Proc. Nati. Acad. Sci. USAVol. 82, pp. 5170-5174, August 1985Medical Sciences

Hemin inhibits internalization of transferrin by reticulocytes andpromotes phosphorylation of the membrane transferrin receptor

(regulation/endocytosis/erythropoiesis)

TIMOTHY M. COX*t, MARTIN W. O'DONNELL*t, PHILIP AISENt, AND IRVING M. LONDON**Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology and Department of Biology, Massachusetts Institute ofTechnology, Cambridge, MA 02139; and *Departments of Physiology and Biophysics and of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461

Contributed by Irving M. London, March 25, 1985

ABSTRACT Addition of hemin to reticulocytes inhibitsincorporation of iron from transferrin [Ponka, P. & Neuwirt,J. (1969) Blood 33, 609-707]. Heme also regulates proteinsynthesis in immature erythroid cells through its effects onphosphorylation of the initiation factor eIF-2. We have there-fore examined its effects on endocytosis of iron-transferrin andphosphorylation of the transferrin receptor. Hemin (10-50gM) reduced iron transport but increased cell-associatedtransferrin. When intracellular iron delivery was inhibited byNH4Cl, no such increase in cell-associated transferrin was seen.During uptake of 125I-labeled transferrin in the steady state, theuse of a washing technique to dissociate bound transferrin onthe cell membrane showed that radioligand accumulated on thesurface of hemin-treated cells. Hemin reduced the initial influxof transferrin, thereby diminishing incorporation of iron.Receptor phosphorylation was investigated by immunoprecip-itation of reticulocyte extracts after metabolic labeling with[32p]p1. In the absence of ligand, phosphorylated receptor waschiefly localized on cell stroma. Exposure to transferrin in-creased cytosolic phosphorylated receptor from 15-30% toapproximately 50% of the total, an effect overcome by hemin.treatment, Addition of hemin in the presence of transferrinenhanced net phosphorylated receptor in the reticulocyte inassociation with a redistribution of phosphorylated receptor tostromal membranes. The findings suggest a possible relation-ship of phosphorylation to endocytosis of the transferrinreceptor in reticulocytes.

rylation (10). Heme regulates a protein kinase that specifi.cally phosphorylates the a subunit of eukaryotic initiationfactor 2 (eIF-2) (11). In heme deficiency, this kinase isactivated and eIF-2 is phosphorylated. The phosphorylatedeIF-2 binds tightly to the reversing factor, a guanine nucleo-tide exchange factor, which is involved in the recycling ofeIF-2. The reversing factor is sequestered in the complex andis unavailable for the recycling of eIF-2, with the result thatchain initiation ceases (12). Thus, in the immature erythroidcell, heme has separate effects on interrelated pathways inthe biosynthesis of hemoglobin.

Transferrin is the normal donor of iron for heme synthesis,by a process of receptor-mediated endocytosis (13). Afterbinding to the cell surface, transferrin is internalized rapidlyas a ligand-receptor complex via coated pits (14). Thereafter,protein ligand and receptor enter an acidic, nonlysosomalcompartment (15). Following removal of iron, theapotransferrin-receptor complex remains intact during cy-cling through the cell to the surface, where apotransferrin isdischarged to the exterior as the vacant receptor is reincor-porated into the plasmalemma (16). The transferrin receptoris a thiol-linked homodimer (Mr 190,000) that contains phos-phate linked covalently to serine residues (17). Accordingly,this paper, which concerns the mechanism by which hemeaffects the iron-transferrin-receptor cycle in erythroid cells,examines the possibility that phosphorylation of the trans-ferrin receptor is involved in this effect of heme.

In immature erythroid cells, heme is of central importance inthe coordination of major biosynthetic pathways for hemo-globin formation. Assimilation of iron, formation of heme denovo, and protein synthesis in these cells are interrelatedprocesses subject to control by the availability ofendogenousheme. In whole animals and intact reticulocytes, heminprofoundly inhibits receptor-mediated iron uptake from plas-ma transferrin by an unknown mechanism (1, 2). Underconditions of suboptimal or inhibited protein synthesis, risingconcentrations of endogenous free heme probably accountfor the progressive decline in the rate of iron uptake byreticulocytes (2, 3). Heme also exercises negative feedbackcontrol on its own synthesis by one or more reactionsinvolved in the formation of 8-aminolevulinic acid inreticulocytes (4, 5). Moreover, hemin directly inhibits theactivity of 5-aminolevulinate synthase purified from thesecells (5, 6).

