Turnover of the Transferrin Receptor Is Not Influenced by Removing ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266,No. 31,Issue of November 5 , pp. 21125-21130,1991 Printed in U.S.A.

Turnover of the TransferrinReceptor Is Not Influenced by Removing Most of the ExtracellularDomain* (Received for publication, April 30, 1991)

Elizabeth A. Rutledge$, Carole A. Mikoryak, andRockford K. Draper8 From the Molecular and Cell Biology Program, University of Texas at Dallns, Richardson, Texas 75083-0688

We treated intact cells with trypsin to remove most domain, and anamino-terminal domain within the cytoplasm of the externaldomain of the transferrinreceptor and (Schneider et al., 1984). The TfR participates in acquiring investigated what effect the absence of the external iron for cells by binding to theiron-carrier protein transferrin domain had on the turnover of the fragment that re- (Tf). The TfR.Tf complex is internalized by the clathrinmained associated with the cells. To detect the cell- coated pit pathway and enters endosomes where the iron is associated tryptic fragment, which contains a small released from Tf in response to thelow endosomal pH. ApoTf amount of the external domain, the transmembrane remains bound to the TfR and recycles to the cell surface domain, and the cytoplasmic domain, we prepared an where apoTf is released, enabling the TfR to repeat the cycle anti-peptide antibody against a segment of the cytoplasmic domain. This antibody specifically immuno- upon binding another molecule of iron-laden Tf in the meprecipitated the intact transferrin receptor as well as dium (Dautry-Varsat et al., 1983; Klausner et al., 1983). The a 2 1-kDa peptide from trypsin-treatedHeLa cells.Sev- binding of Tf to the TfRis not necessary for the receptor to eral lines of evidence indicated that the2 1-kDa peptide be endocytosed and to recycle (Girones and Davis, 1989). We are interested in what structural elements of the TfR was the cell-associated tryptic fragment of the transprovide signals that influence the endocytic itinerary of the ferrin receptor. The fragment was only present in as a dimer receptor, including targeting of the receptor to lysosomes trypsin-treated cells; the fragment migrated in nonreducing sodium dodecyl sulfate-polyacrylamide during normal turnover. One targeting signal in the TW that from has been identified so far is the tyrosine-containing motif in gel electrophoresis, as it should if it were derived the transferrin receptor; a goat antibody prepared to the cytoplasmic domain, Tyr-X-Arg-Phe, which is important the purified human transferrin receptor also precipi- for endocytosis of the receptor by coated pits (Jinget al., 1990; tated the 21-kDa peptide from trypsinized cells. In Collawn et al., 1990). It is not known, however, whether the addition, treating the tryptic fragment with neuramin- extracellular domain is also necessary for endocytosis or idase increased the electrophoretic mobility in sodium whether the extracellular domain contains information redodecyl sulfate-polyacrylamide gels, suggestingthe quired for recycling or for delivery to lysosomes during turnfragment contained 0-linked carbohydrate. When cells over. In this report we removed most of the external domain were trypsinized and then incubated at 37 OC,the halflife of the tryptic fragment(15 f 4 h) was not signifi- of the TfRby cleavage with trypsin and investigated the fate cantly differentthan thehalf-life of the intact receptor of the remaining cell-associated fragment. What happens to (10 f 6 h). This indicates that removing 95% of the the cell-associated fragment is physiologically relevant conexternal domain of the transferrin receptor has little sidering that Shih et al. (1990) recently reported that a sigeffect on processes operating in the turnover of the nificant amount of the external domain of the receptor is cleaved from the TfR inhumans. receptor. To immunoprecipitate the cell-associated fragment in cell lysates, anti-peptide antibodies against the cytoplasmic domain of the TfR were prepared. We reasoned that any influThe transferrin receptor (TfR)’ is a type I1 transmembrane ence of the extracellular domain on the endocytic trafficking glycoprotein comprising an extracellular domain that contains of the receptor would most likely alter the turnover rate of the carboxyl terminus, a single hydrophobic transmembrane the receptor; if endocytosis were blocked, then the receptor * This work was supported in part by National Institutes of Health should be degraded more slowly than normal because endoGrant GM-32042 and by Research Partners Proposal Grant 10690- cytosis is necessary for receptors to reach lysosomes; if the 072 from the University of Texas a t Dallas. The costs of publication receptor were endocytosed but failed to recycle and instead of this article were defrayed in part by the payment of page charges. entered lysosomes,turnover would bemore rapid than normal. This article must therefore be hereby marked “aduertiement” in We report here that turnover of the TfR is little affected by accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $Work was performed in partial fulfillment of the requirements removing 95% of the extracellular domain. This suggests that for the Ph.D. degree in Molecular and Cell Biology. Present address: the absence of the extracellular domain has no major influence Oregon Health Sciences University, CellBiology and Anatomy, on the trafficking of the receptor. We also found that the McKenzie Hall, Rm. 3192, Portland, OR. short extracellular domain of the cell-associated fragment J To whom correspondence should be addressed Molecular and appears to contain 0-linked carbohydrate.

