THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 15, Issue of August 10, pp. 9776-9782,1984 Printed in U.S.A.

Selective Externalization of the Transferrin Receptor by Sheep Reticulocytes in Vitro RESPONSE TO LIGANDS AND INHIBITORS OF ENDOCYTOSIS*

(Received for publication, February 24,1984)

Bin Tao Pan and Rose Johnstone From the Department of Biochemktv, McGill University, Montreal, Quebec, Canada H3G 1 Y6

The transferrin receptor of sheep reticulocytes is released in vesicular form during in vitro incubation of the reticulocytes. A polyclonal antibody against the transferrin receptor slows down the release of the vesicles bearing the receptor, whereas transferrin and calf serum accelerate vesicle release.

Vesicle formation and receptor release are inhibited at low temperatures and by the presence of inhibitors of ATP formation. In addition, lysosomotropic agents or transglutaminase inhibitors block receptor exter- nalization. The externalized receptor has the same mo- lecular size and peptide map as the receptor isolated from the membrane, suggesting that an intact receptor is removed and released from the cell.

An unidentified peptide of 70 kDa is externalized with the transferrin receptor. Peptide maps show that the 70-kDa species is not a degradation product of the receptor. No function has yet been assigned to the 70- kDa peptide.

Transferrin receptors have been shown to be ubiquitous on the surface of eukaryotic cells (1-7). With the exception of reticulocytes, the expression of the transferrin receptor on the cell surface appears to be related to the proliferation stage of the cells, being abundant in rapidly proliferating cells and relatively low or undetectable in resting cells (3-8). Although it has been suggested that the transferrin receptor may be a target molecule for natural killer cells (9), its main function is generally believed to be the mediation of iron uptake from the plasma (1-17). Despite some controversy (lo), most in- vestigators believe that the mechanism of transferrin uptake is via receptor-mediated endocytosis. It is believed that the iron-transferrin-transferrin receptor complex is internalized (endocytosed) followed by iron removal and the recycling of apotransferrin-transferrin complex back to the plasma mem- brane (2, 11-17). Unlike most other endocytic events of this type, apotransferrin is returned intact and the transferrin recycled many times for iron delivery (11-15).

We have previously reported that sheep reticulocytes lose their surface transferrin receptors during maturation into erythrocytes in vitro (18) and that the loss of the receptor is via selective externalization (19). That is, the protein com- position of the vesicles released during maturation differs from the protein profile of the plasma membrane and is

* This work was supported by grants from the Medical Research Council of Canada and the Quebec Department of Education. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

enriched in transferrin receptors. Receptor-enriched domains appear to form buds which are released into the environment.' The externalized transferrin receptors appear to be capable of binding transferrin and anti-transferrin receptor antibody (19). Moreover, there does not appear to be any degradation of the receptor since the externalized transferrin receptors s50w the same electrophoretic mobility on SDSZ-polyacryl- amide gels as those isolated from the plasma membrane (19). In order to gain some insight into the process of reticulocyte maturation, the characteristics of this selective externaliza- tion process were studied and are reported in this communi- cation.

MATERIALS AND METHODS

Isolation of Sheep Reticulocytes-Sheep reticulocytes were isolated as previously described (18). Transferrin-depleted sheep reticulocytes were obtained by a modification of the procedure described by Hem- maplardh and Morgan (21). Briefly, the procedure consists of incu- bating the cells in 150 mM NaC1,5 mM phosphate, and 20 mM sodium bicarbonate (pH 8.0) at 37 "C for three, 20-min intervals with a change of medium after each interval and once in serum-free culture medium at 37 "C.

