Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6406-6410, December 1979
Cell Biology
Identification of transferrin receptors on the surface of human cultured cells (serum factors/ligand binding/glycoproteins)
THOMAS A. HAMILTON, H. GARRETT WADA, AND HOWARD H. SUSSMAN Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Communicated by Robert T. Schimke, August 31, 1979
ABSTRACT We have examined the binding of human transferrin to cultured human choriocarcinoma cell lines and to detergent extracts of such cells. The results indicate the presence of a high-affinity saturable binding site (Ka = 4.25 X 108 M-1) that is specific for transferrin. This receptor has also been detected on three other human cell lines of different phenotypic origin, including Wil-2 (splenic lymphocytes of B-cell origin), RPMI-2650 (a quasi-diploid nasopharyngeal carcinoma), and WI-38 (embryonic lung fibroblasts). By using anti-human transferrin antiserum to immunoprecipitate the receptortransferrin complex from detergent extracts of cells containing saturating levels of transferrin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single polypeptide of 90,000 daltons has been identified as a subunit of the putative transferrin receptor. The protein shows immunochemical identity and coelectrophoreses in sodium dodecyl sulfate gels with a cell surface glycoprotein subunit, previously identified in placental brush border membrane preparations, on all human cultured cell lines examined. These results suggest that the recent demonstration of transferrin dependence of maximal cell growth in culture [e.g., Hutchings, S. E. & Sato, G. H. (1978) Proc. Nat]. Acad. Sci. USA 75, 901-904] is mediated through expression of this glycoprotein receptor.
METHODS Cell Culture. BeWo cells and WI-38 cells were obtained from the American Type Culture Collection (CCL 98 and CCL 75) and grown as described (14, 15). RPMI-2650 cells (CCL 30) were obtained from the same source and grown in minimal essential medium plus 10% fetal calf serum; Wil-2 cells were a gift from David Korn (Stanford University School of Medicine, Department of Pathology) and were grown in RPMI-1640 medium plus 10% fetal calf serum. Transferrin Binding Assays. After two washes with Ca- and Mg-free Earle's balanced salt solution, cells were removed from monolayers by incubation in Earle's solution containing 5 mM EDTA. Cells were then resuspended in Earle's solution containing 1 mg of ovalbumin (twice crystallized, Sigma) per ml for transferrin binding assay. For soluble receptor assay or for immunoprecipitation studies, cells were scraped into phosphate-buffered saline after thorough washing with Earle's solution. The suspension was then made 1% (vol/vol) in Triton X-100 and extracted for 10 min on ice. The extracts were then centrifuged at 140,000 X g (max) for 1 hr. Binding of transferrin to intact cells was measured by using 106 cells per assay in a total volume of 0.5 ml of Earle's solution at pH 7.5 containing 2 mg of ovalbumin per ml. After incubation with 125I-labeled human transferrin [125I-transferrin; labeled by chloramine-T iodination (16)] at 37°C for 30 min, the incubation was stopped by chilling on ice, addition of 1 ml of ice cold phosphate-buffered saline, and centrifugation at 400 X g for 10 min at 0-4°C. Both the supernatant and the cell pellet were assayed for radioactivity and percentage bound to the cell pellet was calculated. Background binding was determined by running controls with no cells, and high-affinity specific binding was determined by subtraction of low-affinity binding obtained with controls containing cells and 1 ,tM unlabeled ferrotransferrin (77 ,ug/ml). Assays of transferrin binding in Triton X-100 extracts of BeWo cells were conducted essentially as described by Wada et al. (13) with approximately 400 ,ug of protein per assay. Human transferrin, albumin, and gamma globulin (Sigma) used for competitive binding studies were further purified by gel filtration through Sephacryl-200 (Pharmacia). Immunoprecipitation of the Solubilized TransferrinReceptor Complex. Surface proteins of BeWo cells and WI-38 cells were radiolabeled with 125I by lactoperoxidase-catalyzed iodination (17). Approximately 2 X 107 cells were labeled, removed by EDTA incubation as described above, and suspended in 1 ml of phosphate-buffered saline. Triton X-100 extracts or cell suspensions were made 10 ,ig/ml in ferrotransferrin and 1 mg/ml in ovalbumin and incubated at 37°C for 10 min. The cells were then extracted with detergent as described above. Triton X-100 extracts were subjected to immunoprecipitation
Several investigations (1-4) have defined the serum dependence of cells in culture to be a requirement for hormones present in serum. Further work demonstrated that the hormonal requirement varies from one cell line to another (5), depending upon the cell type from which the cell line was originally derived (5, 6). In addition to the hormonal requirements of cells in serum-free media, it was noted that transferrin was necessary to maintain maximal growth (5, 6). However, whereas hormonal requirements vary, all types of cells in culture require transferrin (6). Furthermore, the transferrin requirement appears to be related to its iron transport function rather than to a regulatory function because increased levels of FeSO4 can substitute for transferrin in cultures of 3T6 Swiss mouse fibroblasts (7) and simian virus 40-transformed 3T3 cells (8). There is evidence indicating the existence of transferrin receptors on reticulocytes (9, 10) and placental tissue (10, 11), both of which are involved in active iron uptake. In addition, a cell surface receptor for transferrin has been recently demonstrated on cultured human lymphocytes (12). In a previous study, we obtained direct evidence for the presence of a specific transferrin receptor on human placental brush border membranes and identified a placental membrane glycoprotein with high affinity for transferrin (13). The present report presents evidence that this same protein is expressed on the surface of various human cultured cell lines of different phenotypic origin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: NaDodSO4, sodium dodecyl sulfate.
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Proc. Natl. Acad. Sci. USA 76 (1979)
with rabbit anti-human transferrin exactly as described by Wada et al. (13). Staphylococcus aureus was used ax theirMmunoadsorbant according to Kessler (18). 0
E c D 0
6407
Polyacrylamide Gel Electrophoresis. One-dimensional sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was conducted according to Davis (19) with the inclusion of 0.1% NaDodSO4 in the top electrode reservoir, stacking gel, and 8% acrylamide running gel as described (13). Subunit molecular weights were estimated by the method of Weber and Osborn (20) by using, as protein standards, f3-galactosidase (130,000), human transferrin (77,000), placental alkaline phosphatase (64,000), and ovalbumin (43,500). Two-dimensional electrophoresis was conducted according to Wada et al. (21) by using isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis.
C
0
C
0c
Transferrin,
nM
0
10
0
Time, min
cm -,
E c0
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c C
.0
0 C
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C
at
12
11
10
9
7
8
6
5
-log protein concentration (M) FIG. 1.