Cell Biology Program, F03.1, University of Texas at Dallas, P. 0. Box 830688, Richardson, T X 75083-0688. Tel.: 214-690-2512. The abbreviations used are: TfR, transferrin receptor; Tf, transferrin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; SMCC, (maleimidomethy1)cyclohexanecarboxylic acid hydroxysuccinimide ester; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

MATERIALSANDMETHODS

Reagents-Peptide 1 contained 14 amino acids and peptide 2 contained 24 amino acids corresponding to segments of the TfR cytoplasmic domain shown in Fig. 1. In addition, a single cysteine residue was placed at the amino end of peptide 1 and the carboxyl end of peptide 2 for conjugating to carrier proteins. Both peptides

21125

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Turnover of the Trypsinized Transferrin Receptor

were purchased from OCS Laboratories, Inc. (Denton, TX). The following chemicals were purchased from Sigma: bovine albumin, bovine thyroglobulin, (maleimidomethy1)cyclohexanecarboxylicacid hydroxysuccinimide ester (SMCC), 4,4'-dithiodipyridine, 2-mercaptoethylamine-HCl, N,N-dimethylformamide, protein A-Sepharose CL-4B, rabbit serum agarose, IgG agarose, rabbit anti-actin antibody, Freund's complete and incomplete adjuvant, lactoperoxidase, glucose oxidase, tosylamidophenylethyl chloromethyl ketone-treated trypsin, NaBH4,sodium borate, Staphylococcusaureus V8 protease type XVII, soybean trypsin inhibitor, neuraminidase type X, and sodium salicylate. GammaBind G-agarose was from Genex Corp. (Gaithersburg, MD). NalZ5I(100 mCi/ml) was from Amersham Corp. Tran3%-1abel was from ICN Radiochemicals. Anti-TfR monoclonal antibody B3/ 25 was from Hybritech Inc. (San Diego, CA). AcrylAide was from FMC Bioproducts (Rockland, ME). Bicinchoninic acid protein reagent was from Pierce Chemical Co. X-Omat XAR-2 film was from Kodak. Entensify was from Du Pont-New England Nuclear. HEPES was from Calbiochem Corp. Selectamine medium was from GIBCO. Goat antibody to thepurified human TfR was kindly provided by Dr. Howard Sussman (Stanford University). Cells and Cell Culture-HeLa cells were grown in Dulbecco's modified Eagle's medium (Irvine Scientific, Santa Ana, CA), with 5% fetal bovine serum, 40 pg/ml proline, 4.5 mg/ml glucose, 292 pg/ml glutamine in a 10% COn atmosphere at 37 "C. During experiments, 63 pg/ml (100 units/ml) penicillin, 135 pg/ml (100 units/ml) streptomycin, and 2.5 pg/ml amphotericin were usually added. Preparation of Anti-peptide Antibodies-Peptide 1 (see Fig. 1) was conjugated to either BSA or thyroglobulin that had been derivatized with cross-linker SMCC to introduce maleimide groups reactive with the free sulfhydryl of the cysteine present at the amino terminus of the peptide. To derivatize BSA and thyroglobulin, 5 mg of SMCC dissolved in 0.5 ml of dimethylformamide was added dropwise to 10 mgof the carrier protein in 2 ml of PBS at 4 "C until a slight precipitate was visible. After 1 h the solution was centrifuged for 10 min and the supernatant was passed through aSephadex G-25 column equilibrated with 0.1 M sodium phosphate buffer, pH 6.0, at room temperature. Fractions containingthe derivatized protein were combined and, if necessary, concentrated by pressure dialysis. The peptide (10 mg in 0.5 ml of 0.1 M borate buffer, pH 9) was reduced by the addition of 100 pl of 0.1 M NaBH4 on ice for 5 min. The reducing agent was removed by adding 1.0 M HCl to pH 1 to form hydrogen gas, then thepeptide solution was brought to pH6 with 1.0 M NaOH. The reduced peptide was added to thederivatized protein and allowed t o react for 1 h, after which it was passed through a Sephadex G-25 column equilibrated with 0.1 M sodium phosphate buffer, pH 6.0, at room temperature (Michael et al., 1984). For conjugation of peptide 2, 5 mgof peptide were added to 1.0 ml of water and allowed to dissolve for a few hours. The insoluble material was removed by centrifugation, and theabove procedure for peptide 1was carried out except that only BSA was used, and 1 ml of 0.1 M borate buffer was added to the peptide in water and then 200 p1of 0.1 M NaBH4 was added to reduce the peptide. After peptide 2 was coupled to BSA the reaction mixture was passed through a Bio-Gel P30 column. The number of peptides coupled per carrier molecule (BSA or thyroglobulin) was determined by an indirect assay of the number of unreacted maleimide groups (Yoshitake et al., 1982; Grassetti and Murray, 1967). Briefly, the maleimide groups were reacted with a slight excess of mercaptoethylamine, and the amount of unreacted mercaptoethylamine was determined by absorbance at 324 nm after reaction with dithiodipyridine. The difference between the number of maleimide groups on the derivatized intermediate and on the final peptide conjugate indicated the number of peptide molecules coupled to the carrier. BSA, which contains 0.67 SH groups per molecule (King and Spencer, 1970), was used as a control for the detection of sulfhydryl groups by reaction with dithiodipyridine. For peptide 1, 11-23 peptide molecules were coupled to BSA and 114 were coupled t o thyroglobulin. For peptide 2, up to 22 peptides were coupled to each BSA molecule. The conjugation of peptides to carrier proteins was also verified by assessing the electrophoretic mobility of conjugates in SDS-polyacrylamide gels; carrierproteins conjugated to peptides migrated more slowly than the derivatized carriers without attached peptides. To prepare antibodies, each peptide conjugate (2 mg/ml) was emulsified with an equal volume of Freund's complete adjuvant and 1ml of the mixture was injected subdermally into several sites on the back of each of two female New Zealand White rabbits. Four weeks later the rabbits were injected with an additional 1mg of the peptide conjugate in Freund's incomplete adjuvant. The rabbits were boosted