Preparation of Transferrin, Anti-transferrin Receptor Antiserum, and Anti-tranferrin Receptor Antibody-Sheep transferrin, rabbit anti-sheep transferrin receptor antiserum, and purified anti-transfer- rin receptor antibody were prepared as described (18, 19). For im-

activated Sepharose 4B (22). lz5I iodination of the purified antibodies munoaffinity columns, the purified antibody was coupled to CNBr-

was carried out as described previously (19). Incubation of Transferrin-depleted Sheep Reticulocytes and Isola-

tion of the Externalized Vesicles-Incubation of sheep reticulocytes was carried out a t 37 "C using a final cell concentration of 1%. Eagle's Ca2+-free minimal essential medium, supplemented with nonessential amino acids, L-glutamine, penicillin (200 units/ml), streptomycin (0.02 pg/ml), 10 mM inosine, 5 mM adenosine, and 2% fetal bovine serum were used. Specific components were added or removed as indicated in the text. After incubation, the suspension cultures were centrifuged at 12,000 X g for 5 min to remove the cells. Two successive centrifugations at 12,000 X g were carried out to ensure the removal of cells. The externalized veiscles were removed from this postcen- trifugation, cell-free medium either by (a) immunoaffinity chroma- tography or (b) by ultracentrifugation. For immmunoaffinity chro- matography, the supernatant from the culture medium was passed through an immobilized antibody column, the column was washed with buffered saline, and the retained material was eluted with 0.1 M glycine buffer (pH 2.3). The acid-eluate was neutralized, dialyzed overnight against distilled water, lyophilized, and subjected to elec- trophoresis. For isolation by centrifugation, the cell-free supernatant was centrifuged at 100,000 X g for 90 min. The pellets obtained were washed with saline and recentrifuged for a further 90 min. The washed pellets were dialyzed overnight against water, lyophilized, and sub- jected to electrophoresis. Alternatively, the protein content of the washed pellets was determined.

Electrophoresis-The Laemmli method (23) was used for electro-

B. T. Pan, K, Teng, and R. M. Johnstone, unpublished data. The abbreviation used is: SDS, sodium dodecyl sulfate.

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phoresis. Plasma membranes or recovered vesicles were dissolved in electrophoresis buffer in SDS containing 10% mercaptoethanol, heated for 5 min at 100 "C, and subjected to electrophoresis.

1251-Tyrosyl Peptide M~pping-~~~I-Tyrosyl peptide mapping was carried out as described (24). It should be pointed out that the lz5I- tyrosyl peptide map for the transferrin receptor is slightly different from those obtained previously (18), since a different type of electro- phoresis apparatus was used for the separation of the peptides.

The Lowry method (see Ref. 25) was used for protein determina- tion.

The firefly luciferase method (26) was used for ATP determination. Surface Labeling-Lactoperoxidase-catalyzed surface iodination

was carried out as described (27). N a Y was purchased from Frosst Company (Montreal). CNBr-

activated Sepharose 4B was from Pharmacia (Uppsala, Sweden). All the other chemicals were purchased from Sigma.

RESULTS

Identification of the Peptides Released during Maturation of Sheep Reticulocytes: 1251-Tyrosyl Peptide Mapping-An earlier study established that vesicles containing components of the plasma membrane are released during maturation of sheep reticulocytes in vitro (19). The data in Fig. 1 show that the major peptides of the vesicles (93 and 186 kDa) obtained from the cell-free, postincubation medium correspond to the trans- ferrin receptor isolated from intact reticulocytes. Whether the vesicles are isolated by an immunoaffinity column of the anti- transferrin receptor antibody or by ultracentrifugation a t 100,000 X g, their protein profiles are the same (Fig. 1). In addition to the receptor, the protein profiles of the vesicles contain a peptide of 70 kDa which is not found in isolates of the receptor from the plasma membrane.

