Proc. Natl. Acad. Sci. USAVol. 81, pp. 3005-3009, May 1984Biochemistry

Rapid internalization of the transferrin receptor in K562 cells istriggered by ligand binding or treatment with a phorbol ester

(endocytosis/receptor cycling)

RICHARD D. KLAUSNER, JOE HARFORD, AND JOS VAN RENSWOUDELaboratory of Biochemistry and Metabolism, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health,Building 10, Room 9N-119, Bethesda, MD 20205

Communicated by Gilbert Ashwell, January 26, 1984

ABSTRACT Treatment of human K562 cells with 4f3phorbol 12-myristate 13-acetate (PMA) resulted in an approxi-mately 50% reduction in cell surface transferrin receptorswithin 30-45 min as judged by binding of both ligand and anti-receptor antibody. The affinity of the remaining surface re-ceptors for diferric transferrin appeared to be unaltered. Thetime-dependent loss in transferrin receptors was also depen-dent upon PMA concentration, with a half-maximal effect ob-served at approximately 1 nM. The kinetic parameters for thebinding, internalization, intracellular residency, and recyclingof 2'I-labeled transferrin were unchanged by PMA treatment,as were the rate and extent of internalization of anti-receptorantibody. Moreover, despite the decrease in surface receptors,uptake of 59Fe from transferrin proceeded at a rate compara-ble to that seen in untreated cells. Accounting for this observa-tion was the fact that ligand induced a reduction in surfacereceptors in untreated but not PMA-treated cells. Quantitativeimmunoprecipitation of transferrin receptors from surface-io-dinated K562 cells revealed that little receptor internalizationoccurred in untreated cells in the absence of ligand, but inter-nalization of ligand-occupied receptors in these cells was readi-ly detected. In contrast, PMA treatment resulted in the rapidinternalization of surface receptors irrespective of occupancy.Thus, binding of ligand appeared to trigger the internalizationof receptors that were relatively static in their unoccupiedstate, and a signal for receptor internalization was also provid-ed by PMA treatment. The possibility that this signal involvesphosphorylation of the transferrin receptor is discussed.

Phorbol esters have been studied for several decades, largelybecause of their action as tumor promoters (reviewed in ref.1). Cells undergo a wide variety of changes after treatmentwith phorbol esters, including alterations in the synthesisand turnover of phosphatidylcholine, prostaglandins, poly-amines, proteins, DNA, and RNA. Recently, interest hasbeen heightened by the identification of specific phorbol es-ter receptors (2), the discovery that phorbol esters activateprotein kinase C (3), and the likely role of this kinase as thereceptor (4-6). How activation of this kinase leads to themany cellular events triggered by phorbol esters is unknown.Whereas multiple changes in the cell surface upon treatmentwith phorbol esters have been described (1), it is not clearthat all such changes are attributable to phosphorylationevents. Phorbol esters may alter, directly or indirectly, awide variety of cell surface receptors. Exposure to phorbolesters leads to a loss of high-affinity binding sites for epider-mal growth factor (EGF) (7) and somatomedin (8) and hasbeen shown to induce phosphorylation of the receptor forEGF (9), insulin, and somatomedin C (10). In this report, wepresent evidence for a specific alteration in the dynamics ofthe human transferrin (Tf) receptor that accompanies treat-

ment of K562 cells with 43-phorbol 12-myristate 13-acetate(PMA). The effect of treatment with PMA is to shift thesteady-state distribution of Tf receptors so that a smallerpercentage of the total cellular receptors is found on the cellsurface. This effect is indistinguishable from the receptor re-distribution induced by ligand in untreated cells. We proposethat a specific signal is required for the rapid internalizationand cycling of the Tf receptor and that such a signal can beprovided by treatment with either ligand or PMA.