a t 2-week intervals. An IgGfraction of serum from the rabbits injected with BSA-peptide 2 was obtained from a pool of sera from both rabbits by adding 2/3 volume of cold saturated ammonium sulfate. Radiolabeling Cells-HeLa cells (2-3 X lo6 cells) were plated in 10cm dishes the day before the experiment and labeled the following day with either "'I or Trar~~~S-label.label Tocells with '1 (Schneider et al., 19821, cells were washed 4 times with PBS. Two mlof PBS were placed in each dish and 100 pl of lactoperoxidase (1mg/ml), 100 pl of glucose oxidase (1 mg/ml), 0.5-1.0mCiof lZ5I,and 100 plof dextrose (5 mg/ml) were added in that order. Iodination was allowed to proceed for 15 min a t room temperature. KI (2 ml of 1mg/ml) was added to stop the reaction, and the cells were washed 4 times with PBS. To label cells with %, cells were rinsed twice with PBS and 2.5 ml of labeling medium (prepared from a Selectamine kit to contain 1%of the normal amount of methionine and cysteine) containing 0.5-1.0 mCi of Tran3'S-label were added for 8 h at 37 "C. The labeling medium was removed, and thecells were rinsed twice with PBS. Trypsin Cleavage of the TfR-Cells were trypsinized at 0 "Cwith 1 ml of 1 mg/ml trypsin in PBS for 30 min, after which 20 pl of 100 mg/ml soybean trypsin inhibitorwere added. Under these conditions, 99% of radioiodinated TfR molecules on the cell surface were cleaved. Immunoprecipitation-Cells were lysed in 1 ml of Nonidet P-40 (1% in PBS) at 0 "C for 30 min. The lysate was collected and centrifuged in a microcentrifuge for 10 min. The supernatant was either used immediately or frozen at -70 "C. Usually, 20 pl of 0.5 M ATP were added to aliquots of supernatants containing 70pgof protein (Smith et al., 1985) and the lysate volume was brought to 1 ml with 1%Nonidet P-40. The ATP reduced the nonspecific immunoprecipitation of actin (Peltz et al., 1987). Samples at 4 'C were precleared by adding either 100 p1 of rabbit serum agarose or 10 pl of rabbit preimmune IgG for 1 h followed by a 1-h incubation with 100 pl of protein A-Sepharose, a 50% suspension in Triton-NET (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.1% Triton X-100, pH 7.25). In some experiments, preclearing was by incubating samples with rabbit IgG already coupled to agarose. The agarose or Sepharose beads were removed bycentrifugation for 2 min, and the supernatants were recovered. Ten p1 of either rabbit anti-peptide, goat anti-TfR, or monoclonal antibody B3/25 were added to the supernatantfor 112 h. Protein A-Sepharose or protein G-agarose (100-200 pl) suspended in lysates from unlabeled HeLa cells were added for 1 h. The beads were pelleted and washed with 1ml of Triton-NET plus 0.5 M NaCl followed by a wash with 1 ml of Triton-NET plus 0.1% SDS, then a 1-mlwash of 10 mM Tris, pH8. The beads were then incubated with 75-150 pl of double-strength sample buffer (117 mM Tris-HC1, pH 6.8, 5.6% SDS, 0.003% bromphenol blue, and 19% glycerol) without mercaptoethanol for 10 min at 100 "C. After centrifugation the supernatant was removed with an insulin syringe, and 5 pl of mercaptoethanol were added. The samples were electrophoresed through a discontinuous polyacrylamide gel containing SDS. Gels containing 35Swere treated with either 1.0 M sodium salicylate (rinsed 30 min in water, then 30 min in salicylate; Chamberlain (1979)) or Entensify (45 min in solution A, then 45 min in solution B). After drying, the gels wereexposed to preflashed x-ray film a t -70 "C. Gels containing "'1 samples were dried and exposed to film with one or two intensifying screens at -70 "C. S. aureus V8 Protease Digestion-TfR that had been immunoprecipitated from 3'S-labeled cells was electrophoresed in a 7.5% polyacrylamide gel with SDS. Dansylated molecular weight markers (Talbot and Yphantis, 1971) were present in an adjacent lane. Sections of the gel corresponding to 96 kDa (the molecular mass of the TfR), as determined by the dansylated markers, were cut out and placed in V8 buffer (125 mM Tris, pH 6.85,lmM EDTA, 0.1% SDS) plus 20% sucrose for 30 min and then inserted in wells of a 2.5-mm-thick 18% polyacrylamide gel containing AcrylAide as the cross-linker. 100 pl of V8 buffer with 15% sucrose and 20% glycerol wereadded, and then 30 p1 of V8 buffer with 5% sucrose, 10% glycerol, bromphenol blue, and 0.1 pg of V8protease were layered over the slices. Electrophoresis was initiated and temporarily stopped for 40 min when the dye front reached the bottom of the stacker gel to allow proteolytic digestion to proceed in the concentrated sample (Cleveland et al., 1977). Neuraminidase Treatment-The cell-associated tryptic fragment of the TfR was immunoprecipitated from 3SS-labeledcells using goat anti-TfR antibody as already described. Proteins adhering to the Sepharose beads were eluted by heating in 150 pl of 50 mM sodium citrate buffer, pH 5.5, with 0.1% SDS. The supernatantwas divided into aliquots, 20 milliunits (2 pl) of neuraminidase were added to one aliquot, and the samples were placed at 37 "C overnight. An equal volume of double-strength sample buffer with mercaptoethanol was