T o verify that the 186- and 93-kDa peptides are identical whether derived from vesicles or the plasma membranes, Iz5I-

tyrosyl peptide maps of the peptide bands were carried out after SDS-gel electrophoresis. The data in Fig. 2 show that

1 2 3

- - 1 8 6 K

- 9 3 K

- 78) :

- 7 0 K

"

FIG. 1. Peptide profiles of the externalized vesicles. A 1% suspension of transferrin-depleted sheep reticulocytes was incubated in serum-free medium at 37 "C. After 21 h, the culture was centrifuged at 12,000 X g for 20 min to remove cells. The cell-free supernatant was used to isolate the externalized vesicles in two ways: 1) the cell- free supernatant was applied to an anti-transferrin receptor antibody immunoaffinity column, washed with saline, eluted with 0.1 M glycine (pH 2.3) and neutralized; 2) the cell-free medium was centrifuged at 100,000 X g for 90 min. The pellet was washed with saline and recentrifuged at 100,000 X g for 90 min and the pellet collected. The fractions obtained in lanes I and 2 were dialyzed against distilled H,O at 4 "C, lyophilized, and electrophoresed on SDS-polyacrylamide gels. The Commassie Blue-stained gels are shown in lanes I and 2, respectively. Lane 3, transferrin receptor isolated from sheep reticu- locyte plasma membranes by an anti-transferrin receptor antibody immunoaffinity column as described previously (18). In this and subsequent figures, K has been used on the figures to indicate peptides of molecular size 186, 93, 78, and 70 kDa, respectively.

the 93-kDa peptides isolated from both sources are identical. Similar results (not shown) were obtained with the 186-kDa species from vesicles and plasma membrane. We have previ- ously shown (18, 19) that the transferrin receptor of sheep reticulocytes has a monomeric molecular mass of 93 kDa and a dimeric molecular mass of 186 kDa. These data establish the identity of the released receptor with that in the plasma membrane and verify that there is no major modification of the receptor upon externalization. The data confirm our pre- vious conclusion (19, 20) that the transferrin receptor is selectively externalized during sheep reticulocyte maturation. The 70-kDa peptide has a different peptide map from that of the transferrin receptor (Fig. 2c). Thus, this peptide is neither the receptor nor a degradation product of the receptor. The 70-kDa peptide is not albumin since its iodotyrosyl peptide map is entirely different from albumin (not shown).

The Effects of Anti-transferrin Receptor Antibody, Transfer- rin, and Serum on the Selective Externalization of the Trans- ferrin Receptor-Previous work showed that neither the pres- ence of antibody nor transferrin was essential for externali- zation (19). Those studies did not address the question of the quantitative effects of anti-transferrin receptor antibody or transferrin on the selective externalization of the receptor. Since the anti-transferrin receptor antibody has been shown to cause patching (l8-19), it might be reasonable to suspect that the anti-transferrin receptor antibody would have some effect on the selective externalization of the transferrin recep- tor. Also, physiological ligands of cell surface receptors are known to modulate their respective surface receptors. For example, insulin (28, 29) and epidermal growth factor (30- 32) are able to cause down-regulation of their respective receptors. Therefore, transferrin might be anticipated to af- fect the transferrin receptor in some wav. The effects of serum and of anti-transferrin receptor antioody on the selective externalization of the receptor are shown in Fig. 3, A and B. The experiment was done in two ways: (a) by release of receptor protein and (b) by release of surface-bound 1251- labeled anti-receptor antibody which is known to be released as an antibody-receptor complex (19).

(a) To meausre release of receptor protein, the cells were first incubated at 0 "C with 0-50 pllml of the anti-receptor antiserum to allow antibody binding. Then, the cells were washed free from immune serum and incubated a t 37 "C f 2% fetal calf serum. The externalized vesicles were isolated by centrifugation from the cell-free postincubation medium and were subjected to SDS-gel electrophoresis. The Coomas- sie Blue-stained protein scans of the released vesicles are shown (Fig. 3A). In absence of calf serum, (Fig. 3A, left) and in presence of calf serum (Fig. 3A, right) the amount of receptor externalized is inversely related to the concentration of antiserum used in the original treatment.