In contrast, maintenance of active protein synthesis indeveloping erythroid cells is dependent upon the availabilityof intracellular heme (7). In iron-deficient or heme-depletedintact reticulocytes and their lysates, initiation of proteinsynthesis is inhibited (8, 9). Extensive studies indicate thatthis mechanism is controlled by heme-dependent phospho-

MATERIALS AND METHODSCells. Reticulocyte-enriched blood (15-40% on supravital

stain) was collected, into heparin solution, from rabbitsrendered anemic by repeated phlebotomy (hematocrit25-35%, vol/vol). After separation and removal of the buffycoat, the cells were washed three times by centrifugation inreticulocyte saline (RS: 130 mM NaCl/5 mM KCl/7.4 mMMgCl2/10 mM Hepes, pH 7.4).

Proteins. Transferrin was prepared (>95% purity) frompooled rabbit serum (18). Transferrin-free rabbit albumin wasprepared from 10 ml of serum diluted with an equal volumeof RS and passed over a 50-ml column bed of Affi-Gel blueagarose (Bio-Rad) in the same buffer. After extensive wash-ing with RS, the column was pulsed with 3 M NaCl. Theeluted albumin was dialyzed against RS and freed of trans-ferrin by passage over a 2-ml bed of specific guinea pigantibody (5 mg/ml) to rabbit transferrin immobilized onCNBr-activated Sepharose (Pharmacia). This antibody wasprepared from the serum ofimmunized guinea pigs by affinitypurification and desorption chromatography on rabbit trans-ferrin-Sepharose 4B.

Abbreviation: RS, reticulocyte saline.tPresent address: Department of Medicine, Royal PostgraduateMedical School, London, U.K.

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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.

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'251/59Fe-Dual-Labeled Transferrin. The protein was ren-dered iron-free by exhaustive dialysis at 40C against 0.1 Msodium acetate/0.05 M sodium citrate, pH 4.5, followed bydeionized H20. The apotransferrin was labeled with 59Fe(Amersham, 1.5 mmol/Ci; 1 Ci = 37 GBq) as fresh ferricnitrilotriacetate (molar ratio 1:4, pH 7.4), sufficient to satu-rate the protein as judged from the A465/A280 ratio (19). After30 min, the protein was passed over a PD-10 column(Sephadex G-25, Pharmacia) equilibrated in RS. Iron-satu-rated transferrin was further labeled for 30 min at 250C with1251 (New England Nuclear; 17 Ci/mg) using IODO-GEN(Pierce), following the procedure recommended by the man-ufacturer. Free iodine (70% of total iodine) was removed bypassage over a PD-10 column equilibrated in RS and byseveral cycles of dialysis against RS at 4TC. More than 99%of the radioactivity due to 1251 was precipitable by ice-cold10% (wt/vol) trichloroacetic acid, and the specific radioac-tivity was 10 MCi/mg of protein for each isotope.

Incubations. Washed cells were incubated as a 20-30%(vol/vol) suspension in RS at 370C. The medium was sup-plemented with 20 L-amino acids (25-50 AM), 5.5 mMD-glucose, and proteins, as indicated, at 1 mg/ml. Whereshown, aqueous hemin hydrochloride in 1 M NaOH wasadded to incubation mixtures, which were then restored topH 7.4 by addition of acid Hepes. The A610 of 1 mM heminin 0.1 M NaOH was taken as 4.6. NH4Cl and 4,6-dioxoheptanoate were added from neutralized stock solu-tions stored at -70°C. Cells were labeled with 32P by additionof carrier-free H332P04 (50 mCi/ml, New England Nuclear) toa final concentration of 1-2 mCi/ml.