Binding of 1251-transferrin to BeWo cells. (A) Binding
curves for transferrin interaction with BeWo cells. Approximately 20,000 cpm of 1251-transferrin and 1 X 106 cells were used for each point. The concentration of unlabeled ferrotransferrin was varied over the indicated range. Cells were incubated at 371C for 30 min. The pmole of transferrin bound was calculated from the total radioactivity bound by correction for isotope dilution with unlabeled transferrin,
giving
total transferrin bound
(0-0). Low-affinity binding
-) was calculated by taking the slope of the total binding curve
- -
between 0.1 and 1 ,M transferrin and extrapolating through the origin. Specific, high-affinity binding (O- -0) was calculated by taking -
the difference between the total and low-affinity binding curves. (B) Time course of transferrin binding to BeWo cells. Cells were added to the assay medium containing 1251-transferrin at time zero and incubated at 370C. Aliquots were withdrawn immediately after addition of cells and at the indicated time intervals for assay of percentage transferrin specifically bound. 0-0, High-affinity binding (0.1 nM); 0-- 0, low-affinity binding (1 ,uM). (C) Competition by transferrin, albumin, and IgG for 1251-transferrin binding to BeWo cells. The indicated concentrations of ferrotransferrin (0-0), albumin (A.-A- ), and IgG (O--- 0) were tested for their effect on 1251-
transferrin binding to BeWo cells as described above, with a 20-min incubation period. The binding values were normalized by calculating
percentage 1251-transferrin binding remaining by using binding with no additions of competing
protein as the reference value.
RESULTS AND DISCUSSION Binding of transferrin was initially examined by measuring the amount of 125I-transferrin bound to suspensions of cultured BeWo choriocarcinoma cells (22, 23). Low-affinity binding (determined from the slope of the total binding curve taken between transferrin concentrations of 0.1 and 1 AM and extrapolated to 0) was subtracted from the total binding curve to estimate the amount of specific high-affinity binding (Fig. 1A). Fig. 1B, a time course for high-affinity and low-affinity transferrin binding to BeWo cells at low (0.1 nM) and high (1 ,OM) concentrations of transferrin, shows a rapid approach to maximal transferrin binding, achieved in 10 min, and then a slower but steady decrease in transferrin bound to the highaffinity site. After 3 hr of incubation at 370C, this high-affinity binding was decreased to the level observed for low-affinity unsaturable binding. The specificity of transferrin binding to BeWo cells was demonstrated by studies examining the competition of human transferrin, IgG, or albumin for 125I-transferrin binding. Only unlabeled ferrotransferrin competed with the labeled transferrin for binding sites on BeWo cells (Fig. 1C). No difference was observed when apotransferrin was substituted for ferrotransferrin, indicating that the degree of iron saturation has no marked effect on binding specificity. Because transferrin binding to intact cells exhibited a timedependent decrease, the further characterization of the transferrin receptor by Scatchard analysis (24) required the development of an experimental system in which equilibrium binding could be achieved. This was accomplished by examining transferrin binding in Triton X-100 extracts of BeWo cells in a fashion similar to that developed in the course of studies on the transferrin receptor found in placental brush border membranes (13). The soluble binding assay takes advantage of the stability of the receptor-transferrin complex at pH 5.0 and the differential solubility of transferrin in 12% polyethylene glycol, depending upon its association with higher molecular weight structures. Fig. 2A shows a binding curve constructed in a fashion similar to that shown in Fig. 1A but in which transferrin binding was measured by precipitation of the soluble receptor-transferrin complex with polyethylene glycol at pH 5.0. The similarity of Fig. 1A and Fig. 2A is evident and suggests high-affinity interaction of transferrin with a specific receptor. Neither human IgG nor albumin competed in the soluble receptor assay, consistent with the data shown in Fig. 1B. Fig. 2B shows a time course of transferrin binding to lowaffinity or high-affinity components. Equilibrium binding was reached by 30 min and showed no tendency to decrease with further incubation. All studies reported here using the soluble receptor assay were done with a 30-min incubation, and equilibrium binding was assumed. Fig. 2C shows that the binding of transferrin to its soluble high-affinity receptor is fully reversible. Within 1 min after the
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Proc. Natl. Acad. Sci. USA 76 (1979)
c
B
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0
0 D c
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.0
0
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0
--
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30
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a 0 .0 c C
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10 Transferrin, nM
20
30 Time at
40
50
60
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0 c
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60 50 40 Time at 370C, min
75
1.0
2.0
Transferrin, nM
FIG. 2. Binding of 1251-transferrin in detergent extracts of BeWo cells. (A) Equilibrium binding curve for transferrin binding to soluble receptor. To each assay mixture (total volume, 0.5 ml) were added 0.4 mg of detergent-extracted protein and approximately 20,000 cpm of
1251-transferrin. The transferrin concentration was varied over the indicated range by the addition of unlabeled transferrin to the concentrations indicated. The total binding (0-0), low-affinity binding (^-.-^), and high-affinity binding (O--- 0) were determined as in Fig. 1A. (B) Time course for transferrin binding to soluble receptor; 0.4 mg of extracted protein and 20,000 cpm of 1251-transferrin were used for each point. For high-affinity binding (0-0), unlabeled transferrin was present at 0.1 nM. For low-affinity binding (O 0), transferrin was present at 1 juM. (C) Reversibility of transferrin binding to soluble receptor. Each assay was as described for B. Samples first were preincubated at 370C for 15 min and then the experiment was begun by addition of unlabeled transferrin to 1 ,M. (D) Scatchard plot of equilibrium binding. Values for high-affinity binding from A were plotted according to Scatchard (23) by using linear regression to find the line that best fit the experimental values. The correlation coefficient (r2) for this plot was 0.89, and the slope, which is equal to -K,, was 6.2 X 108 M-1. ---
addition of excess unlabeled transferrin, the specific binding was decreased to less than 50% of its equilibrium value. By 30 min the exchange appeared to be complete. Fig. 2D shows a plot of the binding data of Fig. 2A according to Scatchard (24). Plots of bound transferrin/free transferrin versus the concentration of bound transferrin were linear at low concentrations, indicating a single high-affinity binding site. The association constant (Ka) was found to be 4.25 ± 2.1 X 108 M-1 (mean + SD), and the number of binding sites was 2.3 X 1012/mg of protein. When normalized to the number of cells originally extracted, this indicates 3.7 X 105 sites per cell. The Ka of transferrin binding to BeWo cell extracts was found to be an order of magnitude higher than the Ka for binding to receptor solubilized from placental brush border membranes (13). This result does not necessarily indicate different properties of these receptors because the Ka determined for the placentaderived receptor was probably decreased by the presence of endogenous human transferrin bound to such membrane preparations. Because BeWo cells are not grown in human serum, they do not have the complication of endogenous human transferrin; however, some fetal bovine transferrin may be present and influence binding as well. Because iodinated proteins may exhibit properties different from those of their un-
labeled counterparts, the absolute values for binding constants derived in the current study may differ from those reported by others for transferrin binding. The protein nature of the BeWo transferrin-binding site was suggested by the effect of proteolytic digestion of cells on their high-affinity transferrin binding. Incubation at 370C for 10 min with 200 ,.g of purified trypsin per ml decreased the binding capacity of the cells 80% relative to cells incubated without trypsin. A trypsin inhibitor (N-2-tosyl-L-lysyl chloromethyl Table 1. Transferrin binding in various cell lines % specific transferrin Cell line Tissue of origin binding per 106 cells* 19 ± 2.0 Choriocarcinoma BeWo 13 ± 1.0 WI-38 Embryonic lung 14 + 1.0 Wil-2 Splenic lymphocytes 3.6 ± 0.2 RPMI-2650 Nasopharyngeal carcinoma 0.3 + 0.2 Normal adult blood Erythrocytest * Mean ± SD. t Prepared by collection of blood in tubes containing NaEDTA and four washings with phosphate-buffered saline.
