFGF purified fromhuman placenta has a 2-amino acid extension NH2-terminalto the putative initiating methionine (5). Recently, a 25-kDabFGF protein has been identified from guinea pig brainextracts (7). In this report, we demonstrate that multiplemolecular forms ofbFGF (17.8, 22.5, 23.1, and 24.2 kDa) arecoexpressed in the human hepatoma cell line SK-HEP-1. Inaddition, we have used a SK-HEP-1 cDNA to show that bothin vitro transcription-translation and in vivo COS-1 cellexpression experiments result in the synthesis of multiplebFGF protein species. Selective mutagenesis of this cDNAindicates that the 17.8-kDa protein is translationally initiatedat the previously predicted AUG codon (4), while the 22.5-,23.1-, and 24.2-kDa proteins initiate translation at non-AUGcodons.

MATERIALS AND METHODSPreparation of Cell Lysates and Immunoblot (Western)

Analysis. SK-HEP-1 cells (1 x 109) were lysed in 10.0 ml oflysis buffer containing 50 mM Tris HCl buffer (pH 7.5), 400mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, and 1 ,uMphenylmethylsulfonyl fluoride. Nuclei and cell debris wereremoved by centrifugation at 16,000 x g for 10 min at 4°C.

The cell lysate was then purified further by heparin-Sepharose (HS) chromatography as described (8). Fractionsfrom the HS chromatography were analyzed for the presenceof bFGF by 12% sodium dodecyl sulfate/polyacrylamide gelelectrophoresis (SDS/PAGE) (9) and Western blotting tonitrocellulose (10). The Western blots were probed withaffinity-purified rabbit anti-bFGF antibodies raised againsthuman bFGF (5) or against synthetic bFGF peptides. Thesecond antibody used for color development was alkalinephosphatase-conjugated goat anti-rabbit antiserum as de-scribed by the manufacturer (ProtoBlot system, PromegaBiotec).In Vitro Transcription-Translation. The entire 1108-

base-pair (bp) human bFGF cDNA (5) was first subclonedinto the plasmid vector pGEM-4Z at the unique EcoRI site.The orientation of the insert was determined by standardprocedures. mRNA was transcribed according to the manu-facturer's recommendations from Xba I-linearized templateby using the phage SP6 promoter and the pGEM in vitrotranscription system (Promega Biotec). mRNA transcribed invitro was confirmed to be of one size class and to be of thecorrect sense by RNA (Northern) blot-hybridization analysiswith 5'- and 3'-specific oligonucleotides as hybridizationprobes (data not shown). In vitro translation of the mRNAwas performed in wheat germ extracts (Bethesda ResearchLaboratories) in the presence of [35S]methionine as suggestedby the manufacturer. We found it was unnecessary to cap the5' end of mRNA transcribed in vitro prior to in vitro trans-lation in wheat germ extracts. Immunoprecipitations of thereaction mixtures with anti-bFGF antibodies were performedas described (10). Briefly, 1.0 ml ofthe lysis buffer was addedto the in vitro translation reaction mixture; then 5 ,ul of rabbitanti-bFGF antiserum was added, and the mixture was incu-bated for 2 hr at 4°C. Protein A-Sepharose was then added,and the mixture was incubated for an additional 30 min at 4°C.The pellet was washed six times with the same lysis buffer(ice cold), and the immunoprecipitates were eluted fromprotein A-Sepharose with SDS/PAGE sample buffer (9),fractionated by 12% SDS/PAGE, and visualized by fluorog-raphy.

Site-Directed Mutagenesis. Site-directed oligonucleotidemutations at nucleotide positions 199-201 (CTG to CTT) and364-366 (ATG to GCT) (see Fig. 3) were introduced by usinga Bio-Rad mutagen kit and following procedures precisely asdescribed by the manufacturer. The mutation at nucleotidepositions 241-243 (CTG to CTT) was constructed by resyn-thesizing a fragment of DNA (by using four overlappingsynthetic oligonucleotides) between the Xho I site at nucle-otide 192 and the Apa I site at nucleotide 352. The frame-shiftmutation introduced at the unique Apa I site (see invertedtriangle in Fig. 3) was constructed by linker insertion withcomplementary synthetic oligonucleotides so that the nucle-

Abbreviations: bFGF, basic fibroblast growth factor; HS, heparin-Sepharose.tTo whom reprint requests should be addressed.