Transferrin Receptor and Specific Receptor Antibody.Transferrin receptor was purified from reticulocyte stromaby affinity chromatography. Transferrin was linked to CNBr-activated Sepharose 4B (Pharmacia) by following the manu-facturer's recommended procedure. The gel (2 mg ofprotein/ml) was pretreated with 0.5% HAc and, after equil-ibration in 10 mM Hepes/130 mM NaCl/0.1% Triton X-100,pH 7.4 (Hepes/NaCl/Triton), was resaturated with iron byaddition of ferric nitrilotriacetate. Reticulocytes were incu-bated in transferrin-free RS for 20 min at 37°C to vacatereceptors, and stromal membranes were sedimented andwashed by centrifugation at 10,000 x g after hypotonic lysis.The membranes were solubilized in 1% Triton, and a 105,000x g supernatant extract was prepared. The extract waspassed over the 2-ml column of transferrin-Sepharose. Thecolumn was washed extensively with Hepes/NaCl/Tritonbuffer, followed by buffer of this same composition butcontaining 0.5 M NaCl. Receptor was eluted with 5 bedvolumes of 0.5% HAc/0.1% Triton, concentrated bycoprecipitation with trichloroacetic acid and deoxycholate,and washed in ether, acetone, and methanol at -20°C (20).The protein pellet, solubilized in 9 M urea, showed a majorband at 190 kDa under nonreducing conditions and at 90-95kDa under reducing conditions after electrophoresis accord-ing to Laemmli (21). The yield from 40 ml of stroma was 1-2mg of receptor protein.Antiserum was obtained from a sheep after four antigenic

challenges at multiple intradermal sites over 9 weeks. Theovine serum was adsorbed with 4 ml of transferrin-Sepharoseand by incubation at 4°C with washed erythrocyte stroma inHepes/NaCl containing 1 mM phenylmethylsulfonyl fluorideand 10 mM EDTA at pH 7.4. After a similar incubation for 14hr with reticulocyte stroma, specific antibody was elutedfrom the washed membranes with 1% HAc. The antibodywas concentrated, after neutralization and removal of dena-tured protein sediment, by precipitation with 50% saturated(NH4)2SO4. The antibody was finally dialyzed against 130mM NaCl/10 mM Na phosphate, pH 8.0. The yield was 5-10mg of immunoglobulin per 100 ml of serum, and electropho-retic purity exceeded 80%. Rabbit anti-sheep IgG was pre-

pared from the serum of an animal repeatedly immunizedwith sheep IgG and was affinity-purified by acid desorptionfrom ovine IgG-Sepharose 4B.

RESULTSEffect ofHeme on Transferrin-Iron Incorporation. Addition

of fresh hemin to reticulocytes rapidly inhibited overalluptake of iron from [59Fe]transferrin. This effect was accom-panied by a parallel reduction of radioiron incorporation intocellular heme (Fig. 1). The influence of exogenous hemincould be detected at a concentration near 10 ,uM and could bedemonstrated in experiments where reticulocytes were ex-posed to hemin during a preincubation only. To determinewhether this phenomenon is related to an imbalance of hemeand globin synthesis in favor of endogenous heme, a specificinhibitor of heme formation, 4,6-dioxoheptanoate, was used(22). Although delayed addition of dioxoheptanoate rapidlydecreased incorporation of radioiron into heme as expected,the reagent allowed reticulocytes to maintain their initial rateof iron uptake for more than 120 min (Fig. 1 Left). Ironincorporation into heme, however, was measurably de-pressed (Fig. 1 Right), presumably indicating accumulationof iron in a pre-heme pool. Similar results were obtainedwhen dioxoheptanoate was added to cells while proteinsynthesis was inhibited markedly by NaF or cycloheximide(not shown). Diminished incorporation of iron into hemin-treated reticulocytes was accompanied by a persistent in-crease in cell-associated 125I-labeled transferrin (Fig. 2). Nosuch increase in transferrin binding was seen when NH4Clwas used to inhibit iron uptake (23). These findings prompteda more rigorous analysis of the effects of these agents onendocytosis of the receptor-transferrin complex.

Effects on Endocytosis. Reticulocytes acquire transferrin bybinding the protein to cell surface receptors. Hence, heminmay enhance association of the ligand with these cells eitherby stimulating receptor activity on the membrane surface orby inhibiting release of apotransferrin from the cell. In-creased surface membrane receptors could result principallyfrom reduced internalization, recruitment of latent receptors,or both. To discriminate between these possibilities, weexamined the kinetics of 125I-labeled transferrin internaliza-tion. The distribution of 125I was studied during steady-state

Total Incorporation Incorporation into Heme70-

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5172 Medical Sciences: Cox et al.