Cell Biology: Hamilton et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
ketone) was used in the binding studies done on trypsinized, washed cells to prevent any digestion of labeled transferrin which might interfere with the assay. The possibility that the occurrence of transferrin receptors on cultured cells was limited to cells of placental origin was ruled out by the examination of a number of human cell lines for high-affinity transferrin binding. Four phenotypically distinct cell lines (WI-38, Wil-2, BeWo, and RPMI-2650) possessed high-affinity transferrin binding sites (Table 1). Human erythrocytes, which are known to lack transferrin receptors (25), did not bind a significant percentage of labeled transferrin. Using BeWo cells and immunoprecipitation of the solubilized transferrin-receptor complex with anti-transferrin antiserum in the presence of saturating amounts of unlabeled transferrin, we attempted to isolate and identify the surface transferrin receptor on cultured cells. This approach has been used to identify or isolate the receptor from rabbit reticulocytes (26, 27) or placental brush border membranes (13). The transferrin-receptor complex was immunoprecipitated with antihuman transferrin antiserum from 1% Triton X-100 extracts of lactoperoxidase iodinated BeWo cells containing 0.1 JIM unlabeled transferrin. NaDodSO4/polyacrylamide gel electrophoresis of the immune complexes demonstrated an 125I-labeled subunit with a Mr of 90,000 + 5000, which was designated the putative transferrin receptor (Fig. 3, lane 1). This subunit was not transferrin which migrated at a Mr of 77,000 (Fig. 3, lane 6). Controls run either with nonimmune serum (Fig. 3, lane 2) or without preincubation of labeled cells with human transferrin (Fig. 3, lane 3) did not give the 90,000-dalton polypeptide, which indicated that this cell surface labeled component was not binding to the anti-transferrin antibodies
6409
A I
I..
I 1307264-
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WI
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B
I
130-
M-
i*
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ii
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...
43-
130-
a. o 72-
3.8
-U
x 64-
43 -
1
2
3
4
5
6
FIG. 3. NaDodSO4polyacrylamide gel electrophoresis of the immunoprecipitated transferrin-receptor complex. Transferrinreceptor complexes solubilized from BeWo cells (lanes 1-3) or WI-38 cells (lanes 4 and 5) labeled with 125I by lactoperoxidase-catalyzed iodination were precipitated by anti-transferrin antiserum at pH 5.0. Lanes: 1, anti-transferrin antiserum in the presence of 0.1 ,uM transferrin; 2, nonimmune serum; 3, anti-transferrin antiserum; 4, anti-transferrin antiserum; 5, nonimmune serum; 6 125I-transferrin. The gels were dried and autoradiographed for 1-4 days to give optimal exposures.
5.4
6.3 pI
7.4
8.1
FIG. 4. Two-dimensional electrophoresis of the immunoprecipitated transferrin-receptor complex. (A) Triton X-100 extract of 125I-labeled BeWo cells immunoprecipitated with an anti-placental brush border antiserum (26) according to Kessler (17) by using S. aureus. The immunoprecipitate was extracted with 2% NaDodSO4 sample buffer. After reduction with 10%o 2-mercaptoethanol at 1000C for 2 min, the extract was diluted 1:5 with 9 M urea/5 mM NaPO4 pH 8.0/1% Triton X-100/5% 2-mercaptoethanol and concentrated to 100 ,ul for analysis by two-dimensional electrophoresis according to Wada et al. (20). The first dimension was isoelectric focusing in a pH 3.5-10 gradient 4% polyacrylamide gel containing 8 M urea, 1% ampholytes, and 0.5% Triton X-100. The second dimension was polyacrylamide gel electrophoresis in 8% acrylamide gel slabs containing 0.1% NaDodSO4. The dried gels were autoradiographed for 3 days. (B) The transferrin-receptor complex was precipitated from a Triton X-100 extract of 12'I-labeled BeWo cells with anti-transferrin antiserum as in Fig. 3 and analyzed by two-dimensional electrophoresis as described in A. (C) The transferrin-receptor complex was precipitated from a Triton X-100 extract of 125I-labeled placental brush border membrane vesicles with anti-transferrin antiserum by using the same procedure as described for the BeWo cells and was analyzed by two-dimensional electrophoresis. The identification of placental glycoprotein no. 15b as the transferrin receptor is reported elsewhere (13). The most basic spot is 1251-labeled human transferrin bound to the brush border membrane.
