THYROID Volume 2, Number 3, 1992 Mary Ann Liebert, Inc., Publishers

Thyrotropin Modulation of Epidermal Growth Factor (EGF) Binding to Receptors on Cultured Thyroid Cells YUEH-CHU L. TSENG, KENNETH D. BURMAN, SABITA LAHIRI, JUAN D'AVIS, and LEONARD WARTOFSKY

ABSTRACT Previous studies had shown that epidermal growth factor (EGF) will stimulate growth of cultured thyroid cells in vitro, and TSH will stimulate total assayable EGF receptor in cultured porcine thyroid cells. In this study, we report the effect of TSH on EGF binding to human thyroid cells. Addition of bTSH (1 mU/mL) in binding buffer during receptor assay stimulated specific EGF binding to cells, with an increase of 44% observed over the control after 1 h incubation at 37°C. Affinity crosslinking of the [125I]EGF-receptor complex showed a single labeled band with molecular size of 170 kD. No additional band was detected in the presence of TSH. Preincubation of cells with chloroquine, which inhibits lysosomal degradative enzyme activity, caused a continuous accumulation of bound EGF over a 4 h study period at 37°C, and TSH stimulated an increase in internalized EGF. In the presence of chloroquine, total specific bound EGF was linearly correlated to incubation time up to 4 h and can be expressed as

Bound

slope*time + intercept (time,,) Addition of TSH during the binding assay significantly increased the value of the slope when compared to control (p < 0.002). The rate at which prebound [l25I]EGF was released into medium was not affected by the presence of TSH, indicating that TSH-enhanced binding may not be attributed to a reduction in EGF degradation. Coincubation of thyroid cells with EGF at 0 and 1 ng/mL and increasing concentrations of TSH (0-10 mU/mL) indicated that EGF stimulated thymidine incorporation, although TSH failed to synergistically enhance EGF-stimulated cell growth. We conclude that (a) EGF receptor binding in human thyrocytes is enhanced by TSH, (b) the increase results from an enhanced rate of binding, (c) this increased binding rate is related to stimulation by TSH of receptor mediated internalization, (d) TSH-enhanced EGF binding to receptor was not translated into stimulation of cell growth in this cell culture system. =

INTRODUCTION

RECEPTORS prepared

FOR

EGF

have BEEN FOUND

in membranes

from

porcine and human thyroid glands (1,2). In vitro studies employing primary cultured thyroid cells have demonstrated that EGF will stimulate cell growth (3-5), antagonize differentiated thyroid functions mediated by TSH (6,7),

and maintain a normal intracellular thyroglobulin store during long-term culture (8). Several reports also have linked higher EGF binding to thyroid tumors with poorer prognosis (9-11). Binding of EGF to receptors on porcine thyroid cells was foundto be stimulated by TSH (12,13), and one report indicated that TSH also potentiated EGF-stimulated cell growth when cells were cultured in floating gel (12). In this article, we report

Presented in part at the 10th International Thyroid Congress, The Hague, Holland, February 1991. Endocrine-Metabolic Service, Departments of Medicine, Clinical Investigation and Surgery. Walter Reed Army Medical Center. Washington, DC, and the Uniformed Services University of the Health Sciences, Bethesda, Maryland. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

181

182

TSENG ET AL.

studies using human thyroid cells cultured in monolayer in which we characterize the EGF receptor and investigate the effect of TSH on EGF binding, internalization, and degradation as well as on EGF-stimulated DNA synthesis as assessed by

thymidine incorporation.

response variable data explained by the regression. With 2 perfect linear regression fit, the R value is !.