Turnover of the Trypsinized Transferrin Receptor

21127

added, and the samples were electrophoresed througha 10-17% linear face-labeled with '*'I (Fig. 2). The larger of the two proteins gradient gel. had a mobility identical to that of the TfR immunoprecipiHalf-life Determination-HeLacells were metabolicallylabeled tated by the monoclonal antibody B3/25, which is known to with and treated with trypsin to remove the extracellular fragment react with the extracellular domain of the human TfR (Omary of the TfR as described in preceding sections. Prewarmed regular growth medium (15 ml/plate) containing10 times the normal amount et al., 1980). The 66-kDa protein is bovine serum albumin of methionine (10 mM) was added, and the plates were incubated a t from the growth medium that became radiolabeled during the 37 'C. A t 5-h intervals plates were removed, washed twice with PBS, iodination procedure. This protein was not immunoprecipiand lysed in 1% Nonidet P-40 in PBS at0 "C. The samples were then tated after the antiserumwas passed through a column concentrifuged in amicrocentrifugefor 10 min and the supernatants taining immobilized bovine serum albumin to remove antifrozen at -70 "C. As described in previous sections, 70 pg of each sample were used for immunoprecipitation with either antibody to bodies to the carrier protein (data not shown). Preimmune peptide 2 or goat anti-TfR antibody. The immunoprecipitates were serum did not immunoprecipitate eitherof the proteins. T o verify that the 96-kDa protein was the TfR,peptide electrophoresed through a 10-17% linear gradient gel with AcrylAide as the cross-linker. maps of the :%-labeled proteins that were immunoprecipiQuantitation of Fluorographic Rands-To quantitate the intensity tated by eitherantibodytopeptide 2 orthe monoclonal of bands on the fluorograms, it was first necessary to determine the antibody I33125 were compared after the proteins were dilinear range of the film (Laskey and Mills, 1975). This was done by pouring a polyacrylamide gel of several layers, each layer containing gested with S. aureus V8 protease. As seen in Fig. 3, the increasing amounts of ""S-labeled HeLa lysate. The gel was treated peptide patterns from the two proteins were identical, demwith Entensify, dried, and exposed to preflashed film. The absorbance onstrating that the anti-peptide antibody reacted with the of the signal on the film from each layer was measured with anLKB human TfR. Theantibodytopeptide 2 also appearedto Ultroscan enhanced laser densitometer using GelScan software and immunoprecipitate TfR from human A431, mouse the plotted against the amount of radioactivity in the layer. The linear LMTK-, Madin-Darby canine kidney, and Chinese hamster range occurred approximately between absorbances of 0.5 and 2.0. To quantitate the amounts of TfR or the cell-associated tryptic fragment ovary cells (data not shown). Antibody to Peptide2 Reacts with theCell-associated Tryptic of the TfR, the fluorograms were exposed for sufficient times that intact cells withtrypsin the desired bands gave absorbances within the linear range. The dataFragment of the TfR-Treating were expressed as a percentage of the areaof a control band andwere cleaves the TfR toproduce an external fragmentof about 70 corrected forcell growth during the chase period based onthe increase in the amountof protein in the samples. 83/25