6410
Cell Biology: Hamilton et al.
or nonspecifically to IgG. WI-38 cells gave an '25I-labeled polypeptide of the same size (Fig. 3, lane 4) in immunoprecipitates of the transferrin-receptor complex, which was consistent with the detection of high-affinity transferrin binding in these cells. The data provided by the present study do not allow determination of the number of subunits involved in the native receptor molecule. The same 90,000-dalton material was observed whether transferrin was added to cells prior to or after extraction with Triton X-100. This observation is good evidence that the transferrin-binding sites in soluble extracts and on intact cells involve the same component. Two-dimensional electrophoresis of immunoprecipitated transferrin-receptor complexes from 125I-labeled BeWo cells revealed the 90,000 Mr polypeptide to be identical in electrophoretic mobility with a glycoprotein isolated from placental brush border membranes by immunoprecipitation of the transferrin-receptor complex solubilized from this membrane (Fig. 4). The placental receptor is a membrane component that has previously been characterized, by two-dimensional electrophoresis, with respect to its glycoprotein nature, mean pI of 6.6, and Mr of approximately 100,000; it was referred to as no. 15b (21). Fig. 4A shows the pattern of placental cell surface glycoproteins expressed by BeWo cells and isolated by immunoprecipitation with anti-placental brush border antiserum. At Mr of approximately 90,000-100,000, one can see a complex of at least two components, the most basic of which was previously designated no. 15b (21). Fig. 4B is a gel of the immune complex containing anti-human transferrin, transferrin, and the transferrin receptor isolated from the same radiolabeled cell extract as used in Fig. 4A. In the experiment it appears that no. 15a (the more acidic component) and no. 15b are both specifically interacting with transferrin. In addition to their relative positions in the two-dimensional matrix, these data strongly suggest that these components may be differentially glycosylated products of the same polypeptide core. Fig. 4C shows a gel of both the receptor (no. 15b) and transferrin isolated from detergent extracts of placental brush border membranes by the same immunoprecipitation technique. Transferrin is the more basic componenat. In addition, the 90,000-dalton component can be reprecipitated from pH 8.0 buffer eluates of immunoprecipitates by using antisera raised against detergent-extracted placental brush border membranes (data not shown). These observations demonstrate immunochemical and physical similarity of the placenta- and cell culture-derived receptors. This component was also demonstrated, by the use of antiserum against placental brush border membranes, to be absent from human liver and kidney membranes (28) and erythrocyte ghosts (unpublished observation) but present on the cell surfaces of all cultured human cell lines examined, including Wil-2, RPMI-2650 (unpublished data), WI-38, and BeWo (15). The universality of expression of the cell surface receptor for transferrin, glycoprotein no. 15b, is consistent with the universality of the transferrin requirement for cells grown in serum-free medium and suggests the obligatory nature of transferrin-mediated iron transport in growing cells. Interestingly, data indicating a correlation between transferrin binding and growth stimulation were obtained by comparing the Kd (1/Ka) of transferrin binding to BeWo cells (2.2 X 10-9
Proc. Natl. Acad. Sci. USA 76 (1979)
M) to the Km for growth stimulation by transferrin in HeLa cells grown in serum-free medium (1.4 X 10-9 M).* The absence of detectable levels of glycoprotein no. 15b in liver and kidney membranes may be due to the resting state of these tissues and their lower requirement for this form of iron uptake. * The Km for transferrin stimulation of HeLa cell growth was calculated from the data reported by Hutchings and Sato (6). The authors thank Mrs. Agnes W. Tin for assistance with cell culture. This work was supported by Grant CA 13533 from the National Institutes of Health and Contract CB 74086 from the National Cancer Institute.
1. Armelin, H. A. (1973) Proc. Natl. Acad. Sci. USA 70, 27022706. 2. Nishikawa, K., Armelin, H. A. & Sato, G. (1975) Proc. Nati. Acad. Sci. USA 72, 483-487. 3. Samuels, H. H., Tsai, J. S. & Cintron, R. (1973) Science 181, 1253-1256. 4. Sato, G. H. (1975) in Biochemical Actions of Hormones, ed. Litwack, G. (Academic, New York), pp. 391-396. 5. Hayashi, I. & Sato, G. H. (1976) Nature 259, 132-134. 6. Hutchings, S. E. & Sato, G. H. (1978) Proc. Natl. Acad. Sci. USA 75,901-904. 7. Rudland, P. S., Durbin, H., Clingan, D. & Jimenez de Asua, L. (1977) Biochem. Biophys. Res. Commun. 75,556-562. 8. Young, D. V., Cox III, F. W., Chipman, S. & Hartman, S. C. (1979) Exp. Cell Res. 118, 410-414. 9. Laurell, C. B. & Morgan, E. H. (1964) Acta Physiol. Scand. 62, 271-279. 10. Van Bockxmeer, F., Hemmaplardh, D. & Morgan, E. H. (1975) in Proteins of Iron Storage and Transport in Biochemistry and Medicine, ed. Crichton, R. R. (North-Holland, Amsterdam), pp. 111-119. 11. King, B. F. (1976) Anat. Rec. 186, 151-159. 12. Larrick, J. W. & Cresswell, P. (1979) Biochim. Biophys. Acta 583,
483-490. 13. Wada, H. G., Hass, P. E. & Sussman, H. H. (1979) J. Biol. Chem., in press. 14. Hamilton, T. A., Tin, A. W. & Sussman, H. H. (1979) Proc. Natl. Acad. Sci. USA 76,323-327. 15. Hamilton, T. A., Wada, H. G. & Sussman, H. H. (1979) J. Supramol. Struct., in press. 16. Mehdi, S. Q. & Nussey, S. S. (1975) Biochem. J. 145, 105-111. 17. Phillips, D. R. & Morrsion, M. (1970) Biochem. Biophys. Res. Commun. 40, 284-289. 18. Kessler, S. (1975) J. Immunol. 115, 1617-1624. 19. Davis, B. J. (1965) Ann. N. Y. Acad. Sci. 121, 404-427. 20. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 21. Wada, H. G., Gornicki, S. Z. & Sussman, H. H. (1977) J. Supramol. Struct. 6,473-484. 22. Hertz, R. (1959) Proc. Soc. Exp. Biol. Med. 102,77-80. 23. Pattillo, R. A. & Gey, G. 0. (1968) Cancer Res. 28, 12311236. 24. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672. 25. Jandl, J. G. & Katz, J. H. (1963) J. Clin. Invest. 42, 314-426. 26. Sullivan, A. L. & Weintraub, L. R. (1978) Blood 52,436-446. 27. Ecarot-Charrier, B., Grey, U., Wilczynska, A. & Schulman, H. G. (1977) in Proteins of Iron Metabolism, eds. Brown, E. B., Aisen, P., Fielding, J. & Crichton, R. R. (Grune & Stratton, New York), pp. 291-298. 28. Wada, H. G., Hass, P. & Sussman, H. H. (1979) J. Supramol. Struct. 10(3), 287-305.