Precipitation of immunoreactive EGF Immunoreactive [l25I]EGF in incubation

media was determedia with antibody to EGF (raised in rabbit, IBI, New Haven, CT; 1:500 dilution with PBS containing 2 g/L bovine serum albumin) at 4°C for 16 h, followed by precipitation of antibody-EGF complex with a second antibody (goat antirabbit IgG, Peninsula Laboratories, Belmont, CA). After centrifugation at 2000gfor20min, precipitated [l25IjEGF was collected and counted in a gamma counter. Percentages of immunoreactive EGF in media were obtained by comparing precipitable EGF in media with that of stock [ l25I]EGF. mined

MATERIALS AND METHODS

Primary culture of human thyroid cells Surgically removed human thyroid tissues were digested with collagenase (4 mg/mL, Worthington, Freehold, NJ) according to previously described methods (8). After 3 days culture in flasks, thyroid cells were trypsinized and transferred to 24-well plates at a density of 100,000 cells/well in RPMI-1640 medium (GIBCO Lab, Grand Island, NY) containing 10% fetal bovine serum (Hyclone Lab, Logan, UT) and penicillin-streptomycin (100 units/mL-100 |xg/mL, Sigma Chemicals, St. Louis, MO). Primary cultured cells were used in experiments between 3 and 15 days after trypsinization.

[l25l]EGF binding to receptors on thyroid cells

a

by incubating

Effect of TSH on distribution of prebound EGF Cultured cells were incubated with [ ,25I]EGF at 37°C for 2 h. After washing with cold RPMI-1640 medium, cells were incubated in binding buffer with or without TSH ( 1 mU/mL) at 37°C. [125I]EGF remaining bound to cells was measured after various time periods.

[l25I]mEGF (0.1 nmol/L, DuPont, Wilmington, DE) in bind- Affinity crosslinking of [l25l]EGF to receptor buffer (RPMI-1640 medium with 2 g/L bovine serum albumin and 20 mmol/L Hepes), with or without bTSH (Thytro[l25I]EGF (0.2 nmol/L) was incubated with thyroid cells with ing

par, Armour Pharmaceuticals, Kankakee, IL), were added to each well (200 u-L/well), then incubated at temperatures and times specified in text. Nonspecific binding was estimated by coincubating cells with excess unlabeled mEGF (100 nmol/L, Sigma Chemicals). At the end of incubation, cells were washed twice with cold wash buffer (PBS with 2 g/L bovine serum albumin), and cell surface membrane bound and internalized [l25I]EGF were measured as previously described (14,15). To measure EGF binding at 37°C void of any factors contributed by degradation of internalized EGF, cells were preincubated with chloroquine ( 100 p,mol/L, Sigma Chemicals) at 37°C for 30 min to block lysosomal enzyme activity (16). [,25IjEGF (0.1 nmol/L) was then added to each well in the absence or presence of TSH ( 1 mU/mL), and they were further incubated at 37°C for the periods of time indicated. Total specific bound EGF was determined by subtracting the nonspecific binding in cells coincubated with excess unlabeled EGF.

Analysis of binding

data

To determine receptor affinity and the number of binding sites, binding data from cells incubated at 4°C for 6 h with ( l25I]EGF (0.1 nmol/L) in the presence of increasing concentrations of unlabeled EGF were used in Scatchard analysis ( 17) employing the LIGAND program (18). In an attempt to characterize binding at 37°C, at which temperature compartmentalization of EGF occurred, binding

cells were analyzed using a regression model to examine the linear relation between specific bound EGF (response variable) and incubation time (predictor variable). This procedure is used to perform unweighted least square fitting, and the goodness of fit was measured by the R2 value, which determines the proportion of variance in the

data from

chloroquine-pretreated

without TSH (1 mU/mL) at 37°C for 1 h. To assess nonspecific binding, cells were incubated simultaneously with excess unlabeled EGF (100 nmol/L). [,25I]EGF receptor complex was crosslinked chemically by disuccinimidyl suberate (Pierce Chemicals, Rockford, IL) according to previously described methods (19,20). [,25I]-labeled receptors were separated by polyacrylamide gel electrophoresis and detected by exposing dried gel to x-ray film. or

Thymidine incorporation into thyroid cells presence of EGF and TSH Thyroid

in the

cultured in serum-free media (binding or presence of EGF ( 1 ng/mL), with increasing concentrations of TSH (0-10 mU/mL) for 3 days. 3H-thymidine ( 1 p-Ci/well) was added to each well, and the cells were cultured for another 24 h. After removing media, the cells were extracted with cold 10% trichloroacetic acid (0.5 mL/well) for 10 min, followed by washing with cold PBS. The cells were then solubilized with 0.1 mol/L NaOH, and incorporated thymidine was determined by counting solubilized cell fragments in a beta counter. cells

were

buffer) in the absence

RESULTS

[125I]EGF was found to bind to cultured thyroid cells, and the binding was specific since it could be competitively displaced by the presence of unabeled EGF (Fig. 1). After 1 h incubation at 37°C, 25% of specific bound EGF remained on surface membranes, and 75% was internalized into the cells. Differences in distribution of bound EGF were observed at lower incubation temperatures. After 6 h incubation at 4CC, 66% was localized on