pF

A

E

RESULTS

Anti-peptide Antibodies to the Cytoplasmic Domain of the TfR-Fig. 1 shows the amino acid sequence of the first 150 amino acids of the human transferrin receptor (McClelland et al., 1984). The amino-terminal cytoplasmic domain contains approximately the first 68 residues, followed by the putative transmembrane domain underlined inFig. 1. In our first attempt to make anti-peptide antibodies to the cytoplasmic domain, a peptide containing aminoacid residues 2740 (Fig. 1, peptide 1) wascoupled to either bovine serum albumin or to thyroglobulin and injected into rabbits. The antisera failed to immunoprecipitate the TfR, although they reacted with the peptide bound to the carrier proteins by Western blot (data not shown). Thissuggests that peptide 1 conjugated tocarrierproteins may adopt a conformation different than in the native TfR or that the sequence of peptide 1 is sheltered in the receptor and is not available to react with antibodies. We then conjugated peptide 2, which contained the first 24 residues of the TfR, to bovine serum albumin and injected the conjugate into two rabbits. Two proteins of apparent molecularweights 96,000 and 66,000 were observed when antiserum from either rabbit was used to immunoprecipitate a lysate of HeLa cells that had been surPEPTIDE 2 10

96 66

FIG.2. Antibody to peptide 2 immunoprecipitates a protein the sizeof TfR. HeLa cells were labeled with I2'.I as described under "Materials and Methods." Immunoprecipitation of the lysates was with either monoclonal antibody B3/25 (known to react with the TfR), preimmune serum from each rabbit (pre A and pre H ) , or immuneserum from eachrabbit ( A and R ) . Theproteins were electrophoresed in a 7.5% polyacrylamide gel with SDS and detected by autoradiography. Themolecular mass of the proteins in kilodaltons estimated from the mobility of standard markers is shown at the right.

83/25

antipeptide

PEPTIDE 1 20

30

40

50

W R S A F S NLFGGEPLSY TRFSLAR~OW~6DNSHVMLAVOEEENAON 60

70

80

90

NTKANYTKPK RCSGSICYGT IAVIVFFLIGWIGYLGYCK

110

120

130

UGTESPVRE EPGEOFPAARRLYWDOLKRK

140

100

GVEPKTECER 150

LSEKLOSTOF TSTIKLLNEN

FIG. 1. The amino acid sequence (in one-letter code) of the first 150 residues of the human TfR. The cytoplasmic domain comprises aminoacids 1 toabout 68, andthe underlined region indicatestheputativetransmembranedomain.Sequencescorresponding to peptides 1 and 2 are shaded. Both peptidesalso contained a terminal cysteine residue, at the amino terminusof peptide 1 and at the carboxyl terminus of peptide 2. The trypsin cleavage site in the external domain of the TfR is believed to be either a t residues 120-121 ( R R )or possibly a t residues 128-130 ( K R K ) .

FIG.3. The 96-kDa protein immunoprecipitated by antibodies to peptide 2 has a peptide map corresponding to the TfR. V8 protease digestion of the immunoprecipitates obtainedfrom :"'S-labeled cellswas carriedoutusing 0.1 pg of V8 protease as described under"Materialsand Methods."Digestion of theTfR immunoprecipitated by B3/25 produced peptides of identical sizes to those produced by digestion of the 96-kDa protein immunoprecipitated by the antibody to peptide2.