Cell Biology
Identification of transferrin receptors on the surface of human cultured cells (serum factors/ligand binding/glycoproteins)
THOMAS A. HAMILTON, H. GARRETT WADA, AND HOWARD H. SUSSMAN Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Communicated by Robert T. Schimke, August 31, 1979
ABSTRACT We have examined the binding of human transferrin to cultured human choriocarcinoma cell lines and to detergent extracts of such cells. The results indicate the presence of a high-affinity saturable binding site (Ka = 4.25 X 108 M-1) that is specific for transferrin. This receptor has also been detected on three other human cell lines of different phenotypic origin, including Wil-2 (splenic lymphocytes of B-cell origin), RPMI-2650 (a quasi-diploid nasopharyngeal carcinoma), and WI-38 (embryonic lung fibroblasts). By using anti-human transferrin antiserum to immunoprecipitate the receptortransferrin complex from detergent extracts of cells containing saturating levels of transferrin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single polypeptide of 90,000 daltons has been identified as a subunit of the putative transferrin receptor. The protein shows immunochemical identity and coelectrophoreses in sodium dodecyl sulfate gels with a cell surface glycoprotein subunit, previously identified in placental brush border membrane preparations, on all human cultured cell lines examined. These results suggest that the recent demonstration of transferrin dependence of maximal cell growth in culture [e.g., Hutchings, S. E. & Sato, G. H. (1978) Proc. Nat]. Acad. Sci. USA 75, 901-904] is mediated through expression of this glycoprotein receptor.
METHODS Cell Culture. BeWo cells and WI-38 cells were obtained from the American Type Culture Collection (CCL 98 and CCL 75) and grown as described (14, 15). RPMI-2650 cells (CCL 30) were obtained from the same source and grown in minimal essential medium plus 10% fetal calf serum; Wil-2 cells were a gift from David Korn (Stanford University School of Medicine, Department of Pathology) and were grown in RPMI-1640 medium plus 10% fetal calf serum. Transferrin Binding Assays. After two washes with Ca- and Mg-free Earle's balanced salt solution, cells were removed from monolayers by incubation in Earle's solution containing 5 mM EDTA. Cells were then resuspended in Earle's solution containing 1 mg of ovalbumin (twice crystallized, Sigma) per ml for transferrin binding assay. For soluble receptor assay or for immunoprecipitation studies, cells were scraped into phosphate-buffered saline after thorough washing with Earle's solution. The suspension was then made 1% (vol/vol) in Triton X-100 and extracted for 10 min on ice. The extracts were then centrifuged at 140,000 X g (max) for 1 hr. Binding of transferrin to intact cells was measured by using 106 cells per assay in a total volume of 0.5 ml of Earle's solution at pH 7.5 containing 2 mg of ovalbumin per ml. After incubation with 125I-labeled human transferrin [125I-transferrin; labeled by chloramine-T iodination (16)] at 37°C for 30 min, the incubation was stopped by chilling on ice, addition of 1 ml of ice cold phosphate-buffered saline, and centrifugation at 400 X g for 10 min at 0-4°C. Both the supernatant and the cell pellet were assayed for radioactivity and percentage bound to the cell pellet was calculated. Background binding was determined by running controls with no cells, and high-affinity specific binding was determined by subtraction of low-affinity binding obtained with controls containing cells and 1 ,tM unlabeled ferrotransferrin (77 ,ug/ml). Assays of transferrin binding in Triton X-100 extracts of BeWo cells were conducted essentially as described by Wada et al. (13) with approximately 400 ,ug of protein per assay. Human transferrin, albumin, and gamma globulin (Sigma) used for competitive binding studies were further purified by gel filtration through Sephacryl-200 (Pharmacia). Immunoprecipitation of the Solubilized TransferrinReceptor Complex. Surface proteins of BeWo cells and WI-38 cells were radiolabeled with 125I by lactoperoxidase-catalyzed iodination (17). Approximately 2 X 107 cells were labeled, removed by EDTA incubation as described above, and suspended in 1 ml of phosphate-buffered saline. Triton X-100 extracts or cell suspensions were made 10 ,ig/ml in ferrotransferrin and 1 mg/ml in ovalbumin and incubated at 37°C for 10 min. The cells were then extracted with detergent as described above. Triton X-100 extracts were subjected to immunoprecipitation
Several investigations (1-4) have defined the serum dependence of cells in culture to be a requirement for hormones present in serum. Further work demonstrated that the hormonal requirement varies from one cell line to another (5), depending upon the cell type from which the cell line was originally derived (5, 6). In addition to the hormonal requirements of cells in serum-free media, it was noted that transferrin was necessary to maintain maximal growth (5, 6). However, whereas hormonal requirements vary, all types of cells in culture require transferrin (6). Furthermore, the transferrin requirement appears to be related to its iron transport function rather than to a regulatory function because increased levels of FeSO4 can substitute for transferrin in cultures of 3T6 Swiss mouse fibroblasts (7) and simian virus 40-transformed 3T3 cells (8). There is evidence indicating the existence of transferrin receptors on reticulocytes (9, 10) and placental tissue (10, 11), both of which are involved in active iron uptake. In addition, a cell surface receptor for transferrin has been recently demonstrated on cultured human lymphocytes (12). In a previous study, we obtained direct evidence for the presence of a specific transferrin receptor on human placental brush border membranes and identified a placental membrane glycoprotein with high affinity for transferrin (13). The present report presents evidence that this same protein is expressed on the surface of various human cultured cell lines of different phenotypic origin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: NaDodSO4, sodium dodecyl sulfate.
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Proc. Natl. Acad. Sci. USA 76 (1979)
with rabbit anti-human transferrin exactly as described by Wada et al. (13). Staphylococcus aureus was used ax theirMmunoadsorbant according to Kessler (18). 0
E c D 0
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Polyacrylamide Gel Electrophoresis. One-dimensional sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was conducted according to Davis (19) with the inclusion of 0.1% NaDodSO4 in the top electrode reservoir, stacking gel, and 8% acrylamide running gel as described (13). Subunit molecular weights were estimated by the method of Weber and Osborn (20) by using, as protein standards, f3-galactosidase (130,000), human transferrin (77,000), placental alkaline phosphatase (64,000), and ovalbumin (43,500). Two-dimensional electrophoresis was conducted according to Wada et al. (21) by using isoelectric focusing and NaDodSO4/polyacrylamide gel electrophoresis.
C
0
C
0c
Transferrin,
nM
0
10
0
Time, min
cm -,
E c0
._
c C
.0
0 C
4V
C
at
12
11
10
9
7
8
6
5
-log protein concentration (M) FIG. 1.