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FIG. 2. Uptake of dual-labeled transferrin. Cells were incubatedin nutrient medium containing 59Fe/251I-labeled transferrin (1.1mg/ml). Hemin (50 /AM) or NH4Cl (20 mM) was added and theseparate radioactivities were measured in washed cell pellets.

transferrin uptake: surface-bound ligand and internalizedligand were assessed by measuring the cellular radioactivityresistant to, or removed by, washing in ice-cold buffercontaining 100 jLM desferrioxamine at pH 5.0 or pH 7.4 (24)(Fig. 3). This technique depends on the ability of desfer-rioxamine to complex iron removed by exposure of transfer-rin to mildly acidic conditions. The low affinity of theapotransferrin for its receptor at pH 7.4 allows this ligand tobe dissociated from the cell surface by subsequent washingsat neutral pH in the presence of desferrioxamine to preventrebinding of iron (24). Under steady-state conditions, hemintreatment led to an increased association with ligand whichwas largely accounted for by a progressive accumulation of1251-labeled transferrin on the cell surface. In cells incubatedwith 125I-labeled transferrin under control conditions or in thepresence of NH4Cl, the ligand was internalized rapidly at theexpense of the surface component, which decreased slightly.To determine whether transferrin accumulates on the cell

surface as a primary effect of hemin treatment or as a resultof inhibition of the endocytotic process within the cell, theinfluence of hemin and NH4Cl on the endocytosis of a single

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cohort of surface receptors was analyzed by following theintracellular passage of dual-labeled transferrin.

59Fe/'251-labeled transferrin was added to the cells at 0C,and unbound ligand was washed away before further prein-cubation with hemin or NH4Cl in the cold. Internalization ofradioactivity was measured at intervals after rapid warmingto 370C in the presence of unlabeled transferrin (Fig. 4). Incontrol incubations, 59Fe was rapidly internalized, reachingan early plateau of radioactivity resistant to desferrioxaminetreatment. The fate of "25I-labeled transferrin differed in thatmaximal incorporation at 2 min was followed by exponentialdischarge from the cells. Internalization of transferrin in thepresence of NH4Cl was unaffected but dissociation of 59Fe/125I-labeled transferrin failed to occur and both labels wereretarded in their efflux back to the medium. In contrast,hemin inhibited the influx of transferrin but did not affect itssubsequent discharge from the cells. These effects reducedthe rate of cellular iron influx and the extent of iron retentionfrom a single cycle of endocytosis. Furthermore, only whenhemin was preincubated with reticulocytes did it inhibit thefirst cycle of internalization of labeled transferrin. Thus,internalization is more rapid than the onset of the effect ofhemin, which therefore appears not to be exerted directly atthe cell surface.

Phosphorylation of the Receptor. Reticulocytes were la-beled metabolically with 32p for 90 min and isotope incorpo-ration into transferrin receptor was analyzed after immuno-precipitation with specific antibody. Antibody, but notpreimmune IgG, specifically precipitated a 92-kDa phospho-protein (electrophoresis under reducing conditions) fromreticulocyte extracts. This phosphoprotein was coincidentwith a single 125I-labeled species in stromal extracts from cellslabeled in situ with IODO-GEN (Pierce) and with stainedreceptor protein obtained from columns of transferrin-Seph-arose. Phosphorylated receptor was detected in stromal andcytosolic fractions, after electrophoresis of immunopre-cipitates, by radioautography (Fig. 5). In three experiments,labeled receptor was quantified by laser densitometry. In thepresence of transferrin, the ratio of phosphorylated receptorin stroma to that in cytosol was 0.7-1.4; addition of heminmarkedly altered phosphorylation in favor of the stromalcomponent (ratio 2.5-7). In the absence of transferrin (cells

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FIG. 3. Steady-state internalization of 125I-labeled transferrin. Intwo separate experiments, with hemin (50 zM) or NH4Cl (20 mM),cells were incubated with labeled transferrin (1 mg/ml) and afterrapid washing in RS at 0CC, radioactivity in pellets and two furtherwashes was determined. Washes with 150mM NaCI/25 mM NaOAc,pH 5.0/100 ALM desferrioxamine and 150 mM Tris Cl, pH 7.4/50mMNaCI/100 ;LM desferrioxamine at 0C were used (24).