MATERIALS AND METHODSK562 cells were grown in RPMI 1640 medium containing10% fetal bovine serum at densities of between 2 and 6 x 10cells per ml. The PMA (Sigma) was dissolved in dimethylsulfoxide (Me2SO) at a concentration of 20 ,ug/ml and storedat -20°C, and dilutions (1:1000) of this stock were added togrowth medium. Control cells were treated with 0.1%Me2SO. Human Tf (Calbiochem) was made diferric accord-ing to published procedures (11) and was iodinated by usinglactoperoxidase/glucose oxidase immobilized on beads (En-zymobeads, BioRad). After 30 min of iodination at roomtemperature according to the supplier's instructions, the re-action mixture was passed through a Sephadex G-25 column.The protein peak fractions were dialyzed at 4°C against 500vol of 20 mM Tris HCI/0.15 M NaCl, pH 7.4. The specificactivity of the ligand employed in these studies was approxi-mately 550 cpm/ng of protein.Monoclonal antibody to Tf receptor (OKT9, Ortho Diag-

nostics) was purified by passage through DEAE-Sephacel(Pharmacia), and this preparation consisted largely of IgG(>90%) as judged by sodium dodecyl sulfate/polyacrylam-ide electrophoresis. The antibody was iodinated by using theprocedure described above for Tf. Specific activities of l251_labeled IgG (125I-IgG) preparations varied between 1100 and1600 cpm/ng of protein. To assess binding of radiolabeled Tfor antibody, K562 cells were centrifuged through a cushionof dibutyl phthalate (12). Values for specific binding wereobtained by comparing the binding of radiolabeled Tf or anti-body to that seen in the presence of an excess of unlabeledligand or antibody. The amount of surface-bound ligand wasdetermined by using the acid wash technique of Haigler et al.(13) as described previously (12).The K562 cells were labeled by iodination, using lactoper-

oxidase at 0°C. Cells (2-4 x 107) were washed three timeswith 50 ml of ice-cold phosphate-buffered saline and resus-pended in 1.5 ml of cold phosphate-buffered saline. To thissuspension of cells was added lactoperoxidase (Sigma) at 100,ug/ml, 1 mCi (1 Ci = 37 GBq) of carrier-free 1251, and0.001% H202. After 30 min of gentle shaking at 0°C, equiva-lent amounts of lactoperoxidase and H202 were added againand the incubation was continued for another 30 min at 0°C.Cells were then washed three times with 50 ml of ice-cold

Abbreviations: Tf, transferrin; PMA, 4,l3phorbol 12-myristate 13-ac-etate; Me2SO, dimethyl sulfoxide; LDL, low density lipoprotein.

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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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phosphate-buffered saline, three times with 50 ml of ice-cold0.15 M NaI/10 mM sodium phosphate, pH 7.2, and threetimes with 50 ml of ice-cold RPMI 1640 medium containing0.1% bovine serum albumin, (Pentex, Kankakee, IL) and 25mM Hepes, pH 7.2 (RPMI/0.1% albumin). Labeled washedcells were resuspended to 1-3 x 106 per ml in RPMI/0.1%albumin and 1-ml aliquots were placed in 12-ml plastic coni-cal tubes (Falcon). To assess the effect of added Tf, 50 lug ofdiferric Tf was added to some samples and all tubes wereincubated for 20 min at 0C. Designated cell samples werewarmed by transfer to a 370C water bath for 3 min. All cellsamples were diluted 1:10 with ice-cold RPMI/0.1% albu-min. Immunoprecipitation of surface I251-labeled receptorwas accomplished as follows: OKT9 IgG (25 Mg/ml) wasadded to designated cell suspensions and, after 30 min at40C, cells were washed three times with 10 ml of ice-coldRPMI/0.1% albumin. The cell pellets were solubilized in0.25 ml of ice-cold phosphate-buffered saline containing 1%Triton X-100, 0.5% sodium deoxycholate, and bovine serumalbumin at 0.1 mg/ml (solubilization buffer). After 30 min at0°C, the solubilized cells were centrifuged at 11,000 x g for 5min and 0.1 ml of a 5% (wt/vol) suspension of staphylococ-cal protein A-Sepharose (Pharmacia) was added to the su-pernatant fluid. To assess total immunoprecipitable recep-tor, identical cell suspensions were carried through all incu-bations and washes but OKT9 (25 ,ug/ml) was added afterthe solubilization step. In each case samples were tumbledfor 1 hr at 4°C with the protein A-Sepharose. Subsequently,the gel was washed three times with 1 ml of solubilizationbuffer. The resultant pellet was boiled for 5 min in the sodi-um dodecyl sulfate sample buffer of Laemmli (14), the Seph-arose was removed by centrifugation, and identical volumesof the supernatant fluids were subjected to electrophoresison 12.5% polyacrylamide slab gels (14). After electrophore-sis gels were fixed, dried, and exposed to X-Omat film (Ko-dak) at -70°C, using an intensifying screen (DuPont CronexLightning Plus). The autoradiograms were quantitatively an-alyzed by using a Hoeffer scanning densitometer attached toa chart recorder (LKB). In preliminary experiments, areas