(6) To measure the release of '*'I-labeled anti-transferrin receptor antibody, 0-40 pg/ml of purified "'I-anti-transferrin receptor antibody was bound to sheep reticulocytes a t 0 "C. The cells were washed free from unbound "'1 antibody and incubated f 2 % fetal calf serum a t 37 "C. The cells were recovered after centrifugation a t 12,000 x g, and the exter- nalized vesicles were collected by centrifugation of the cell- free medium. The results (Fig. 3B) show that as a fraction of l2'1-antibody bound, less "'I is released as the antibody con- centration increases. Thus, in both experiments, the presence of increasing concentrations of anti-transferrin receptor an- tibody reduces the selective externalization of the transferrin receptor. In contrast, fetal calf serum increases receptor re- lease a t all antibody concentrations examined. At high anti- body concentrations (40 pglml) the relative effect of serum is

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9778

A

1 8 6 ~

9 3 K -

7 0 %

5 3 K

Selective Externulization of the Transferrin Receptor

FIG. 2. '261-tyrosyl peptide mapping. The peptides at 93 and 70 kDa (corresponding to those in Fig. 1) were cut from the gel, iodinated as described under "Materials and Methods" and electrophoresed. a, the 93-kDa peptide of the transferrin receptor isolated from sheep reticulocyte plasma membranes by an immunoaffinity column of the anti-transferrin receptor antibody. b, the 93-kDa and c , 70-kDa peptides of the externalized vesicles isolated by centrifugation a t 100,000 X g. It can be seen that '251-tyrosyl peptide patterns in a and b are similar to each other, but are different from those in c.

. L. "q 1 2 3 4 1 2 3 4 "'1 B

1 8 6 K

9 3 K

7 OK 7 8 K

5 3 K

r

I L

ABY Conc

HOURS

FIG. 3. Effects of serum and anti-transferrin receptor antibody on the selective externalization of the receptor. A, release of receptor protein. A 10% suspension of transferrin-depleted sheep reticulocytes was incubated for 90 min a t 0 "C with 50, 25.8, and 0 pl per ml (lanes 1-4, respectively of both left and right) of rabbit antiserum against sheep transferrin receptor. After the incubation, the cells were washed with saline, resuspended in media without (left) or with 2% fetal calf serum (right) to give a 1% cell suspension, and reincubated at 37 "C. After 21 h, the cultures were centrifuged to remove the cells (12,000 X g), and the cell-free medium was recentrifuged a t 100,000 X g for 90 min to obtain the externalized vesicles. The vesicles were dialyzed overnight against distilled H20, lyophilized, electrophoresed, and stained with Coomassie Blue. Note that with increasing concentrations of antiserum (lanes 1-4) in both left and right) externalization of the transferrin receptor is gradually reduced. After pretreatment with antiserum, immunoglobulin heavy chain (53 kDa) is detected with the vesicles. Cells incubated in serum (right) also show the presence of transferrin (78 kDa) in the vesicles as well as the 70-kDa peptide. B, Release of iodinated antibody. One ml of a 10% suspension of transferrin-depleted sheep reticulocytes was incubated with different concentrations of purified '251-anti-transferrin receptor antibody (-lo5 cpmlpg) a t 0 "C for 90 min. The cells were washed, resuspended to give a 1% suspension in incubation medium (+2% fetal calf serum), and incubated a t 37 "C. At various times, aliquots were removed and centrifuged a t 12,000 X g to remove the cells. The cell pellet was counted. The supernatant was applied to a Sepharose 4B column to separate the externalized vesicles from the medium and contaminating free I&, and the fraction in the void volume was counted (18). The degree of externalization was calculated as follows.

cDm in vesicle fraction (void volume) cpm of vesicle fraction + cpm of cell fraction (12,000 X g pellet)

Solid symbols, with serum. Open symbols, without serum.