Turnover of the Trypsinized Transferrin Receptor

21128

cells were treated with and without trypsin, and the electrophoretic mobility of proteins immunoprecipitated with the anti-peptide antibody was compared in SDS-polyacrylamide gels with and without mercaptoethanol (Fig. 5A). The outside lanes contained asa standard radiolabeled transferrin, which 96 has a slightly greater mobility when reducing agent is absent (lane 6 ) than when present (lane 1). Under reducing conditions, the 21-kDa bandwas present in trypsinizedcells (lane 3 ) but was absent in untrypsinized cells (lane 2 ) . However, the 21-kDa band was also absent under nonreducing conditions in trypsinizedcells (lane 4 ) , consistent with the prediction that the mobility of the 21-kDa band should be altered in the absence of reducing agent. Moreover, aband withlower 21 mobility appeared in lane 4 that was specific to trypsinized 21 cells (compare lanes 4 and 5). The new band (markedfragment dimer in lane 4 ) is probably a dimer of the 21-kDa fragment. FIG.4. A 21-kDa trypsin-specific fragment is immunopreconclusion that the21-kDa cipitated by antibody to peptide 2 and by goat antibody to Altogether, these data support the purified TfR. Cells were metabolically labeled with "'S and treated fragment is thecell-associated tryptic fragment of the TfR. with or without 200 pg/ml trypsin a t room temperature for 15 min. Evidence That the Cell-associated Tryptic Fragment ConProteins were immunoprecipitated and electrophoresed ina 10-1776 tains0-Linked Oligosaccharides-The trypsin-specific 21gradient polyacrylamide gel with SDS asdescribed under "Materials kDa fragment identified inFig. 4 has the propertiesexpected and Methods." A, immunoprecipitation was with antibody to peptide 2: lane I , no trypsin; lane 2, with trypsin. B, cells were treated with of the cell-associated tryptic fragment of the TfR with one trypsin and were lysed with detergent without removing the trypsin major exception: the apparent molecular weight is too high. solution so that the external tryptic fragment as well as the cell- The fragment should contain either 120 or 130 amino acids, associated fragment would be present in the lysate. Immunoprecipi- depending on exactlywhere trypsin cleaves (see Fig. l ) , which tation was with goat antibodyto purified human TfR. The estimated should give anelectrophoretic mobility in SDS-polyacrylmolecular mass of the proteins is indicated inkilodaltons. amide gels corresponding toa calculated molecular weight of 13,300-14,500. However, this discrepancy could be explained kDaand a cell-associated fragmentcontainingthecytoif the fragment contained carbohydrate that retarded mobilplasmic and transmembrane domains anda small portion of ity. The carbohydrate would have to be 0-linked since there the externaldomain. The site of trypsin cleavage is reported are no sitesfor N-linked carbohydrate in the external portion t o be a t arginine residues 120-121 (Turkewitz et al., 1988), of the tryptic fragment. Two recent reports indicate that the although itcould alternatively be at theLys-Arg-Lys sequence TfR contains 0-linked oligosaccharides (Neefjes et al., 1988; at residues 128-130 (see Fig. 1).T o determine if the antibody to peptide 2 could immunoprecipitate thecell-associated trypA tic fragment, HeLacells were metabolically labeled with 35S, @-ME + + + - - - + + - trypsin treated with or without trypsin, washed, lysed, and the TfR was immunoprecipitated (Fig. 4A).In the absenceof trypsin ( l a n e 1 ), a single majorprotein corresponding to the TfR was present. After trypsin treatment(lane Z ) , intact TfRwas still fragment dimer present, but in reduced amounts, consistent with observations that only about 30% of the TfR is on the surface of HeLa fragment cells and shouldbe accessible to external trypsin(Stoorvogel et al., 1989): In addition, trypsin treatment resulted in a 1 2 S 4 5 8 protein of apparent molecular mass of 21 kDathat was absent when cells were not treated with trypsin, suggesting that it was thecell-associated tryptic fragmentof the TfR. In another B experiment, 35S-labeled cells were treated with trypsin and NNRAUINIDASE were lysed without washing away the external TfR fragment - + so that both the external and cell-associated the fragments of 2 1 - 1 the TfR should be present. The TfR was then immunoprecipitated with goat antibody to intact purified human TfR. This FIG.5. Evidence that the 21-kDa cell-associated fragment antibody has the potential to react with both the external isand a disulfide-linked dimer and also contains 0-linked carbocytoplasmic domains of the TfR. Three major proteins of 96, hydrate. A , cells labeled with '"S were treated with (+) or without 70, and 21 kDa were immunoprecipitated, corresponding to (-) trypsin, and proteins were immunoprecipitated with antibody to peptide 2 as described under "Materials and Methods." The immuthe intact receptor, the external tryptic fragment, and the cell-associated tryptic fragment, respectively (Fig. 4B). Thus, noprecipitates were treated either with (+) or without (-) mercaptoethanol ( & M E ) and electrophoresed through a 10-17% gradient both antibody to peptide 2 and the goat antibody to the intact polyacrylamide gel containing SDS.'"I-Tf was used as a reference in TfR precipitated the21-kDa protein. lanes 1 and 6. The positions of TfR, Tf, fragment, and fragment The TfR is a dimer in the Dlasma membrane with the dimer are indicated. The dimerof intact TfR (lanes 4 and 5 ) is not monomersconnected by disulfide bonds betweenresidues shown because under these conditions it migrates near the top of the Cys-89 and Cys-98 (Jing andTrowbridge, 1987). The disulfide gel. B, the cell-associated fragment was immunoprecipitated from bonds remain intact after trypsin cleavage, so the cell-asso- ""S-labeled cells using the goat antibody topurified TfR. The immuciated tryptic fragment should also be a dimer. To test this, noprecipitate was then treated with (+) or without (-) 20 milliunits A