under the peaks in scans of this type were shown to be pro-portional to the amount of radioactivity contained in the90,000-dalton Tf-receptor subunit.

RESULTSEffect of PMA on the Number and Distribution of Tf Recep-

tors. We noted initially that the treatment of K562 cells withPMA at 20 ng/ml resulted in 50-60% loss of surface Tf re-ceptors as assessed by comparison of the binding of humandiferric Tf to treated and control cells at 0°C. The loss ofbinding was due to a decrease in surface binding sites withno change in the affinity of the receptor for Tf (Fig. LA). Thereduction in binding sites was maximal after 30-60 min ofincubation with PMA and persisted for several hours (Fig.1B). An identical change was observed in the binding of twodifferent monoclonal anti-receptor antibodies, directedagainst the human Tf receptor (Fig. 18). The number of sur-face receptors determined by ligand binding was in excellentagreement with that determined by antibody binding. Thedecrease in surface receptors never exceeded 50%, eitherupon longer incubations with PMA or with higher PMA con-centrations. The loss of surface receptors as a function ofPMA concentration is shown in Fig. 1C. Employing a quanti-tative soluble receptor assay that measures total (surfaceplus intracellular) Tf receptors (12), we found that untreatedand PMA-treated cells displayed identical numbers of totalbinding sites. This finding suggested a redistribution of theTf receptor rather than a decrease in the total Tf receptorsper cell. The surface receptors made up 36% of the total re-ceptors in control cells and 17% in PMA-treated cells. The4a form of the phorbol ester had no effect on receptor distri-bution.

Effect of PMA on Receptor Dynamics. A drop in the per-centage of total receptors found on the surface might be ex-plained by a perturbation of the rate constants of internaliza-tion and recycling of receptors. To test this, we examinedthe rate parameters of the Tf cycle in control and PMA-treat-ed cells. Cells were incubated at 0°C with a saturating con-

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FIG. 1. Surface Tf receptors. (A) Scatchard analysis ofTf binding to control and PMA-treated cells. K562 cells at 5 x 105 cells per ml weretreated with either Me2SO (0.1%) or PMA at 20 ng/ml in Me2SO at 37°C for 2 hr. The cells were then washed twice and resuspended inRPMI/0.1% albumin to a density of 107 cells per ml. The washed cells were incubated with different concentrations of "251-labeled diferric Tf(380 cpm/ng) for 1 hr at 0C before samples (0.15 ml) were centrifuged through dibutyl phthalate. The cell-associated radioactivity in the cellpellet was assessed and nonspecific binding of radiolabeled ligand was determined in the presence of a 20-fold excess of unlabeled diferric Tf. e,Control cells; o, PMA-treated cells. Data were analyzed according to Scatchard (15). (B) Time course of loss ofsurface Tf binding. K562 cellsat 5 x 10' cells per ml were treated with either Me2SO (0.1%) or PMA at 20 ng/ml in Me2SO at 37C. After the indicated times samples were