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Selective Extermlization of the Transferrin Receptor 9779

less (Fig. 3B). Like serum, the presence of sheep transferrin in the incubation medium appears to accelerate the external- ization of the transferrin receptor (Fig. 4). The data show that as the transferrin concentration is increased, more recep- tor is externalized. Indeed, it is likely that much of the serum effect is due to the transferrin in serum since 2% serum would yield approximately 80 pg of transferrin/ml. However, precise comparisons cannnot be made because the relative effects of sheep and bovine transferrin on sheep cells would have to be taken into account. Note that when transferrin or serum is present in the medium, a peptide of 78 kDa, characteristic of transferrin, appears with the vesicles (Figs. 3 and 4).

Kinetics of Receptor Externulization-To follow the kinetics of the effects of antibody and transferrin, the externalization of the receptor was followed during a 40-h incubation period in the presence of anti-transferrin receptor antibody, trans- ferrin, and serum. The externalized vesicles, isolated by cen- trifugation of the cell-free postincubation medium, were sub- jected to gel electrophoresis, and the Coomassie Blue-stained gels are shown (Fig. 5A). It is evident that serum and trans- ferrin increase while antibody decreases receptor release. The total protein in the externalized vesicles was also determined. The data in Fig. 5B show that 25 pg/ml of antibody reduces vesicle protein release by -50%, whereas serum and transfer- rin increase vesicle protein release by -60 and -30%, respec- tively. In addition, the release of '251-labeled vesicles from 1251- surface-labeled cells was measured in the presence of antibody and calf serum. The presence of 40 pg of antiserum reduced

release by 60%, and 2% serum increased 1251 release by 80% in 36 h, consistent with the data in Fig. 5A. The data are consistent with the conclusion that the specific antibody inhibits externalization of the vesicles, whereas sheep trans- ferrin and fetal bovine serum accelerate the process. In all cases, the rate of externalization decreases with time, but does

1 2 3

-1 86K

-93K

- 70K

FIG. 4. Effect of transferrin concentration on the selective externalization of the receptor. A 1% suspension of transferrin- depleted sheep reticulocytes was incubated in serum-free medium with: lane I , 75 pglml; lane 2, 25 pglml; and lane 3, 0 pg of sheep transferrin. Externalized veiscles were isolated, washed, and subjected to SDS-gel electrophoresis. The 78-kDa peptide (transferrin) is found with vesicles when transferrin is added to the incubation medium.

not follow strictly first order kinetics (Fig. 5B). Externulization of 70-kDa Peptide from '251-surface-lubeled

Cells-Since the 70-kDa peptide appears to be released along with the transferrin receptor, it seemed important to deter- mine whether the 70-kDa peptide is exposed at the external surface of the plasma membrane. For these studies, the cells were surface-labeled with 1251 and lactoperoxidase and were cultured for 36 h. The vesicles of the cell-free postincubation medium were collected by centrifugation and show the pres- ence of the 70-kDa peptide by Commassie Blue staining (Fig. 6). Significantly, the 70-kDa peptide is totally absent on the autoradiograms, suggesting that this peptide is not exposed at the external surface of the cell (Fig. 6, right). These data also show that when the cell surface is labeled, the released receptor is labeled (Fig. 6, right). The amount of radioactivity released parallels the protein released, serum and transferrin stimulating 1251 release, and the antibody reducing the release.