183

TSH INDUCES EGF INTERNALIZATION

37°C/1

4°C/6 hr

hr

2.5

Buffer

ES +TSH

Surface membrane bound

O

Internalized

"o

Z

~g

O

o

m CJ

2.0

w

i 1.5

m

CL CO 'S

0.5

0

TEMPERATURE

1

0.1

UNLABELLED EGF

(nmol/L)

FIG. 1. Specific binding of [,2SI]EGF (0.1 nmol/L) to thyroid cells ( 100,000 cells/well) in the presence of increasing concentration of unlabeled EGF, after incubation at 37°C for 1 h or 4°C for 6 hr. Surface membrane bound and internalized EGF were assayed as described in Materials and Methods.

surface membranes and 34% was internalized. Scatchard analysis of 4°C/6 h binding data showed that EGF receptor affinity varied in different tissues, with Kd ranging from 0.2 to 0.7 nmol/L and binding sites ranging from 0.6 to 3.5 fmol/100,000 cells in the thyroids studied (Table 1). EGF binding to thyroid cells decreased with declining temperature. After 1 h incubation, specific bound was 0.47%, 0.80%, and 1.4% of total added EGF at 4°C, 23°C, and 37°C, respectively (Fig. 2). Preincubation of cells with sodium cyanide (5 mmol/L) did not inhibit EGF internalization at 37°C. Addition of TSH to the incubation mixture promotes EGF binding to receptors on thyroid cells at 37°C. At 1 mU/mL, TSH significantly stimulated EGF binding by 44% over the control (Fig. 2). Similar stimulation by TSH was observed in cells preincubated with sodium cyanide (data not shown). TSHstimulated binding remained significant after 2 h at 37°C. However by 4 h, no difference could be detected (Fig. 3A). Affinity crosslinking of [ IJEGF receptor complex on intact

Table 1. Scatchard Analysis of Receptor Binding Data Obtained From Competitive EGF Binding to Thyroid Cells at 4°C for 6 h

Binding K,,

Thyroid tissues" Whole cell" Surface membrane1 Whole cell Surface membrane Whole cell Surface membrane

0.71 0.50 0.26 0.15 0.22 0.08

nmol/L nmol/L nmol/L nmol/L nmol/L nmol/L

M

37°C

23°C

4°C

FIG. 2. Specific binding of [l25I]EGF (0.1 nmol/L) to cultured thyroid cells(100,000 cells/well) after incubation for 1 hat 4°C, 23°C, and 37°C, with or without TSH (1 mU/mL) in binding buffer. Each data point was calculated from results of three samples (average ± SEM), p values were calculated from student's i-test. cells showed a single band of labeled protein with molecular size of 170 kD (Fig. 4). Excess unlabeled EGF completely abolished the labeling of this 170 kD protein. No additional radiolabeled band was observed when TSH was added to incubation buffer. When cells were preincubated with chloroquine at 100 p,mol/L to inactivate lysosomal enzymes, total bound EGF continued to rise with incubation time. By 4 h, chloroquine stimulated an 81% increase in total bound EGF (Fig. 3B). In comparison, TSH stimulated an even greater accumulation when chloroquine was present, with an increase of 148% over

*

*

A-û —

-t-Chloroquine +TSH + chloroquine

00

sites

100,000 cells 3.54 2.00 0.59 0.28 1.68 0.66

fmol fmol fmol fmol fmol fmol

"All thyroid cells were derived from multinodular goiter. Thyroid tissues 1 and 2 here represent same source as tissues 5 and 4 in Table 2. 'Total specific bound data (surface membrane bound + internalized) were used in Scatchard analysis. cOnly surface membrane bound data were used in Scatchard analysis.