removed, chilled to 0°C, and washed twice with ice-cold RPMI/0.1% albumin. The cells were resuspended and binding of 125I-labeled diferric Tf(20 ,ug/ml) was determined as for A. Binding of 125I-IgG (OKT9) was similarly assessed. Washed cells were incubated with 125I1IgG (1 ,g/ml)for 45 min at 0C and then centrifuged through dibutyl phthalate. Values for nonspecific binding of radiolabeled Tf and OKT9 IgG weredetermined in the presence of a 75- to 100-fold excess of unlabeled ligand or antibody. e, Tf binding to control cells; A, OKT9 binding to controlcells; o, Tf binding to treated cells; and A, OKT9 binding to treated cells. Results comparable to those seen with OKT9 125I-IgG were alsoobtained with a different radiolabeled anti-receptor monoclonal antibody (B3/25, Boehringer Mannheim). (C) Dependence of loss of surfacebinding on PMA concentration. Cells were incubated at 37°C for 3 hr in the presence of the indicated concentrations of PMA. The finalconcentration of Me2SO in all incubations was 0.1%. Experiments to determine binding of 251I-Tf were performed as described for B.

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centration of 125I-Tf and washed free of unbound ligand. Thecells were then rapidly warmed and internalization was as-sessed by measuring the rate at which surface-bound ligandbecame resistant to removal by an acid/high-salt wash (12).This experiment, shown in Fig. 2A, revealed identical ratesof internalization of Tf in control and PMA-treated cells (tk= 2.0-2.5 min). The total cycling time was determined byobserving the release of apo-Tf from the cells after warming(Fig. 2B). A delay of 2-4 min was noted before any releasewas observed; loss of ligand from the cell then occurred witha half time of 7-10 min. Again, no difference was observedbetween control and PMA-treated cells. The ratio of the rateof internalization to the rate of externalization was calculat-ed to be 0.2-0.3.

If the cycling times were the same and the number of sur-face receptors were reduced by a factor of 2, then the rate ofiron uptake at saturating concentrations of Tf might be ex-pected to be decreased by a factor of 2. To our surprise, the

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FIG. 2. Tf dynamics in control and PMA-treated cells. (A) Inter-nalization of Tf. Cells were treated with PMA at 20 ng/ml for 2 hr at37°C before being washed as for Fig. 1A. Cells (107) were resus-

pended in 1 ml of RPMI/0.1% albumin at 0°C and incubated for 40min with 251I-Tf at 10 ,ug/ml with or without unlabeled Tf at 1mg/ml. After washing three times with 15 ml of ice-cold RPMI/0.1%albumin and resuspension in 1.5 ml of this medium at 37°C, sampleswere taken at the indicated times. Cells were either centrifuged di-rectly through dibutyl phthalate or subjected to the acid-wash proto-col before centrifugation. Shown is the loss of sensitivity to dis-placement for control (o) and PMA-treated (o) cells. (B) Cycling ofTf. Cells were treated as for A except that when cells were warmedto 37°C, unlabeled Tf (50 ,ug/ml) was included. At the indicatedtimes cell-associated 1251-Tf was determined by centrifuging samplesof the cells through dibutyl phthalate.

rate of cellular accumulation of 59Fe from Tf was not re-duced in the PMA-treated cells (Fig. 3). Our previous studieswith K562 cells had demonstrated that during continuous ex-posure to saturating concentrations of Tf, a rapid and dra-matic redistribution of receptors occurred, with receptorsshifting from the cell surface to an internal pool. During max-imal ligand uptake, a 50-60% reduction in the steady-statelevel of cell surface receptors was observed. This ligand-in-duced redistribution was complete within 8 min and, utilizingthis new level of surface receptors, the cells accumulatediron. We reasoned that the PMA-treated cells, without Tf,might already be in this redistributed state and would dem-onstrate no further receptor redistribution upon incubationwith ligand. This possibility was tested by loading cells at0C for 5 min with a saturating concentration of 12'I-Tf fol-lowed by rapidly warming the cells to 370C without removingthe excess unbound 125I-Tf. The level.of surface binding wasdetermined as a function of time by the acid-wash techniquedescribed above. The results are shown in Fig. 4. Ligandinduced a 50% reduction in cell surface receptors in controlcells but failed to induce any redistribution in PMA-treatedcells. Furthermore, the total cell-associated Tf in PMA-treated cells reached 88% of that in control cells, therebysuggesting that most of the redistributed receptors partici-pate in Tf recycling in both control and PMA-treated cells.Receptor Internalization Triggered by Ligand or PMA