Effects of Chloroquine, Methylamine, Momdansylcadaver- ine, and Protease Inhibitors on the Selective Externalization of the Transferrin Receptor-Lysosomotropic agents such as chloroquine (33) and transglutaminase inhibitors (34, 35) have been shown to have inhibitory effects of receptor-medi- ated endocytosis (36, 37). Since endocytosis and selective externalization both involve membrane processing, the selec- tive externalization of the transferrin receptor was examined in presence of agents known to affect endocytosis. Reticulo- cytes were incubated in culture medium in the presence of a number of these reagents, and the externalized vesicles were collected and their protein composition analyzed (Fig. 7). It can be seen that the presence of chloroquine, monodansylca- daverine, and methylamine all decrease the selective exter- nalization of the transferrin receptor relative to the control. Additional work with '251-anti-transferrin receptor antibody showed that the release of '251-labeled antibody responded similarly to these agents (data not given). Moreover, since ATP-dependent protease activity has been demonstrated in reticulocytes (38), and these enzymes may be involved in maturation of reticulocytes (391, the effects of various pro- tease inhibitors on the selective externalization have also been examined. I t appears that the presence of a variety of protease inhibitors such as antipain, chymostatin, pepstatin, and leu- peptin under the present conditions does not change the amount of transferrin receptor externalized (Fig. 7, lunes 5- 8). The absence of an effect may be due to poor uptake of these antiproteolytic agents by the cells.

Effects of Energy Metabolism on the Externulization-En- docytosis of transferrin is known to be an energy-dependent process (for review, see Ref. 12). Inhibition of metabolism and inhibitors of ATP production appear to inhibit the selective externalization of the receptor as well. Incubation at 37 "C in the presence of NaCN, NaF, NaF + NaCN or Na2As0, (Fig. 8), all of which cause dramatic depletion of intracellular ATP (Table I), reduces externalization of receptor. Incubation at 0 "C completely reduces externalization (19) despite the fact that the intracellular ATP level is only moderately reduced. Similar results are obtained if the release of '251-labeled anti- body is followed (not shown). The data indicate that the selective externalization is temperature-dependent and en- ergy-dependent. Incubation at 37 "C with transferrin-immune or -nonimmune serum does not significantly alter cellular ATP levels (Table I).

DISCUSSION

'251-Tyrosyl peptide mapping of the vesicle-associated pep- tides released from sheep reticulocytes confirms our previous conclusion that the transferrin receptor is released intact into

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9780 Selective Externalization of the Transferrin Receptor

- 1 8 6 K

-93 K

- 7 8 K -70 Y,

601 B

40-

30-

20-

10-

- FIG. 5. Kinetics of the selective externalization of the transferrin receptor. A, effects on peptide

components of the vesicles. A 1% suspension of transferrin-depleted sheep reticulocytes was incubated at 37 "C in the presence and absence of anti-transferrin receptor antibody, sheep transferrin, or fetal bovine serum (2%). At the indicated times, the externalized vesicles were isolated from the cell-free postincubation medium by centrif- ugation and processed for electrophoresis. Lanes 1-3, a 10% cell suspension was preincubated with 25 pl/ml of anti-transferrin receptor antiserum at 0 "C for 90 min, following which the antibody-labeled cells were washed and then incubated in serum-free medium for 3, 18, and 40 h, respectively. Lanes 4-6, control cells were incubated in serum-free medium for 3, 18, and 40 h, respectively. Lanes 7-9, cells were incubated in serum-free medium with 30 pg/mg of sheep transferrin for 3, 18, and 40 h, respectively, Lanes 10-12, cells were incubated with 2% fetal bovine serum for 3, 18, and 40 h, respectively. In all cases, vesicles were harvested by centrifugation, washed, and subjected to SDS-gel electrophoresis. B, total protein content of the vesicles. Experimental conditions were as in Fig. 5A, but the externalized vesicles were collected and total protein determined. The released protein is expressed as micrograms of protein released per ml of packed reticulocytes used for incubation.

1 2 3 4 4 3 2 1

+Serum

+Transferrin - Serum

+Antibody

- 1 8 6 K -186 K

-0-

FIG. 6. T h e 70-kDa peptide is inaccessible to lactoperoxi- dase-catalyzed '261 iodination of reticulocytes. Transferrin-de- pleted sheep reticulocytes were surface-labeled with '%I using lacto- peroxidase (27). The radiolabeled cells were then incubated at 37 "C under the following conditions. Lane 1, the cells were preincubated with 25 pl/ml of anti-receptor antiserum (see Fig. 5A on treatment with antiserum) prior to incubation at 37 "C. Lane 2, control cells - serum-free medium. Lane 3, medium contained 2% fetal calf serum. Lane 4, medium contained 75 pg/ml of transferrin. Twenty-one hours later, the cells were removed and the externalized vesicles isolated by centrifugation at 100,000 X g and prepared for electrophoresis as described above. Right, Coomassie Blue staining. LRft, radioautogra- phy of gels. Only the transferrin receptor (93K) is significantly radiolabeled.