Binding of 1251-transferrin to BeWo cells. (A) Binding
curves for transferrin interaction with BeWo cells. Approximately 20,000 cpm of 1251-transferrin and 1 X 106 cells were used for each point. The concentration of unlabeled ferrotransferrin was varied over the indicated range. Cells were incubated at 371C for 30 min. The pmole of transferrin bound was calculated from the total radioactivity bound by correction for isotope dilution with unlabeled transferrin,
giving
total transferrin bound
(0-0). Low-affinity binding
-) was calculated by taking the slope of the total binding curve
- -
between 0.1 and 1 ,M transferrin and extrapolating through the origin. Specific, high-affinity binding (O- -0) was calculated by taking -
the difference between the total and low-affinity binding curves. (B) Time course of transferrin binding to BeWo cells. Cells were added to the assay medium containing 1251-transferrin at time zero and incubated at 370C. Aliquots were withdrawn immediately after addition of cells and at the indicated time intervals for assay of percentage transferrin specifically bound. 0-0, High-affinity binding (0.1 nM); 0-- 0, low-affinity binding (1 ,uM). (C) Competition by transferrin, albumin, and IgG for 1251-transferrin binding to BeWo cells. The indicated concentrations of ferrotransferrin (0-0), albumin (A.-A- ), and IgG (O--- 0) were tested for their effect on 1251-
transferrin binding to BeWo cells as described above, with a 20-min incubation period. The binding values were normalized by calculating
percentage 1251-transferrin binding remaining by using binding with no additions of competing
protein as the reference value.
RESULTS AND DISCUSSION Binding of transferrin was initially examined by measuring the amount of 125I-transferrin bound to suspensions of cultured BeWo choriocarcinoma cells (22, 23). Low-affinity binding (determined from the slope of the total binding curve taken between transferrin concentrations of 0.1 and 1 AM and extrapolated to 0) was subtracted from the total binding curve to estimate the amount of specific high-affinity binding (Fig. 1A). Fig. 1B, a time course for high-affinity and low-affinity transferrin binding to BeWo cells at low (0.1 nM) and high (1 ,OM) concentrations of transferrin, shows a rapid approach to maximal transferrin binding, achieved in 10 min, and then a slower but steady decrease in transferrin bound to the highaffinity site. After 3 hr of incubation at 370C, this high-affinity binding was decreased to the level observed for low-affinity unsaturable binding. The specificity of transferrin binding to BeWo cells was demonstrated by studies examining the competition of human transferrin, IgG, or albumin for 125I-transferrin binding. Only unlabeled ferrotransferrin competed with the labeled transferrin for binding sites on BeWo cells (Fig. 1C). No difference was observed when apotransferrin was substituted for ferrotransferrin, indicating that the degree of iron saturation has no marked effect on binding specificity. Because transferrin binding to intact cells exhibited a timedependent decrease, the further characterization of the transferrin receptor by Scatchard analysis (24) required the development of an experimental system in which equilibrium binding could be achieved. This was accomplished by examining transferrin binding in Triton X-100 extracts of BeWo cells in a fashion similar to that developed in the course of studies on the transferrin receptor found in placental brush border membranes (13). The soluble binding assay takes advantage of the stability of the receptor-transferrin complex at pH 5.0 and the differential solubility of transferrin in 12% polyethylene glycol, depending upon its association with higher molecular weight structures. Fig. 2A shows a binding curve constructed in a fashion similar to that shown in Fig. 1A but in which transferrin binding was measured by precipitation of the soluble receptor-transferrin complex with polyethylene glycol at pH 5.0. The similarity of Fig. 1A and Fig. 2A is evident and suggests high-affinity interaction of transferrin with a specific receptor. Neither human IgG nor albumin competed in the soluble receptor assay, consistent with the data shown in Fig. 1B. Fig. 2B shows a time course of transferrin binding to lowaffinity or high-affinity components. Equilibrium binding was reached by 30 min and showed no tendency to decrease with further incubation. All studies reported here using the soluble receptor assay were done with a 30-min incubation, and equilibrium binding was assumed. Fig. 2C shows that the binding of transferrin to its soluble high-affinity receptor is fully reversible. Within 1 min after the
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Cell Biology: Hamilton et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
c
B
50
0
0 D c
E
.0
0
E
0-
0
--
40 0 ~ ~~~~~~~~~-
30
c
a 0 .0 c C
0 4-
20 _____
._
__
___
---0
---
10 '|,
L
10 Transferrin, nM
20
30 Time at
40
50
60
370C, min
0 c
n c 0
m
0
60 50 40 Time at 370C, min
75
1.0
2.0
Transferrin, nM
FIG. 2. Binding of 1251-transferrin in detergent extracts of BeWo cells. (A) Equilibrium binding curve for transferrin binding to soluble receptor. To each assay mixture (total volume, 0.5 ml) were added 0.4 mg of detergent-extracted protein and approximately 20,000 cpm of
1251-transferrin. The transferrin concentration was varied over the indicated range by the addition of unlabeled transferrin to the concentrations indicated. The total binding (0-0), low-affinity binding (^-.-^), and high-affinity binding (O--- 0) were determined as in Fig. 1A. (B) Time course for transferrin binding to soluble receptor; 0.4 mg of extracted protein and 20,000 cpm of 1251-transferrin were used for each point. For high-affinity binding (0-0), unlabeled transferrin was present at 0.1 nM. For low-affinity binding (O 0), transferrin was present at 1 juM. (C) Reversibility of transferrin binding to soluble receptor. Each assay was as described for B. Samples first were preincubated at 370C for 15 min and then the experiment was begun by addition of unlabeled transferrin to 1 ,M. (D) Scatchard plot of equilibrium binding. Values for high-affinity binding from A were plotted according to Scatchard (23) by using linear regression to find the line that best fit the experimental values. The correlation coefficient (r2) for this plot was 0.89, and the slope, which is equal to -K,, was 6.2 X 108 M-1. ---
addition of excess unlabeled transferrin, the specific binding was decreased to less than 50% of its equilibrium value. By 30 min the exchange appeared to be complete. Fig. 2D shows a plot of the binding data of Fig. 2A according to Scatchard (24). Plots of bound transferrin/free transferrin versus the concentration of bound transferrin were linear at low concentrations, indicating a single high-affinity binding site. The association constant (Ka) was found to be 4.25 ± 2.1 X 108 M-1 (mean + SD), and the number of binding sites was 2.3 X 1012/mg of protein. When normalized to the number of cells originally extracted, this indicates 3.7 X 105 sites per cell. The Ka of transferrin binding to BeWo cell extracts was found to be an order of magnitude higher than the Ka for binding to receptor solubilized from placental brush border membranes (13). This result does not necessarily indicate different properties of these receptors because the Ka determined for the placentaderived receptor was probably decreased by the presence of endogenous human transferrin bound to such membrane preparations. Because BeWo cells are not grown in human serum, they do not have the complication of endogenous human transferrin; however, some fetal bovine transferrin may be present and influence binding as well. Because iodinated proteins may exhibit properties different from those of their un-
labeled counterparts, the absolute values for binding constants derived in the current study may differ from those reported by others for transferrin binding. The protein nature of the BeWo transferrin-binding site was suggested by the effect of proteolytic digestion of cells on their high-affinity transferrin binding. Incubation at 370C for 10 min with 200 ,.g of purified trypsin per ml decreased the binding capacity of the cells 80% relative to cells incubated without trypsin. A trypsin inhibitor (N-2-tosyl-L-lysyl chloromethyl Table 1. Transferrin binding in various cell lines % specific transferrin Cell line Tissue of origin binding per 106 cells* 19 ± 2.0 Choriocarcinoma BeWo 13 ± 1.0 WI-38 Embryonic lung 14 + 1.0 Wil-2 Splenic lymphocytes 3.6 ± 0.2 RPMI-2650 Nasopharyngeal carcinoma 0.3 + 0.2 Normal adult blood Erythrocytest * Mean ± SD. t Prepared by collection of blood in tubes containing NaEDTA and four washings with phosphate-buffered saline.