Treatment. The observations described above indicate thatthere is no demonstrable difference in the dynamics of the Tfreceptor in PMA-treated and untreated cells exposed to lig-and. However, treatment of cells with PMA renders theminsensitive to ligand-induced receptor redistribution. Indeed,the extent to which the number of surface receptors is re-duced by PMA treatment is indistinguishable from the extentof reduction brought about by Tf in untreated cells. Since Tf-induced receptor redistribution is temporally correlated withand quantitatively equivalent to ligand internalization (12) asimple model is suggested by these data. Receptors appearto remain on the cell surface unless occupied with Tf. Uponbinding of Tf the Tf cycle is initiated and receptor redistribu-tion occurs. The triggering of the Tf cycle by receptor occu-pancy might be mimicked by treatment with PMA. To test

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pended to a density of 107 cells per ml. [59Fe]diferric Tf (1600cpm/fig) was added to a final concentration of 100 ,ug/ml and themixtures were warmed to 370C. At the indicated times, 150- I sam-ples were centrifuged through dibutyl phthalate and the cell-associ-ated radioactivity in the cell pellet was measured. *, Control cells;o, PMA-treated cells.

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FIG. 4. Redistribution of Tf receptors. Control and PMA-treatedcells were incubated with 125I-Tf at 50 gg/ml for 5 min at 0C beforebeing rapidly warmed (still in the presence of the ligand). Other ex-perimental parameters were as described for Fig. 2. At the indicatedtimes, total cell-associated and acid-sensitive 125I-Tf were deter-mined. Shown are total cell-associated radioactivity for control (e)or PMA-treated (E) cells and acid-sensitive radioactivity for control(0) or PMA-treated (m) cells.

directly this hypothesis, K562 cells were surface-radioiodi-nated and the amount of immunoprecipitable Tf receptor onthe plasma membrane was quantitated as described underMaterials and Methods (Fig. 5). In cells that were main-tained at 0C, we found that the ratio of surface to total re-ceptors was 0.74 ± 0.04 (mean + SEM, n = 4). The fact thatthis ratio is less than 1 is probably due to a composite ofvariables. For example, if the radioiodination were 90% re-stricted to the cell surface, if the degree of binding of anti-receptor antibody to intact cells were 90% efficient, and ifthis binding were 90% irreversible during the wash-out ofunbound antibody and solubilization of the cells, then a sur-face-to-total ratio of 0.73 (0.9 x 0.9 x 0.9) would be expect-ed. When radiolabeled cells were warmed to 370C for 3 minthe surface-to-total ratio in our immunoprecipitation assay

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FIG. 5. Receptor internalization triggered by receptor occupan-cy or phorbol ester treatment. Cells were treated for 2 hr at 370Ceither with PMA at 20 ng/ml in 0.1% Me2SO or with 0.1% Me2SOalone. Each cell population was then surface iodinated and quantita-tive immunoprecipitation was carried out. Where indicated, sampleswere warmed for 3 min to 370C either without (-) or with (+) prioroccupation of surface receptors by ligand. At the top of each panel isshown an autoradiograph of the 90,000-dalton subunit of the 12511labeled receptor immunoprecipitated from the surface (left memberof each pair) or the total solubilized cells (right member of each

pair). In the lower portion of the figure, surface-to-total ratios basedon densitometric analysis of the autoradiographs are presented.

was found to be 0.76 ± 0.04 (n = 4). However, when surfacereceptors were occupied with Tf prior to warming to 370C for3 min, this ratio dropped to 0.40 ± 0.04 (n = 3). Thus, itappears that surface Tf receptors were relatively static dur-ing the warming period unless the receptors were occupiedwith ligand (Fig. 5 Left).