B

1

-

2

'5'''

E. A. Rutledge, C. A. Mikoryak, and R. K. Draper, unpublished results.

of neuraminidase a t 37 "C overnight as described under "Materials and Methods" and electrophoresed in a 10-17% gradient polyacrylamide gel containing SDS. The location of the 21-kDa trypsin-specific fragment before neuraminidase treatment isindicated.

Turnover Trypsinized of the Transferrin Receptor

21129

TARLE I Do et al. 1990), but it is notyet knownwhich serineor S u m m a r y of experiments to determine the half-lifrof the T f R and threonine residues are glycosylated. Thetrypticfragment the cell-associated tryptic fragmentof the 7 ’ f R contains three potential sites for 0-linked glycosylation, ThrHalf-lives were measured asdescribed under “Materials and Meth96, Thr-104, and Ser-106 (Fig. 1). If one or more of these residues contained 0-linked sugars that terminated in sialic ods.” Goat anti-TfR was used in Experiments 1 and 3. Antibody to peptide 2 was used in Experiment 2. acid, then the electrophoretic mobility of the fragment in SDS Half-life gels should increase after treatment with neuraminidase. As Fragment/Intact Fragment Intact TfR seen in Fig. 5B, the mobility of the fragment increased after neuraminidase treatment. Thissuggests that at least one, and h /l 7; possibly more, of the Ser or Thr residues were glycosylated Exp. 1 19 13 68 11 100 and that the apparent molecular weight of the tryptic frag11 Exp. 2 21 81 26 Exp. 3 ment estimated from its mobility in SDS gels is an over15 & 4 83 c 19 Average & S.D. 19 f 6 estimate due to 0-linked glycosylation.

The TfR and the Cell-associated Tryptic Fragment Are Turned Over at Similar Rates-To compare the half-lives of the intact TfR and the tryptic fragment, metabolically labeled cells were trypsinized and replated. At 5-h invervals over the next 30 h, the intact and trypsinized forms of the receptor were immunoprecipitated. The amountof radioactivity in the proteins was determined by quantitating the fluorograms as described under “Materials and Methods.” Portions of the fluorograms from one experiment are shown in Fig. 6, and graphic analysis of the data is presented in Fig. 7. Table I summarizes the data from three independent experiments. The variation in half-life among experiments was unexpectedly high, ranging from 11 to 26 h for the intact receptor and from 11 to 21 hfor the fragment. However,for any given experiment, thehalf-life of the fragmentwas close to thatfor the intact receptor, being on average 83 f 13% of the intact

receptor. We conclude from this data that thehalf-life of the receptor is not markedly affected by the absenceof the extracellular tryptic fragment. We also determined that the halflife of the intact receptor in untrypsinizedcells was 23 f 4 h (data not shown), similar to that in trypsinized cells. This demonstrated that thecells were not so adversely affected by the trypsinization process that the turnover of the intact receptor was significantly altered. DISCUSSION