3978

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 86 (1989) 3979

otide sequence at this site, after insertion, would be 5'-GGCCTCTAGAGCCGGCC-3'. This mutation keeps the up-stream putative CUG start codons unchanged, but as trans-lation proceeds past the oligonucleotide insertion, the readingframe changes by plus one. Since the antibodies used in all ofthe experiments described were directed against the 17.8-kDaprotein, which initiates translation at the AUG codon down-stream from the insert, no immunoreactive signal would bedetected for the higher molecular weight proteins (22.5, 23.1,or 24.2 kDa) if their translation were initiated upstream fromthe linker insertion. If the source of the multiple highermolecular weight proteins was 3' to the inserted oligonucle-otide, then the immunoreactive signal would remain thesame.COS Cell Expression. The bFGF "wild-type" and muta-

genized cDNAs were subcloned into the hybrid expressionvector pJC119, and COS-1 cells were transfected as de-scribed (11, 12). Briefly, the expression vector pJC119 uti-lizes the simian virus 40 late promoter and polyadenylylationsignal. Plates (60 mm) of COS-1 cells were transfected with10 ,ug of DNA by using 500 tzg of DEAE-dextran per ml for30 min, followed by a 2-hr incubation in medium supple-mented with 100 ,uM chloroquine; 48 hr after transfection,cell lysates were prepared as described above. COS-1 celllysates were then incubated with HS for 2 hr at 40C. Aftercentrifugation, the HS pellets were washed three times withbuffer containing 20 mM Tris HCl (pH 7.5), 0.5 M NaCl, and1 ,uM phenylmethylsulfonyl fluoride and then three timeswith the same buffer containing also 1 M NaCl. Finally,protein was released from the HS resin with SDS/PAGEsample buffer, fractionated by 12% SDS/PAGE, and ana-lyzed for the presence of bFGF proteins by Western blottingas described above.

Bioassay. COS-1 cell lysates were prepared as describedabove except that heparin-bound protein was eluted from theHS resin in buffer containing 3M NaCl instead ofSDS/PAGEsample buffer. Aliquots from these 3 M NaCl eluates werethen diluted into phosphate-buffered saline containing 0.01%gelatin and assayed for stimulation of 3T3 cell DNA synthe-sis. This 3T3 cell mitogenicity assay, as described (7), mea-sures the incorporation of [3H]thymidine into CCl3COOH-precipitable radioactive material.

RESULTS

SK-HEP-1 Cells Synthesize Multiple Forms of bFGF. Weexamined SK-HEP-1 cell extracts for the presence of multi-ple forms ofbFGF by Western blot analysis (Fig. 1). The datashow that SK-HEP-1 cells contain at least three immunore-active species of bFGF that bind to heparin. All threeimmunoreactive proteins were eluted from HS by >1 MNaCl, a characteristic of all bFGFs previously isolated (13)(Fig. LA, lanes 1-3). We then wanted to determine whichamino acid sequences (domains) were conserved among thethree immunoreactive bFGF proteins observed. To do thiswe carried out three competitive Western blot experiments:(0) with anti-bFGF antiserum alone or after preincubationwith human recombinant bFGF (Fig. 1B, lanes 1 and 2,respectively); (ih) with anti-peptide antiserum specific for anNH2-terminal domain of bFGF alone or after preincubationwith the corresponding synthetic peptide (Fig. 1C, lanes 1and 2, respectively), and (iii) with anti-peptide antiserumspecific for a COOH-terminal domain alone and after prein-cubation with the corresponding synthetic peptide (Fig. 1C,lanes 3 and 4, respectively). In each case, immunoreactivitywas eliminated after preincubating the affinity-purified anti-sera with either recombinant bFGF or the correspondingsynthetic peptide, respectively. The data show that all three