Treatment of cells for 2 hr with PMA resulted in virtuallyno change in the surface-to-total ratio (0.68) in the immuno-precipitation assay when cells were maintained at 0C. How-ever, warming such cells for 3 min to 370C resulted in a dra-matic redistribution of radiolabeled receptors independent ofreceptor occupancy. This was discerned as a drop in surface-to-total ratios in the presence or absence of ligand to 0.24and 0.23, respectively (Fig. 5 Right).An experimental design conceptually similar to ours has

been utilized by Bleil and Bretscher (16) to assess Tf recep-tor internalization in HeLa cells. Although these investiga-tions did not address the question of the effect of ligand, noTf was added in their experiments. They concluded that thereceptors enter the cells with a half-time of -5 min. Theseresults would appear to be in disagreement with our conclu-sion that Tf receptors do not enter K562 cells when not occu-pied with ligand. However, Bleil and Bretscher (16) did in-ternalization experiments in HeLa cell growth medium thatwas 5% in calf serum. We suspected that the serum might betriggering receptor internalization since, among other things,serum contains transferrin. To test this hypothesis, we com-pared the surface-to-total ratio in our immunoprecipitationassay by performing the experiments in RPMI 1640 mediumwith 10% fetal calf serum (K562 growth medium) and, as be-fore, in medium containing 0.1% bovine serum albumin. We;ound that serum indeed did appear to induce receptor inter-nalization during 3 min of warming to 370C, albeit to a lesserextent than added Tf. The surface-to-total ratios were 0.80,0.64, and 0.44, respectively, for unwarmed cells, cellswarmed in serum-containing medium, and cells warmed inserum-containing medium with added Tf.

DISCUSSIONReceptors that mediate endocytic events are dynamic mole-cules migrating in the course of these events from one cellu-lar locus to another. Moreover, it is clear that in several en-docytic systems pools of receptors exist in a number of cellu-lar organelles. The mechanisms by which receptor dynamicsare regulated and receptor pools are maintained remain fun-damental questions in receptor-mediated endocytosis. Insystems exhibiting ligand-induced receptor down-regulation,occupation of the receptor's binding site modulates cellulartraffic of receptors, leading to receptor degradation. Howev-er, this phenomenon is not observed in all receptor systems.For example, the low density lipoprotein (LDL) receptor hasbeen estimated to make one round trip to and from the cellsurface every 12 min or 150 round trips during its 30-hr lifespan (17). We have previously estimated the round trip forthe Tf receptor to take about 6 min (12). Do ligands, in sys-tems such as these, influence receptor dynamics or do recep-tors go their way irrespective of occupancy? Brown et al.(18) have used the analogy of elevators and escalators to il-lustrate this distinction. Escalators move independent of oc-

cupancy, whereas elevators require the pushing of a button.Which analogy best fits cellular receptors? If elevators is theanswer to this question, what is the cellular button?The use of radiolabeled ligand has provided evidence that

receptor-ligand complexes are internalized. Direct evidencefor movement of receptors in the absence of ligands is moreelusive. Whereas ligands can be easily tagged with radioiso-topes or conjugated to electron-dense particles or enzymesfor cytochemistry, the specific and covalent labeling ofreceptors in situ is technically more difficult. Attempts to

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approach ligand-independent receptor movement moreobliquely have taken the form of treating cells with agents

such as amines or proton ionophores that result in the loss ofsurface receptors. This effect has been observed for the re-

ceptors for asialoglycoproteins (19, 20), lysosomal enzymes(21), mannose-conjugated proteins (22), a2-macroglobulin(23), and LDL (24). Generally, the loss of cell surface recep-

tors, as judged by a reduction in ligand binding, has beeninterpreted to reflect a lower rate of return to the cell surfaceof receptors that had moved into the cell without ligand. Amore complex situation is suggested by two recent investiga-tions. Chinese hamster ovary cells treated with NH4Cl (25)and rat hepatocytes treated with monensin (26) were foundto bind significantly less lysosomal enzymes and asialogly-coproteins, respectively, as compared to untreated cells.However, in both instances, quantitation of surface recep-