A. Specific binding of [,25I]EGF (0.1 nmol/L) to cultured thyroid cells after incubation at 37°C for various time periods, with or without TSH ( I mU/mL) in binding buffer. B. Thyroid cells were preincubated with chloroquine (100 p,mol/L) at 37°C for 30 min, followed by addition of [125I]EGF (0.1 nmol/L) to binding buffer with or without TSH ( 1 mU/mL), and further incubated at 37°C for various time periods. Each data point was calculated from results of four samples (average ± SEM), p values were calculated by student's f-test with (*) and (**) indicating data points with p < 0.01 and p < 0.05, respectively, when compared with controls of respective incubation periods (TSH 0).

FIG. 3.

receptors

on

=

TSENG ET AL.

184

•

-200

-116 97 m

n—66

Fig. 4. Affinity crosslinking of (I25I]EGF to receptors on thyroid cells. Materials added in binding buffer: lane 1, [l25I]EGF(0.2

nmol/L); lane 2, [l25I]EGF and unlabeled EGF (100 nmol/L); lane 3, and unlabeled EGF. the control. Dissociation of surface-bound EGF with mild acidic solution showed that TSH stimulation of binding could be attributed to increases in internalized EGF (Fig. 5). At 37°C, accurate characterization of EGF receptor affinity and estimation of binding site number in cultured cells cannot be achieved by Scatchard analysis, which requires competitive binding data obtained during steady-state equilibrium. To exam+TSH

(1 mil/ml)

Fig. 5. Thyroid cells were pretreated with chloroquine (100 p.mol/L) at 37°C for 30 min, then incubated with [l2 T]EGF (0.1 nmol/L) EGF

at

were

37°C. Surface membrane bound and internalized

assayed as described in Materials and Methods.

[l25I]EGF and TSH (1 mU/mL); lane 4, [l25I]EGF and TSH

ine TSH influences on the rate of EGF binding (bound/time) to cell receptors, data from chloroquine treated cells were used to minimize loss through EGF degradation. Specific bound EGF appeared to be linearly related to incubation time (Fig. 3B), as demonstrated in the following model.

Bound (% total added EGF) slope*time(h) + intercept (at time0) =

Thus, Bound/time (binding rate)

=

slope

+

intercept/time

Statistical analysis of binding data during 0.5-4 h incubation showed a highly linear correlation between total bound and incubation time. R2 values are above 0.92 in all thyroid cells examined (Table 2). Binding data of 6 h incubation deviated from the linear line and appeared to approach the plateau, possibly due to depletion of free receptors or inactivation of chloroquine. Although the slopes of this binding rate model differed between thyroid tissues, addition of TSH during binding assay universally increased the slopes, with a ratio of 1.4-3.5:1 compared to control (Table 2). The TSH effect on slopes is significant according to the paired /-test (p < 0.002) or Wilcoxon signed rank test (p < 0.02). Immunoprecipitation of media from cells incubated at 37°C indicated that concentrations of immunoreactive EGF declined with prolonged incubation time, but addition of TSH did not affect immunoprecipitable EGF when compared to control during the 6 h incubation period.

TSH INDUCES EGF INTERNALIZARON Table 2. EGF Binding

to

185

Thyroid Cells Pretreated AT 37°C FOR 30 MINa Bound

(B)

vs

time

with

(Tf

TSH B

1

B B B B B B B B

+

2 +

3 +

4 +

5

B B B B

+

6 +

7

B B B

+

8 +

=

=

= =

=

=

= = = = =

Slope-ratio

R2

(h)

(%)

(I mUlmL)

Tissue

Chloroquine (100 p.mol/L)

1.225*T + 0.071 1.835*T- 0.056 1.059*T + 0.295 1,500*T + 0.625 0.934*T + 0.163 1,444*T + 0.271 .808*T + 0.338 2.121*1 + 0.277 0.892*T 0.467 1.938*T 0.108 0.826*T 0.240 2.902*T 0.955 1.439*T 0.067 2.060*T 0.862 0.539*T 0.239 1.545*T 0.441

TSH ( + )/TSH (-)