FIG. 7. Effects of lysosomotropic agents, transglutaminase inhibitors, and protease inhibitors on the selective externali- zation of the transferrin receptor. A 1% suspension of transfer- rin-depleted sheep reticulocytes was incubated in serum-free media with the additions indicated below. After 21 h at 37 "C, the external- ized vesicles were isolated by centrifugation at 100,000 X g and electrophoresed. Lane 1, 0.1 mM monodansylcadaverine; lane 2, 20 mM methylamine; lane 3, 2% dimethyl sulfoxide; lane 4, control, no additions; lane 5, 20 p~ antipain; lane 6, 20 p~ chymostatin; lane 7, 20 p M pepstatin; lane 8, 20 p M leupeptin; and lane 9, 10 p M chloro- quine.

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1 2 3

186K

r. -4

-0

FIG. 8. Inhibition of ATP production and selective exter- nalization of the transferrin receptor. The experimental condi- tions were described in Fig. 7. The media used were as follows. Lane I , complete medium, serum-free; lane 2, as lane 1 but without 10 mM inosine and 5 mM adenosine; lane 3, as lane 1 plus 10 mM NaCN; lane 4, as lane 1 plus 10 mM NaF; lane 5, cells were pretreated with 10 mM NaCN for 15 min at 37 “C, then washed, and reincubated in medium as in lane 1; lane 6, as lane 1 plus 10 mM NaCN + 10 mM NaF; lane 7, as lane 1 plus 10 mM NaAsO,; lane 8, as lane 1 plus 0.1% rotenone; lane 9, as lane 1 plus 1% dimethyl sulfoxide; lane 10, as lane 1 but incubated at 0 “C.

TABLE I ATP levels in reticulocytes incubated in vitro

A 1% suspension of transferrin-depleted sheep reticulocytes was incubated as described in Fig. 7. The ATP level was measured after 2 h of incubation at 37 “C. The control medium was supplemented with amino acids, 10 mM inosine, and 5 mM adenosine (see “Materials and Methods”).

Conditions ATP mM

Control medium (serum-free) 2.4 Minus (inosine + adenosine) 1.3 10 mM NaCN 0.01 10 mM NaF 0.70 10 mM NaCN + 10 mM NaF Not detectable

10 mM Na2AsOI 0.4 0.1% rotenone (in 1% dimethyl 1.9

1% dimethyl sulfoxide 2.8 0 “C incubation-control medium 1.0

Control 2.0 10 pM chloroquine 1.6 20 mM methylamine 1.9 120 pM monodansylcadaverine 2.1

(CO.01 mM)

sulfoxide)

the environment during sheep reticulocyte maturation in vitro (19). The data show that the 12sII-tyrosyl peptide maps of the 186- and 93-kDa peptides in the released vesicles are not detectably different from the 93-kDa monomer of the trans- ferrin receptor isolated from the plasma membranes by im- munoaffinity columns.