Cell Biology: Hamilton et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
ketone) was used in the binding studies done on trypsinized, washed cells to prevent any digestion of labeled transferrin which might interfere with the assay. The possibility that the occurrence of transferrin receptors on cultured cells was limited to cells of placental origin was ruled out by the examination of a number of human cell lines for high-affinity transferrin binding. Four phenotypically distinct cell lines (WI-38, Wil-2, BeWo, and RPMI-2650) possessed high-affinity transferrin binding sites (Table 1). Human erythrocytes, which are known to lack transferrin receptors (25), did not bind a significant percentage of labeled transferrin. Using BeWo cells and immunoprecipitation of the solubilized transferrin-receptor complex with anti-transferrin antiserum in the presence of saturating amounts of unlabeled transferrin, we attempted to isolate and identify the surface transferrin receptor on cultured cells. This approach has been used to identify or isolate the receptor from rabbit reticulocytes (26, 27) or placental brush border membranes (13). The transferrin-receptor complex was immunoprecipitated with antihuman transferrin antiserum from 1% Triton X-100 extracts of lactoperoxidase iodinated BeWo cells containing 0.1 JIM unlabeled transferrin. NaDodSO4/polyacrylamide gel electrophoresis of the immune complexes demonstrated an 125I-labeled subunit with a Mr of 90,000 + 5000, which was designated the putative transferrin receptor (Fig. 3, lane 1). This subunit was not transferrin which migrated at a Mr of 77,000 (Fig. 3, lane 6). Controls run either with nonimmune serum (Fig. 3, lane 2) or without preincubation of labeled cells with human transferrin (Fig. 3, lane 3) did not give the 90,000-dalton polypeptide, which indicated that this cell surface labeled component was not binding to the anti-transferrin antibodies
6409
A I
I..
I 1307264-
9:
WI
43-
B
I
130-
M-
i*
4
T 72° 64x
43-
ii
J C
1307264-
...
43-
130-
a. o 72-
3.8
-U
x 64-
43 -
1
2
3
4
5
6
FIG. 3. NaDodSO4polyacrylamide gel electrophoresis of the immunoprecipitated transferrin-receptor complex. Transferrinreceptor complexes solubilized from BeWo cells (lanes 1-3) or WI-38 cells (lanes 4 and 5) labeled with 125I by lactoperoxidase-catalyzed iodination were precipitated by anti-transferrin antiserum at pH 5.0. Lanes: 1, anti-transferrin antiserum in the presence of 0.1 ,uM transferrin; 2, nonimmune serum; 3, anti-transferrin antiserum; 4, anti-transferrin antiserum; 5, nonimmune serum; 6 125I-transferrin. The gels were dried and autoradiographed for 1-4 days to give optimal exposures.
5.4
6.3 pI
7.4
8.1
FIG. 4. Two-dimensional electrophoresis of the immunoprecipitated transferrin-receptor complex. (A) Triton X-100 extract of 125I-labeled BeWo cells immunoprecipitated with an anti-placental brush border antiserum (26) according to Kessler (17) by using S. aureus. The immunoprecipitate was extracted with 2% NaDodSO4 sample buffer. After reduction with 10%o 2-mercaptoethanol at 1000C for 2 min, the extract was diluted 1:5 with 9 M urea/5 mM NaPO4 pH 8.0/1% Triton X-100/5% 2-mercaptoethanol and concentrated to 100 ,ul for analysis by two-dimensional electrophoresis according to Wada et al. (20). The first dimension was isoelectric focusing in a pH 3.5-10 gradient 4% polyacrylamide gel containing 8 M urea, 1% ampholytes, and 0.5% Triton X-100. The second dimension was polyacrylamide gel electrophoresis in 8% acrylamide gel slabs containing 0.1% NaDodSO4. The dried gels were autoradiographed for 3 days. (B) The transferrin-receptor complex was precipitated from a Triton X-100 extract of 12'I-labeled BeWo cells with anti-transferrin antiserum as in Fig. 3 and analyzed by two-dimensional electrophoresis as described in A. (C) The transferrin-receptor complex was precipitated from a Triton X-100 extract of 125I-labeled placental brush border membrane vesicles with anti-transferrin antiserum by using the same procedure as described for the BeWo cells and was analyzed by two-dimensional electrophoresis. The identification of placental glycoprotein no. 15b as the transferrin receptor is reported elsewhere (13). The most basic spot is 1251-labeled human transferrin bound to the brush border membrane.