A

US

1 2 3 1 2

B C

24.2-s.22.5--_117.8--W

'.-m

1 2 3 4

FIG. 1. Western blot analysis of bFGF proteins from SK-HEP-1cells. (A) SK-HEP-1 lysates probed with affinity-purified anti-bFGFantibodies. Lanes: 1, cell lysate before HS chromatography; 2, 1 MNaCI HS eluate; 3, 3 M NaCl HS eluate. (B) Identical SK-HEP-1lysates probed with affinity-purified bFGF antibodies alone (lane 1)or after preincubation with 25 ,ug of recombinant bFGF (lane 2). (C)SK-HEP-1 lysates probed with affinity-purified anti-peptide-(40-63)antibodies alone (NH2-terminal) (lane 1) and after preincubation with25 ,g of peptide-(40-63) (lane 2) or probed with affinity-purifiedbFGF anti-peptide-(147-153) antibodies alone (COOH-terminal)(lane 3) and after preincubation with 25 Ag of peptide-(147-153) (lane4). Molecular masses are given in kDa.

immunoreactive proteins share similar, if not identical, NH2-terminal and COOH-terminal domains.

Multiple Species of-bFGF Are Synthesized in Vitro. Wewanted to determine if the multiple bFGF species wereencoded within a previously characterized bFGF cDNA thathad been cloned from SK-HEP-1 poly(A)+ RNA (5). To dothis, we subcloned that cDNA into a standard in vitrotranscription system. The single class size mRNA tran-scribed in vitro was then used for translation by using a wheatgerm in vitro translation system followed by immunoprecip-itaton and analysis by SDS/PAGE. The data in Fig. 2 showthat the cDNA used to generate mRNA in vitro contains thecoding information required for the synthesis of at least threeimmunoprecipitable species (17.8, 22.5, and 24.2 kDa) ofbFGF. The immunoprecipitable band at 16 kDa is most likelya bFGF degradation product.DNA Sequence Predicts Translation Initiation at Upstream

CTG Codons. We examined the possibility that codons otherthan AUG were used to initiate translation of the highermolecular weight forms of bFGF because (0) sequence anal-ysis of the bFGF cDNA clone showed that it contained onlyone putative ATG translational initiator methionine codon (4,5), (ii) alternative RNA splicing was not required to generatethe multiple forms ofbFGF (data presented in Fig. 2), and (iii)pulse-chase experiments with SK-HEP-1 cells indicated thatthere is no precursor-product relationship between any ofthemultiple bFGF species observed (data not shown).

Fig. 3 shows the relevant nucleotide sequence of the bFGF

68 -

34 w-9

25 -

18 W-

i-24.2*: -22.5

-*o_17.8. 16

2

FIG. 2. In vitro translation ofmRNA transcribed from the wild-type bFGF cDNA. RNA-depen-dent in vitro translations were per-formed in wheat germ extracts andimmunoprecipitated as described.Immunoprecipitated samples wereeluted from protein A-Sepharose,resolved on 12% SDS/PAGE, andvisualized by fluorography. Lanes:1, protein standards; 2, immuno-precipitated proteins from in vitrotranslation. Molecular masses aregiven in kDa.

Biochemistry: Florkiewicz and Sommer

3980 Biochemistry: Florkiewicz and Sommer

181 OTT]CGG CCG AGC GGC TCG AGG CTG GGG GAC CGO

LeuGGG CGC GGC CGC GCG CTG COG GGC GGG AGG[Leu] 250LCTTJ25CTG GGGGGCCGG GGCCGG GGCCGTCCC CCGLOu 300GAG CGG GTC GGA GGCOCGG GGCCGG GGCOCGG