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FIG. 4. Pulse-chase internalization kinetics of dual-labeled trans-ferrin. Cells were incubated at 0C for 30 min with 59Fe/1251-labeledtransferrin (0.26 mg/ml). After two washes in RS, the cells wereresuspended in four volumes of nutrient medium containing hemin(50 .uM) or NH4Cl (20 mM) and kept on ice for 20 min. One volumeof 40o rabbit plasma in RS was added to promote release of labeledtransferrin from surface receptors, and after warming to 370C,samples were removed for measurement of radioactivity in cellpellets resistant to desferrioxamine washing, as in Fig. 3.

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FIG. 5. Phosphorylation of transferrin receptors. Cells wereincubated in nutrient medium with various additions (indicatedbelow) and [32p]p; (1 mCi/ml) for 90 min at 370C. Labeling wasstopped with ice-cold 150 mM NaF/20 mM Na phosphate, pH 8.0,and the cells were lysed in six volumes ofH20 followed by two-thirdsvolume of 500 mM NaF/100 mM EDTA/1 mM vanadate, pH 7.5.The 10,000 x g supernatants were mixed with one-ninth volume of10% Triton X-100/10%o sodium deoxycholate/1% NaDodSO4.Stromal pellets were washed twice in 50mM NaF/10mM EDTA/0.1mM vanadate, pH 7.5, and solubilized in this containing 1%Triton/1% sodium deoxycholate/0.1% NaDodSO4. Samples (eachequivalent to 75 Al ofpacked cells) of 105,000 x g supernatants wereimmunoprecipitated overnight on ice with 50 Aug of receptor anti-body, followed by rabbit anti-sheep IgG. Washed precipitates (eachequivalent to 12 ,ul of cells) were electrophoresed, and the dried gelwas exposed to Kodak X-Omat AR5 film for 2 days. Stromal andcytosolic receptor (TR) are shown in lanes 1-5 and 6-10, respec-tively. Additions to incubation medium were as follows. Lanes 1 and6: rabbit serum albumin (1 mg/ml). Lanes 2 and 7: apotransferrin (1mg/ml). Lanes 3 and 8: transferrin (1 mg/ml). Lanes 4 and 9:transferrin plus 250 ,uM dioxoheptanoate. Lanes 5 and 10: transferrinplus 50 AM hemin.

incubated with apotransferrin or transferrin-free albumin),stromal receptor phosphorylation predominated (ratio 3-6).Phosphorylation of cellular transferrin receptors was in-

creased by hemin treatment (Fig. 5) in the presence oftransferrin. Since hemin is required for active protein syn-thesis in reticulocytes, the effect on de novo synthesis ofmembrane receptors was examined by metabolic labelingwith L-[35S]methionine. Under the conditions used for phos-phorylation in the presence of transferrin, no appreciableincrease in the synthesis ofmembrane receptors was inducedby hemin (data not shown). Phosphorylation of transferrinreceptors was not reduced by incubation of the cells withcycloheximide (40 ,g/ml), indicating that phosphorylation isnot restricted to nascent receptors. To discriminate betweenenhanced receptor phosphorylation and redistribution ofphosphorylated receptors, whole-cell extracts and immuno-precipitates from these extracts were examined (Fig. 6).Phosphoprotein profiles and measurement of trichloroaceticacid-insoluble 32p in whole extracts revealed no changes inoverall radioactivity. Immunoprecipitation of whole extractsconfirmed that in the presence of transferrin, hemin, and toa lesser degree NH4Cl, enhanced overall phosphorylation oftransferrin receptors. Net phosphorylation of the cellulartransferrin receptors was similarly stimulated when rabbitalbumin or apotransferrin was substituted for the normalligand (Fig. 6). Under conditions of absent or inhibitedendocytosis, the stimulation was estimated in two experi-

1 2 3 4 5 6

FIG. 6. Phosphorylation of total cellular transferrin receptor(TR). Cells were incubated in nutrient medium and [32P]Pj asdescribed in the legend to Fig. 5. After quenching and washing at 00C,the cells were lysed in 1% Triton/1% sodium deoxycholate/0.1%NaDodSO4/50 mM NaF/10 mM EDTA/0.1 mM vanadate/20 mMTris Cl, pH 7.5. Immunoprecipitates from high-speed supernatantswere analyzed by electrophoresis and radioautography. Each trackrepresents the equivalent of 6 u1l of cells. Additions to incubationmedium were rabbit serum albumin (1 mg/ml, lane 1), rabbit serumalbumin plus 50 gM hemin (lane 2), apotransferrin (1 mg/ml, lane 3),apotransferrin plus hemin (lane 4), transferrin (1 mg/ml, lane 5), andtransferrin plus hemin (lane 6).

ments to be 1.5 and 2.6 times that of the transferrin control.No differences in phosphopeptides generated from mem-brane or cytosolic receptor or in response to hemin treatmentwere detected by partial proteolytic mapping (data notshown).