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Cell Biology: Hamilton et al.
or nonspecifically to IgG. WI-38 cells gave an '25I-labeled polypeptide of the same size (Fig. 3, lane 4) in immunoprecipitates of the transferrin-receptor complex, which was consistent with the detection of high-affinity transferrin binding in these cells. The data provided by the present study do not allow determination of the number of subunits involved in the native receptor molecule. The same 90,000-dalton material was observed whether transferrin was added to cells prior to or after extraction with Triton X-100. This observation is good evidence that the transferrin-binding sites in soluble extracts and on intact cells involve the same component. Two-dimensional electrophoresis of immunoprecipitated transferrin-receptor complexes from 125I-labeled BeWo cells revealed the 90,000 Mr polypeptide to be identical in electrophoretic mobility with a glycoprotein isolated from placental brush border membranes by immunoprecipitation of the transferrin-receptor complex solubilized from this membrane (Fig. 4). The placental receptor is a membrane component that has previously been characterized, by two-dimensional electrophoresis, with respect to its glycoprotein nature, mean pI of 6.6, and Mr of approximately 100,000; it was referred to as no. 15b (21). Fig. 4A shows the pattern of placental cell surface glycoproteins expressed by BeWo cells and isolated by immunoprecipitation with anti-placental brush border antiserum. At Mr of approximately 90,000-100,000, one can see a complex of at least two components, the most basic of which was previously designated no. 15b (21). Fig. 4B is a gel of the immune complex containing anti-human transferrin, transferrin, and the transferrin receptor isolated from the same radiolabeled cell extract as used in Fig. 4A. In the experiment it appears that no. 15a (the more acidic component) and no. 15b are both specifically interacting with transferrin. In addition to their relative positions in the two-dimensional matrix, these data strongly suggest that these components may be differentially glycosylated products of the same polypeptide core. Fig. 4C shows a gel of both the receptor (no. 15b) and transferrin isolated from detergent extracts of placental brush border membranes by the same immunoprecipitation technique. Transferrin is the more basic componenat. In addition, the 90,000-dalton component can be reprecipitated from pH 8.0 buffer eluates of immunoprecipitates by using antisera raised against detergent-extracted placental brush border membranes (data not shown). These observations demonstrate immunochemical and physical similarity of the placenta- and cell culture-derived receptors. This component was also demonstrated, by the use of antiserum against placental brush border membranes, to be absent from human liver and kidney membranes (28) and erythrocyte ghosts (unpublished observation) but present on the cell surfaces of all cultured human cell lines examined, including Wil-2, RPMI-2650 (unpublished data), WI-38, and BeWo (15). The universality of expression of the cell surface receptor for transferrin, glycoprotein no. 15b, is consistent with the universality of the transferrin requirement for cells grown in serum-free medium and suggests the obligatory nature of transferrin-mediated iron transport in growing cells. Interestingly, data indicating a correlation between transferrin binding and growth stimulation were obtained by comparing the Kd (1/Ka) of transferrin binding to BeWo cells (2.2 X 10-9
Proc. Natl. Acad. Sci. USA 76 (1979)
M) to the Km for growth stimulation by transferrin in HeLa cells grown in serum-free medium (1.4 X 10-9 M).* The absence of detectable levels of glycoprotein no. 15b in liver and kidney membranes may be due to the resting state of these tissues and their lower requirement for this form of iron uptake. * The Km for transferrin stimulation of HeLa cell growth was calculated from the data reported by Hutchings and Sato (6). The authors thank Mrs. Agnes W. Tin for assistance with cell culture. This work was supported by Grant CA 13533 from the National Institutes of Health and Contract CB 74086 from the National Cancer Institute.
1. Armelin, H. A. (1973) Proc. Natl. Acad. Sci. USA 70, 27022706. 2. Nishikawa, K., Armelin, H. A. & Sato, G. (1975) Proc. Nati. Acad. Sci. USA 72, 483-487. 3. Samuels, H. H., Tsai, J. S. & Cintron, R. (1973) Science 181, 1253-1256. 4. Sato, G. H. (1975) in Biochemical Actions of Hormones, ed. Litwack, G. (Academic, New York), pp. 391-396. 5. Hayashi, I. & Sato, G. H. (1976) Nature 259, 132-134. 6. Hutchings, S. E. & Sato, G. H. (1978) Proc. Natl. Acad. Sci. USA 75,901-904. 7. Rudland, P. S., Durbin, H., Clingan, D. & Jimenez de Asua, L. (1977) Biochem. Biophys. Res. Commun. 75,556-562. 8. Young, D. V., Cox III, F. W., Chipman, S. & Hartman, S. C. (1979) Exp. Cell Res. 118, 410-414. 9. Laurell, C. B. & Morgan, E. H. (1964) Acta Physiol. Scand. 62, 271-279. 10. Van Bockxmeer, F., Hemmaplardh, D. & Morgan, E. H. (1975) in Proteins of Iron Storage and Transport in Biochemistry and Medicine, ed. Crichton, R. R. (North-Holland, Amsterdam), pp. 111-119. 11. King, B. F. (1976) Anat. Rec. 186, 151-159. 12. Larrick, J. W. & Cresswell, P. (1979) Biochim. Biophys. Acta 583,
483-490. 13. Wada, H. G., Hass, P. E. & Sussman, H. H. (1979) J. Biol. Chem., in press. 14. Hamilton, T. A., Tin, A. W. & Sussman, H. H. (1979) Proc. Natl. Acad. Sci. USA 76,323-327. 15. Hamilton, T. A., Wada, H. G. & Sussman, H. H. (1979) J. Supramol. Struct., in press. 16. Mehdi, S. Q. & Nussey, S. S. (1975) Biochem. J. 145, 105-111. 17. Phillips, D. R. & Morrsion, M. (1970) Biochem. Biophys. Res. Commun. 40, 284-289. 18. Kessler, S. (1975) J. Immunol. 115, 1617-1624. 19. Davis, B. J. (1965) Ann. N. Y. Acad. Sci. 121, 404-427. 20. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 21. Wada, H. G., Gornicki, S. Z. & Sussman, H. H. (1977) J. Supramol. Struct. 6,473-484. 22. Hertz, R. (1959) Proc. Soc. Exp. Biol. Med. 102,77-80. 23. Pattillo, R. A. & Gey, G. 0. (1968) Cancer Res. 28, 12311236. 24. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672. 25. Jandl, J. G. & Katz, J. H. (1963) J. Clin. Invest. 42, 314-426. 26. Sullivan, A. L. & Weintraub, L. R. (1978) Blood 52,436-446. 27. Ecarot-Charrier, B., Grey, U., Wilczynska, A. & Schulman, H. G. (1977) in Proteins of Iron Metabolism, eds. Brown, E. B., Aisen, P., Fielding, J. & Crichton, R. R. (Grune & Stratton, New York), pp. 291-298. 28. Wada, H. G., Hass, P. & Sussman, H. H. (1979) J. Supramol. Struct. 10(3), 287-305.