GGG ACG GCG GCT CCC CGC GCG GCT CCA GCG, ~~~35C

GCT CGG GGA TCC CGG CCG GGC 0CC GCA GGGA/l1

.GCT]ACO ATGGCA bFGF TGA

Met Stop

FIG. 3. bFGF cDNA sequence analysis. The relevant sequenceof the wild-type bFGF cDNA clone is shown with the position ofsite-directed mutations indicated in bold letters. The frame-shiftmutation is indicated by the inverted triangle. The construction of allmutations is described in Materials and Methods.

cDNA used to generate mRNA for in vitro translation. Whenwe considered the literature describing alternative transla-tional initiation codons (14-17), synthesis of a 24.2-kDa and22.5-kDa bFGF protein could initiate translation at CTGcodons in nucleotide positions 199-201 and 241-243, respec-tively, whereas synthesis of the 17.8-kDa bFGF would beginat the ATG codon in nucleotide positions 364-366. All threecodons are contained within a nucleotide sequence describedby Kozak (14) as most preferred for eukaryotic translationalinitiation, while another in-frame CTG codon at nucleotideposition 228, which could initiate translation of a 23.1-kDabFGF protein, is considered less preferred. Mutations weremade as described in Materials and Methods.

Mutagenesis and in Vivo COS-1 Expression Show MultipleCTG Translation Initiations. To test the hypothesis that CTGcodons could initiate translation of the 24.2-kDa and 22.5-kDa bFGF proteins, we conducted in vivo expression exper-iments using COS-1 cells transfected with the hybrid expres-sion vector pJC119 containing the following bFGF cDNAs:(i) a wild-type cDNA previously used for in vitro translation(see Fig. 2); (ii) a wild-type cDNA inserted in the incorrectorientation; (iii) a frame-shift mnutant with an altered (+1)reading frame beginning at nucleotide position 353 (betweenthe CTG codons and the first ATG codon); (iv) a Guo-201 --

dThd mutation changing CTG codon to CTT; (v) a Guo-241-+ dThd mutation changing CTG codon to CTT; and (vi)mutations at positions 364-366 changing ATG (methionine)to GCT (alanine).

Extracts from COS-1 cells transfected with these bFGFcDNA/pJC119 constructs were analyzed by Western blottingwith anti-bFGF antibodies (Fig. 4A). A standard recombinantbFGF 17.8-kDa marker is presented in lane 1. COS-1 cellstransfected with the wild-type cDNA (inserted into pJC119 inthe correct orientation) synthesized at least three forms ofbFGF (lane 5) with electrophoretic mobilities identical to thebFGF proteins expressed by the human SK-HEP-1 cells (seeFig. 1). This result supports the conclusion derived from thein vitro transcription-translation data, that the wild-typebFGF cDNA, although containing only one putative trans-lational initatior methionine codon, also contains the infor-mation required for the synthesis of at least two additionalmolecular forms of bFGF. COS-1 cells transfected with thewild-type cDNA inserted into the expression vector in theincorrect orientation (lane 7) or COS-1 cells mock-transfected (lane 8) do not synthesize bFGF. COS-1 cellstransfected with the frame-shift mutation described in Fig. 3

1 2 3 4 5 6 7 8

0T-

x 12E

0

-B

bFGF, ng

FIG. 4. COS-1 cell expression (A) and bioactivity (B) of thewild-type and mutagenized bFGF cDNA. Lysates from the trans-fected cells were partially purified by HS chromatography andanalyzed by SDS/PAGE and Western blotting with affinity-purifiedanti-bFGF antibodies (A). Lanes: 1, recombinant bFGF 17.8-kDamarker; 2, Guo-201 - dThd mutation (codon CTG to CTT at 199-201); 3, Guo-243 -> dThd mutation (codon CTG to CTT at 241-243);4, frame-shift mutation; 5, wild-type cDNA in the correct orientation;6, ATG to GCT mutation at nucleotides 364-366; 7, wild-type cDNAin the incorrect orientation; and 8, mock-transfected COS-1 cells.Molecular masses are given in kDa. (B) HS-bound protein was elutedwith 3 M NaCl and then assayed by the 3T3 cell mitogenicity assay.Quantitative Western blot analysis of these samples indicated abFGF concentration of 1 ng/Al of 3 M NaCl eluate. *, Wild-typecorrect orientation; *, wild-type incorrect orientation; A, frame-shiftmutation; a, ATG to GCT mutation; o, control COS-1 cells non-transfected.

express only the 17.8-kDa bFGF protein (Fig. 4A, lane 4).This result indicates that the 22.5-kDa and 24.2-kDa proteinsare translationally initiated 5' to the ATG codon (nucleotidepositions 364-366) and that the synthesis of the 17.8-kDaprotein is, in fact, initiated at ATG at 364-366.