Apolypeptidewith an electrophoretic mobilityin SDSpolyacrylamide gels corresponding toa molecular mass of 21 kDa was immunoprecipitated from trypsin treated HeLacells by antibody to peptide 2. Several lines of evidence indicate that this polypeptide is the cell-associated tryptic fragment derived from the TfR. 1) The fragment was only observed in CHASE (h) trypsin treatedcells. 2) Goat antibody topurified human TfR 5 10 15 20 25 30 also immunoprecipitated a trypsin-specific 21-kDa fragment. 3) The 21-kDa fragment migrated as a higher molecular mass species in nonreducing polyacrylamide gels, consistent with the prediction that the fragment shouldbe a disulfide-linked dimer. 4)Thefragmentcontained0-linkedcarbohydrate, FIG. 6. Example of a fluorogram used inassessing the halfevidenced by the increased mobility in SDS-polyacrylamide life of the intact TfR and the 21-kDa cell-associated tryptic fragment. Cells labeled with ‘“Swere treated with trypsin, washed, gels after neuraminidase treatment, consistent with reports 0(Neefjes et al., 1988; Do et al., 1990) that the TfR contains and incubated in medium a t 37 “C in the absence of radioactivity. At chase intervals of 5 h as indicated, a sample of cells was lysed and linked carbohydrate.Yoshimori et al. (1988) also immunopreproteins were immunoprecipitated with goat antibody to purified TfR. cipitated a protein with apparent molecular mass of 25 kDa A , 22-h exposure to show intact TfR.R, 5-day exposure of the same from trypsin-treated human amnion cells using a monoclonal gel to show the 21-kDa fragment. antibody thatrecognized the cytoplasmic domain of the TfR. The average half-life we found for the TfR was 19 f 6 h. The reason there was such alarge variation among three independent experiments, from 11 to 26 h, is not clear, but may reflect the technically demanding nature of the experiments. Nevertheless, 19h is ingood agreement with half-lives reported in the literature for the intact TfR: 14 h in HeLa cells (Ward et al., 1982), 8 h (Weissman et al., 1986) and 15 h (Snider and Rogers, 1985) in K562 cells, 16 h in HL-60 cells (Enns et al., 1988), and 10-12 h in mouse AKRl cells (Lesley and Schulte, 1985). An unusually longhalf-life of 60 h in CCRF-CEM cells was reported by Omary and Trowbridge (1981). The averagehalf-life of the 21-kDa cell-associated 5 15 25 trypticfragment was 15 +- 4 h. This is not significantly CHASE (h) different than the half-life of the intact receptor; however, FIG. 7. Graphical analysis of densitometric data to assess the half-life of t h e TfR and the 21-kDa cell-associated frag- given the uncertainty in the data we cannot resolve small ment. Intensities associated with the bands in Fig. 6 were determined differences in half-lives,on the orderof a few hours, thatmay as described under “Materials and Methods” and were plotted as a exist between the intact receptor and the fragment. Neverpercentage of the 5-h time point. The 5-h time point was chosen as theless, the data is good enough to conclude that removing the reference to allow the cells to recover from trypsin treatment. about 95% of the external domain of the TfR neither increased The percentages are correctedfor cell growth during the chaseperiod according to the increase in total protein in each lysate. The lines nor decreased the turnover rate of the receptor to any major represent the best tit to the data for the cell-associated fragment extent. Factors regulating the turnoverof membrane proteins are (filled circles, r = 0.981) and the intact TfR (open circles, r = 0.904) The data in this figure correspond to Experiment 1 in Table I. not well understood; however, it is generally accepted that

21130

Turnover of the Trypsinized Transferrin

turnover of most plasma membrane proteins occurs in lysosomes (Hare, 1990; Hare and Huston, 1985). Access to lysosomes normally depends on whethera plasma membrane protein is endocytosed, so a critical parameter in the rate of turnover should be the rate of endocytosis. There is experimental evidence to support the importance of endocytosis in the rapid turnover of plasma membrane proteins; membrane receptors that areefficiently endocytosed have half-lives generally less than 22 h, whereas most other plasma membrane proteins have half-lives between 30 and 100 h (Hare, 1990). The fact that the half-life of the TfR did not increase upon removing most of the extracellular domain suggests that the tryptic fragment is still efficiently endocytosed. The last 639 amino acid residues of the TfR, missing in the tryptic fragment, probably contain no structural feature thatis required for efficient endocytosis. Once within endosomes the TfR is either recycled or transported to lysosomes. The balance between these two processes should be an important determinant of the rate of turnover; TfFts that are not recycled presumably enter lysosomes and are degraded. Since the 21-kDa fragment was not degraded markedly faster than the intact TfR, the fragment is apparently not transferredfrom endosomes to lysosomes to a much greater extent than the intact TfR. We infer from this that the absence of 95% of the external domain has littleinfluence on the process that regulates the rate at which the TfR is transferred from endosomes to lysosomes as partof the turnover process. It is likely that the21-kDa fragment is recycled normally from within endosomes, suggesting that themissing external domain is not essential for recycling; however, with the data available we cannot exclude the possibility that truncated receptors are retained within endosomes where they are neither rapidly degraded nor recycled. Although the absence of the external domain of the T W had no major effect on theturnover of the receptor, this does not imply that theexternal domain is incapable of influencing turnover. For example, if the external domain were damaged or denatured in some way, there might be quality control mechanisms to recognize damaged or denatured regions and either keep the aberrant receptors from recycling or divert them to lysosomes. This would be analogous in concept to retention and degradation of misfolded proteins in the endoplasmic reticulum. Acknowledgment-We thank Howard Sussman (Stanford University) for providing goat anti-human transferrin antibodies.

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