0.987 0.990 0.968 0.988 0.923 0.965 0.962 0.931 0.973 0.986 0.984 0.980 0.995 0.932 0.980 0.990

1.498

1.416 1.546 1.508 2.173 3.513 1.432 2.866

"Specific binding data (% of total added EGF) from 0.5, 1, 2, 4 h incubation at 37°C with or without TSH ( 1 mU/mL) used in fitting the first order, linear regression model. 'Tissues 1-5 are from multinodular goiters. Each incubation time point was assayed in triplicate. Tissues 6-8 are from normal thyroids. Each incubation time point was assayed in duplicate. 'Bound (% total added EGF) slope * time (h) + intercept (at time,,). Slopes of control and TSH-treated groups are, significantly different according to paired i-test (p < 0.002) or Wilcoxon signed rank test (p < 0.02). dR2 measures goodness of fit to the proposed mathematical model, with perfect fit as 1.0. were

=

To examine whether the apparent TSH-enhanced EGF bind-

ing could be attributed, at least in part, to decreased degradation

of internalized EGF, cells with prebound [ ,25I]EGF were incubated with or without TSH at 37°C over a 24 h period. Radioactivity released into buffer gradually increased, whereas cell bound radioactivity decreased with time. Addition of TSH did not alter the distribution profile of prebound EGF over the period of time studied, suggesting that TSH did not affect the processing of internalized EGF (Fig. 6). Precipitation of radioactivity released into the media with antibody to EGF showed that only 20% remained immunoreactive.

EGF promotes thymidine incorporation into cultured thyroid cells. After 3 days exposure to 1 ng/mLEGF, an increase of 35% was obtained. Under similar conditions of culture, TSH (0.001-10 mU/mL) had little effect on DNA synthesis. Moreover, TSH did not potentiate EGF-stimulated thymidine incorporation in thyroid cells (Fig. 7).

DISCUSSION We have found that EGF binds specifically to receptors on cultured human thyroid cells. Receptor affinity assayed at 4°C »EGF

Buffer

100

+TSH

Ing/ml

(1 i ,U/ml)

X 2

Z3 O

o

m

24

3

TIME

(hr)

Fig. 6. Disappearance of prebound [l25I]EGF after incubating

cells at 37°C in the absence various time periods.

or

presence of TSH ( 1 mU/mL) for

0

0.001

0.01

TSH

0.1

1.0

10

(mU/ml)

Fig. 7. Thymidine incorporation (24 h labeling) into thyroid cells that were preexposed to EGF at 0 or 1 ng/mL and TSH (0-10 mU/mL) for 3 days in serum-free media. Each data point represents the average ± SEM of four samples.

TSENG ET AL.

186 is similar to results in previous reports on thyroid cells, although a much lower number of binding sites was determined in our study (21-22). At physiologic temperature, most EGF was internalized after binding to receptor. Removal of EGF-receptor complex from media into cells appears to continuously drive the reaction toward formation of EGF-receptor complex, precluding achievement of a binding equilibrium. Although EGF binding to receptor reached a plateau after 4 h incubation at 37°C, binding to cells pretreated with chloroquine continued to rise with incubation time. Apparently, the plateau was reached not as a result of equilibrium among receptors, free EGF, and EGF-receptor complex but rather as a balance between the formation of EGF-receptor and degradation of internalized EGF. Due to limitations in Scatchard analysis that allow application only to data gathered at steady-state equilibrium, calculation of rates of EGF binding in active viable cells (as described here) is a logical technique to analyze hormone binding to cultured cells. Binding rate is directly related to several factors, including receptor affinity, the number of binding sites, and the internalization rate of hormone-receptor complex. Although our data demonstrated that the rate of EGF binding to cultured cells varied with thyroid tissue sources, TSH stimulated the binding rate in all tissues studied. Examination of the same data by Scatchard analysis indicated that TSH treatment increased receptor affinity, with no significant change in the number of binding sites. Our studies demonstrate that the observed TSH stimulation in EGF binding is the direct result of enhanced EGF internalization rather than a decreased EGF degradation rate in media or the decreased release of internalized EGF. We have observed that ANP internalization in cultured thyroid cells may be similarly stimulated by TSH (23). The exact mechanism underlying this action of TSH is unclear. EGF can be shown to stimulate thymidine incorporation in human thyroid cells cultured in monolayer, but under similar conditions, TSH does not affect DNA synthesis. In our studies, simultaneous addition of TSH to incubation media containing EGF did not enhance the EGF action on DNA synthesis despite the observed increased in receptor binding. One possible explanation is that the receptor number on cells and EGF concentration, which were similar during assay, are the only decisive factors in EGF-stimulated DNA synthesis. Additionally, the TSH effect on EGF binding might be transient, that is, a result of changes in the cell membrane. TSH has been shown to modulate membrane lipid fluidity of rat thyroid cells (24), to induce a transient increase in diacylglycerol and a decrease in phosphatidylinositol of pig thyroid follicles (25) and human thyroid (26), and to stimulate hydrolysis of phosphoinositides in FRTL-5 cells