DISCUSSIONHemin rapidly inhibits the incorporation of transferrin ironinto reticulocytes. The sensitivity of iron incorporation tochanges in intracellular heme, including those that occurduring perturbed formation of heme or globin, indicates thatendogenous heme may be a natural modulator of transferrinendocytosis in reticulocytes. Kinetic studies have shown thathemin affects endocytosis by a primary action on the influxof ligand across the surface membrane, thereby reducing thedelivery of iron. Unlike NH4Cl, a weak base which impairsacidification and prevents the intracellular dissociation of theiron-transferrin complex (23), hemin does not affect the latterphases of endocytosis. Hemin treatment in the presence ofligand thus leads to an accumulation of transferrin receptorson the reticulocyte surface.

In this study, care was taken to minimize artifactualchanges in phosphorylation which we recognize may occurduring prolonged processing for immunoprecipitation analy-sis of receptors in cell extracts. Given this qualification,during inhibition of transferrin internalization or inhibition ofthe dissociation of iron from internalized transferrin, a netincrease in phosphorylation of the transferrin receptor waspromoted. Under these circumstances and also in cellsincubated in the absence of ligand, an increased proportion ofphosphorylated receptor was detected in the stromal com-partment.The effect of hemin in increasing phosphorylation of total

cellular receptors and on the distribution of phosphorylatedreceptor within cell compartments was dependent on the

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presence of transferrin. This finding suggests that whenhemin inhibits receptor-mediated endocytosis of transferrin,the influence of transferrin itself on phosphorylation of thereceptor may be observed. Our data are consistent with thehypothesis that a cycle of phosphorylation and dephospho-rylation may be involved in the mechanism of receptorinternalization. Since phosphorylation of total immuno-precipitable receptors is increased when endocytosis isinhibited in the absence ofligand or treatment with hemin, theprocess of internalization may be associated with dephos-phorylation of the receptor.These effects of hemin in normal reticulocytes differ from

those observed in human erythroleukemia K562 cells and inseveral cultured lines of nucleated cells in which hemindown-regulates surface expression of transferrin receptors(25, 26). In K562 cells, hemin induces erythroid differentia-tion and synthesis of hemoglobin (27), and the erythroiddifferentiation is associated with increased phosphorylationof the transferrin receptor and greater internalization of thesurface receptors (26). Treatment of K562 and HL60 cellswith active phorbol ester also induces rapid internalization ofsurface receptors without alteration of the total receptorcomplement and leads to increased phosphorylation of thereceptors (28, 29). But neither hemin nor phorbol ester affectsthe uptake of iron by K562 cells (29, 30). These diversefindings indicate that the effects of hemin on endocytosis ofthe receptor-transferrin complex and on phosphorylation ofthe receptor are not uniform and appear to vary with the typeof cell and the stage of its differentiation.The increased phosphorylation of the receptor brought

about by hemin in the presence oftransferrin may result fromrecruitment of more receptors to the surface membrane orfrom a stimulation of net kinase activity. It is thus of interestthat in vitro phosphorylation of the transferrin receptor inmembranes from sheep reticulocytes does not show directdependence on the presence of transferrin but that thereceptor can undergo phosphorylation in isolated immuno-precipitates (31). Further clarification of the mechanism ofincreased phosphorylation will require measurement of thespecific activity of phosphorylated receptors in the differentcellular compartments. The present observations point to arelationship ofphosphorylation to the cycle ofreceptor-trans-ferrin endocytosis. The effect of hemin on internalization ofthe receptor-transferrin complex affords an opportunity toprobe the possible link between phosphorylation andendocytosis in the interaction of transferrin with the reticu-locyte.Note. After completion of this work and during preparation of themanuscript, a study by lacopetta and Morgan (32) appeared whichalso presents evidence that hemin inhibits internalization of thereceptor-transferrin complex in reticulocytes.

We thank Dr. A. Ciechanover for vigorous discussion and advice.The work was supported by the award of a Wellcome Senior ClinicalResearch Fellowship to T.M.C. and by Public Health Service GrantAM16272.

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