Direct mutagenesis of this ATG to GCT supports thisinterpretation, resulting in synthesis ofonly the 22.5-kDa and24.2-kDa bFGF proteins (Fig. 4A, lane 6). The mutant cDNAcontaining a mutation at nucleotide position 201 giving codonCTT and eliminating the CTG expected to initiate synthesisof the 24.2-kDa protein expresses only the 22.5-kDa and17.8-kDa proteins (Fig 4A, lane 2). This result implicates theCTG codon (positions 199-201) in the synthesis of the24.2-kDa protein species of bFGF. Mutagenesis of codonCTG at nucleotide position 243, expected to eliminate syn-thesis of the 22.5-kDa protein, revealed a previously uniden-

A

24.2-m

23.1/22.5 [17.8-

Proc. Natl. Acad. Sci. USA 86 (1989)

."No: 4'm am,W40 40 "m

.40""Am a* a*

Proc. Natl. Acad. Sci. USA 86 (1989) 3981

tified immunoreactive protein species-actually the top bandof a 22.5/23.1-kDa doublet (Fig. 4A, lane 3). DNA sequenceanalysis of this mutant confirmed that the only nucleotidechange was, as by design, at position 243, changing the codonat 241-243 from CTG to CTT. We suggest that, although theCTG at nucleotide position 226-228 (see Fig. 3) has a lesspreferred Kozak consensus sequence than that at 199-201 orat 241-243 or ATG at 364-366, it nonetheless appears to berecognized as a functional translational initiator codon (invivo) for the synthesis of a 23.1-kDa bFGF protein species.During review of this manuscript we demonstrated thatsimultaneously mutagenizing both CTG codons at nucleotidepositions 226-228 and 241-243 to CTJ followed by COS-1cell expression results in the synthesis of only the 24.2-kDaand 17.8-kDa proteins as suggested above.

Bioactivity. The bFGF protein species synthesized bytransfected COS-1 cells are bioactive in a 3T3 cell mitoge-nicity assay (Fig. 4B). All cell lysates tested were firstpurified by HS chromatography. The 3 M NaCl eluate ofHS-purified lysates from control (nontransfected) COS-1cells or from COS-1 cells transfected with the bFGF cDNAinserted in the incorrect orientation were not stimulatory inthe mitogenicity assay. All other 3 M NaCl eluates from wildtype as well as mutants synthesizing only the higher molec-ular weight proteins (24.2,23.1, and 22.5 kDa) or synthesizingonly the 17.8-kDa protein stimulated nearly identical levels of[3H]thymidine incorporation. For all samples tested, mito-genic activity was neutralized by preincubation with anti-bFGF antibodies (data not shown).

DISCUSSIONWe have identified four molecular forms of human bFGFcoexpressed by the human hepatoma cell line SK-HEP-1.Based on SDS/PAGE analysis and the predicted amino acidsequence, the four proteins have apparent molecular weightsof 24.2, 23.1, 22.5, and 17.8 kDa. We have shown by in vivoexpression of frame-shift and oligonucleotide-directed muta-tions that the synthesis of bFGF 24.2, 23.1, and 22.5-kDaproteins begins at non-AUG translational initiation codons.Thus, we demonstrate a normal animal cell gene encodingmultiple colinear extended forms of the same protein in vivo,that these proteins are synthesized from one mRNA species,and that some initiate translation at non-AUG codons. Al-though the CTG codons described have reasonably goodcontext in the +4 and -3 positions as described by the classicKozak consensus sequence, it is not clear what nucleotidesequence around certain CTG codons affects the associatedribosome scanning model and translational initiation. We arebeginning a more extensive mutagenesis program in an effortto understand this particular exception to the rules. In relatedexperiments, primarily using in vitro translations, one form of

the MYC protooncogene has also been shown to utilize anon-AUG codon for translational initiation (16).