ACKNOWLEDGMENTS We are indebted to Dr. Audrey Chang and Ms. Robin Howard for their advice on statistical analysis and to Ms. Estelle Coleman-Fraser for expert editorial assistance. This work was supported by funding from the Walter Reed Army Medical Center, Department of Defense, protocols 1399-87.

REFERENCES 1. Atkinson S, Smith PA, Taylor JJ, Kendall-Taylor P 1983 Specific epidermal growth factor receptors on porcine thyroid cell membrane. FEBS Lett 153:88-92. 2. Humphries H, MacNeil S, Munro DS, Tomlinson S 1984 Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes. J Endocrinol 102:57-61. 3. Roger PP, Dumont JE 1982 Epidermal growth factor controls the proliferation and the expression of differentiation in canine thyroid cells in primary culture. FEBS Lett 144:209-212. 4. Westermark K, Westermark B 1982 Mitogenic effect of epidermal growth factor on sheep thyroid cells in culture. Exp Cell Res

138:47-55. 5. Ollis CA, Davies R, Munro DS, Tomlinson S 1986 Relationship between growth and function of human thyroid cells in culture. J Endocrinol 108:393-398. 6. Eggo MC, Bachrach LK, Fayet G, et al 1984 The effects of growth factors and serum on DNA synthesis and differentiation in thyroid cells in culture. Mol Cell Endocrinol 38:141-150. 7. Errick JE, Eggo MC, Burrow GN 1985 Epidermal growth factor inhibits thyrotropin-mediated synthesis of tissue-specific proteins in cultured ovine thyroid cells. Mol Cell Endocrinol 43:51-59. 8. Tseng YL. Burman KD, Schaudies RP, et al 1989 Effects of epidermal growth factor on thyroglobulin and adenosine 3',5'monophosphate production by cultured human thyrocytes. J Clin Endocrinol Metab 69:771-775. 9. Duh QY, Gum ET, Gerend PL, Râper SE, Clark OH 1985 Epidermal growth factor receptors in normal and neoplastic thyroid tissue. Surgery 98:1000-1005. 10. Masuda H, Sugenoya A, Kobayashi S, Kasuga Y, Iida F 1988 Epidermal growth factor receptor on human thyroid neoplasms. World JSurg 12:616-622. 11. Kanamori A. Abe Y. Yajima Y. Manabe Y, Ito K 1989 Epidermal growth factor receptors in plasma membranes of normal and diseased human thyroid glands. J Clin Endocrinol Metab 68:899-903. 12. Westermark K, Westermark B, Karlsson FA, Ericson LE 1986 Location of epidermal growth factor receptors on porcine thyroid follicle cells and receptor regulation by thyrotropin. Endocrinology 118:1040-1046. 13. Atkinson S, Kendall-Taylor P 1986 The effect of TSH on the binding of EGF to thyroid cell monolayers. J Endocrinol

(27).

Finally, our results support earlier reports indicating that TSH was not a mitogen to cultured thyroid cells in monolayer (28,29). Methodologie factors may underlie differences in experimental observations, with Westermark et al. (12) reporting that TSH potentiates an EGF effect on cell growth when cells are cultured in floating gel, and Kraiem et al. (30) observing that TSH was mitogenic only in early in vitro culture. Also, the mitogenic action of TSH may be species specific, as reported by Roger et al. (31) in dog thyroid cells. Clarification of these differences in TSH effect must await future investigation.