The identity of the 70-kDa peptide in the isolated vesicles has not been established. This peptide, which does not appear with the receptor isolated by immunoaffinity chromatography or immunoprecipitation from the cellular plasma membrane,

appears to coexist with the transferrin receptor in the same vesicle since it is co-isolated with the transferrin receptor whether the vesicles are isolated by anti-transferrin receptor antibody columns or by ultracentrifugation. The distinctive- ness of the 1251-tyrosyl peptide maps of the transferrin receptor and the 70-kDa peptide also argues against the possibility that the 70-kDa peptide is a degradation product of the receptor. Furthermore, this 70-kDa peptide is probably not exposed at the external surface since it does not become labeled during iodination of the reticulocyte surface (Fig. 6) nor of the released vesicles (not shown). Most interestingly, this 70-kDa peptide appears to be released in approximately the same quantity as the transferrin receptor (as indicated by the apparent density of the Coomassie Blue stain) under all conditions examined. The fact that the release of the 70-kDa peptide and the 93-kDa peptide (transferrin receptor) re- sponds similarly to agents which either increase or decrease receptor release, also suggests that their externalizations are linked. Although a role for the 70-kDa peptide is unknown, the possibility exists that the 70-kDa peptide is responsible for binding the receptor to the cytoskeleton and is released when the receptor is released.

It is well known that surface receptors can be modulated by their ligands (40). Down-regulation of receptors has been reported in many cases as, for example, with the insulin receptor (28,29) and the receptor for epidermal growth factor (30-32). In these cases, down-regulation is attributed to the stimulation of receptor-mediated endocytosis by the ligands. The demonstration of enhanced selective externalization of the transferrin receptor by transferrin may be viewed as a type of down-regulation since the cell loses transferrin recep- tor in response to transferrin.

Selective externalization of receptor appears to be ATP- dependent, indicating that the modification of the membrane is an active process under the control of intracellular events. Externalization is also inhibited by chloroquine, monodan- sylcadaverine, and methylamine, agents which affect mem- brane recycling (32, 41). Our earlier studies (18, 19) demon- strated that the transferrin receptor of sheep reticulocytes is internalized (endocytosed) and that internalized receptors are eventually released into the medium. We proposed (19) that the transferrin receptor continues to be recycled until the receptor becomes “aged (i.e. undergoes some change which has not yet been established) and then is removed by selective externalization. We have not yet been able to distinguish whether metabolic inhibitors or lysosomotropic agents (or transglutaminase inhibitors) have a direct effect on external- ization of the receptor or whether these agents reduce exter- nalization because endocytosis and processing are reduced, i.e. externalization is dependent on some aspects of endocy- tosis.

Although no evidence was found for any chemical change in the externalized receptor, it is tempting to suggest that some alteration has occurred which designates the receptor for removal. This change (if it exists) must be more subtle than loss of transferrin or antibody binding activity or a change in molecular size since the externalized receptor binds antibody and transferrin (Figs. 3 and 4) as well as maintaining its molecular size. Perhaps, the change occurs in a protein associated with the receptor, releasing the receptor from its “moorings” in the membrane at the cytoplasmic surface. This may be the origin of the 70-kDa peptide. Alternatively, since the receptor is known to be phosphorylated (41), acylated (42), and glycosylated (41), some change in any of these post- translational modifications may be the trigger for externali- zation. I t is not known whether the transferrin receptors of

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other cells which are not undergoing a developmental change experience a similar fate to that of sheep reticulocytes, i.e. selective externalization.

In conclusion, the transferrin receptor appears to be in a dynamic state in sheep reticulocytes, undergoing internaliza- tion (receptor-mediated endocytosis), recycling, and selective externalization. The antibody-induced redistribution of the receptor on the surface (18, 19) suggests that the surface distribution of the receptor is under the control of cytoskel- eton. Since mammalian reticulocytes are simple in structure, nucleus-free, and appear to lack intermediate filaments and microtubules (20,43), they appear to be a useful model system to study the molecular interactions between transmembrane proteins and the intracellular mechanisms which mediate endocytosis, surface clustering, and selective loss of mem- brane proteins.

Acknowledgments-We thank Anoush Cotchikian for excellent technical assistance and Kathy Teng for the photographic work.

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B T Pan and R JohnstoneResponse to ligands and inhibitors of endocytosis.

Selective externalization of the transferrin receptor by sheep reticulocytes in vitro.

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