Since cell extracts containing the higher molecular weightforms of bFGF appear to be as bioactive as extracts con-taining only the 17.8-kDa protein species, the physiologicalfunction of the multiple molecular weight bFGF proteinsremains to be determined. Differences in their intracellularsorting, transport, or targeting may be one potential function.The multiple molecular forms ofbFGF could be differentiallystored, each to be released from the cell in response tospecific physiological signals; alternatively, the multiple bF-GFs may individually localize to different subcellular com-partments and modulate cell growth and differentiation. Wehave described a system that will allow the generation ofmutants that produce only one specific molecular form ofbFGF; such mutants can be used to determine the intracel-lular location and the physiological function of the individualmolecular forms of bFGF.

We thank D. Abbott-Brown for establishing the bioassay used inour lab; T. Gleason and R. Weaver for oligonucleotide synthesis; B.Cooley for peptide synthesis and affinity purification of anti-bFGFantisera; R. Green for valuable discussions regarding in vitro trans-lations; T. Klein, C. Worland, and D. Higgins for preparation of themanuscript; and D. Hirsh, R. Thompson, R. Majack, and A. Flexerfor critical review of this report.

1. Gospodarowicz, D. (1987) Nucl. Med. Biol. 14, 421-434.2. Moscatelli, D., Presta, M. & Rifkin, D. B. (1986) Proc. NatI.

Acad. Sci. USA 83, 2091-2095.3. Folkman, J. & Klagsbrun, F. M. (1987) Science 235, 442-447.4. Abraham, J. A., Whang, J. L., Tumolo, A., Mergia, A., Fried-

man, J., Gospodarowicz, D. & Fiddes, J. (1986) EMBO J. 5,2523-2528.

5. Sommer, A., Brewer, M. T., Thompson, R. C., Moscatelli, D.,Presta, M. & Rifkin, D. B. (1987) Biochem. Biophys. Res.Commun. 144, 543-550.

6. Mergia, A., Eddy, R., Abraham, J., Fiddes, J. & Shows, T. B.(1986) Biochem. Biophys. Res. Commun. 138, 644-651.

7. Moscatelli, D., Joseph-Silverstein, J., Manejias, R. & Rifkin,D. B. (1987) Proc. Natl. Acad. Sci. USA 84, 5778-5782.

8. Squires, C. H., Childs, J., Eisenberg, S. P., Poverini, P. J. &Sommer, A. (1988) J. Biol. Chem. 263, 16297-16302.

9. Laemmli, U. K. (1970) Nature (London) 263, 789-798.10. Florkiewicz, R. Z., Smith, A., Bergmann, J. E. & Rose, J. K.

(1983) J. Cell Biol. 97, 1381-1388.11. Machamer, C. E., Florkiewicz, R. Z. & Rose, J. K. (1985)

Mol. Cell. Biol. 5, 3074-3983.12. Rose, J. K., Adams, G. A. & Gallione, C. J. (1984) Proc. Natl.

Acad. Sci. USA 81, 2050-2054.13. Lobb, R. R., Harper, J. W. & Fett, J. W. (1986) Anal. Bio-

chem. 154, 1-14.14. Kozak, M. (1986) Cell 44, 283-292.15. Peabody, D. S. (1987) J. Biol. Chem. 262, 11847-11851.16. Hann, S. R., King, M. W., Bentley, D. L., Anderson, C. W. &

Eisenman, R. N. (1988) Cell 52, 185-195.17. Curran, J. & Kolakofsky, P. (1988) EMBO J. 7, 245-251.

Biochemistry: Florkiewicz and Sommer