108(suppl):174. Haigler HT, Maxfield FR, Willingham' MC, Pastan I

1980 Dansylcadaverine inhibits internalization of 25I-epidermal growth factor in Balb 3T3 cells. J Biol Chem 255:1239-1241. 15. Tseng YL, Sellitti DF, Ahmann AJ, Burman KD, D'Avis JC, Wartofsky L 1989 Thyrotropin modulates receptors for atrial natriuretic peptide on intact human thyroid cells. Am J Med Sei 298:15-19. 16. De Duve C 1983 Lysosomes revisited. Eur J Biochem 137:391-397. 17. Scatchard G 1949 The attraction of proteins for small molecules and ions. Ann NY Acad Sei 51:660-672. 14.

TSH INDUCES EGF INTERNALIZATION 18. Munson PH, Rodbard D 1980 Ligand: A versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220-239. 19. Meloche S, Ong H, Cantin M, DeLean A 1986 Molecular characterization of the solubilized atrial natriuretic factor receptor from bovine adrenal zona glomerulosa. Mol Pharmacol 30:537-543. 20. Tseng YL, Lahiri S, Sellitti DF, Burman KD, D'Avis JC, Wartofsky L 1990 Characterization by affinity cross-linking of a receptor for atrial natriuretic peptide in cultured human thyroid cells associated with reduction in both adenosine 3',5'-monophosphate production and thyroglobulin secretion, J Clin Endocrinol Metab 70:528-533. 21. Bachrach LK, Eggo MC, Mak WW, Burrow GN 1985 Phorbol esters stimulate growth and inhibit differentiation in cultured thyroid cells. Endocrinology 116:1603-1609. 22. Atkinson S, Kendall-Taylor P 1987 The mechanism of TSH induced increase in binding of EGF to porcine thyroid cell monolayers: The role of thyroid hormones. Acta Endocrinol 23.

281(suppl):260-263. Tseng YL, Burman KD, Lahiri S, Abdelrahim MM, D'Avis JC, Wartofsky L 1991 Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid

cells. J Clin Endocrinol Metab 72:669-674. Beguinot F, Beguinot L. Tramontano D, et al 1987 Thyrotropin regulation of membrane lipid fluidity in the FRTL-5 thyroid cell line. Its relationship to cell growth and functional activity. J Biol chem 262:1575-1582. 25. Igarashi Y, Kondo Y 1980 Acute effect of thyrotropin on phosphatidylinositol degradation and transient accumulation of diacylglyc-

24.

187

26.

27.

28.

29.

30.

31.

erol in isolated thyroid follicles. Biochem Biophys Res Commun 97:759-765. Laurent E, Mockel J, Van Sande J, Graff I, Dumont JE 1987 Dual activation by thyrotropin of the phospholipase C and cyclic AMP cascades in human thyroid. Mol Cell Endocrinol 52:273-278. Bone EA. Ailing DW, Grollman EF 1986. Norepinephrine and thyroid-stimulating hormone induce inositol phosphate accumulation in FRTL-5 cells. Endocrinology 119:2193-2200. Westermark B, Karlsson FA. Walinder O 1979 Thyrotropin is not a growth factor for thyroid cells in culture. Proc Nati Acad Sei USA 76:2022-2026. Valente WA, Vitti P, Kohn LD, et al 1983 The relationship of growth and adenylate cyclase activity in cultured thyroid cells: Separate bloeffects of thyrotropin. Endocrinology 112:71-79. Kraiem Z, Sadeh O, Sobel E 1990 Thyrotropin, acting at least partially via adenosine 3',5'-monophosphate, exerts both mitogenic and antimitogenic effects in cultured human thyroid cells. J Clin Endocrinol Metab 70:497-502. Roger PP, Servais P, Dumont JE 1987 Induction of DNA synthesis in dog thyrocytes in primary culture: Synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin. J Cell Physiol 130:58-67.

Address reprint requests to: Col. Leonard Wartofsky, M.D., Department of Medicine Walter Reed Army Medical Center Washington DC 20307-5001