tors by immunological means rather than by ligand bindingindicated near-normal numbers of surface receptor mole-cules. Although there is no satisfactory explanation for theseimmunologically detected surface receptors that apparentlylack the ability to bind ligands, these findings render suspectthe conclusions regarding receptor movement in drug-treat-ed cells that are based only on ligand binding.We have examined receptor movement more directly by

iodination of the surface of K562 cells followed by differen-tial immunoprecipitation to quantitate radiolabeled Tf recep-tors that remain on the surface. Our data indicate that in theabsence of added ligand little, if any, internalization of sur-face receptors occurs during 3 min at 370C. On the basis ofpreviously published ligand internalization data (12), we cal-culate that approximately 60% of the surface receptorswould be internalized in 3 mm if this system operated in theescalator mode. Although the receptors appear relativelystatic in the absence of ligand, they rapidly and extensivelyredistribute when cells with occupied receptors are warmedto 370C for 3 min. These results strongly suggest that bindingof Tf delivers a signal that is translated into receptor internal-ization. These findings are consistent with those of Enns etal. (27), who found that Tf added to cells at 20'C induced anaggregation of fluorescent anti-Tf receptor Fab fragments.In another endocytic system, Ciechanover et al. (28) demon-strated that asialoglycoprotein uptake by hepatoma cells re-sulted in a transient decline in the number of surface recep-tors for asialoglycoproteins with no decrease in Tf receptors.These results are most readily explained by an increase inreceptor internalization triggered by ligand binding. A simi-lar ligand-induced loss was observed with a portion of theLDL receptors on human fibroblasts (24).A clue to the nature of the signal for internalization is here-

in provided by our findings that PMA treatment of K562cells results in changes in Tf receptor dynamics that mimicthose observed upon ligand binding. Half of the surface re-ceptors were found shifted to an internal pool upon PMAtreatment. Moreover, PMA treatment resulted in ligand-in-dependent receptor internalization as judged by our quanti-tative immunoprecipitation experiments. Because of theconnection between phorbol esters and receptor phosphoryl-ation (9, 10) we sought to determine whether PMA treatmentor ligand binding led to Tf-receptor phosphorylation. Wehave observed a 9-fold increase in Tf-receptor phosphoryl-

ation in PMA-treated cells (unpublished data). These datalead us to propose that phosphorylation of the Tf receptor isthe elevator button in the Tf cycle. This hypothesis can betested by determining whether ligand binding in the absenceof PMA leads to receptor phosphorylation.

We thank Drs. J. E. Neidel and P. J. Blackshear for their encour-agement and useful discussions.

1. Weinstein, I. B., Lee, L. S., Fisher, P. B., Mufson, R. A. &Yamasaki, H. (1979) J. Supramol. Struct. 12, 195-213.

2. Dreidger, P. E. & Blumberg, P. M. (1980) Proc. Natl. Acad.Sci. USA 77, 567-571.

3. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa,U. & Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851.

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5. Sando, J. J. & Young, M. C. (1983) Proc. Nati. Acad. Sci.USA 80, 2642-2646.

6. Kikkawa, U., Takai, Y., Tanaka, Y., Miyake, R. & Nishizuka,Y. (1983) J. Biol. Chem. 258, 11442-11445.

7. Shoyab, M., DeLarco, J. E. & Todaro, G. J. (1979) Nature(London) 279, 387-391.

8. Osborne, R. & Tashjian, A. J. (1982) J. Cancer Res. 42, 4375-4382.

9. Iwashita, S. A. & Fox, C. F. (1983) Fed. Proc. Fed. Am. Soc.Exp. Biol. 42, 1902a (abstr.).

10. Jacobs, S., Sahyoun, N. E. & Cuatrecasas, P. (1983) Proc.Natl. Acad. Sci. USA 80, 6211-6213.

11. van Renswoude, J., Bridges, K. R., Harford, J. B. & Klaus-ner, R. D. (1982) Proc. Natl. Acad. Sci. USA 79, 6186-6190.

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