Characterization of activin A binding sites on the human leukemia cell line K562

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 844-849

December 15, 1988

C H A R A C T E R I Z A T I O N OF ACTIVIN A BINDING SITES ON THE HUMAN L E U K E M I A C E L L LINE K562 Carolyn A. Campen and Wylie Vale The Clayton Foundation Laboratories for Peptide Biology, Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037 Received November 8, 1988

SUMMARY: Recombinant activin A was radioiodinated to a high specific activity with maintenance of bioactivity using the Bolton-Hunter method. The human leukemia cell line K562, known to differentiate in response to activin A, was found to possess high affinity [125I]BH-activin A receptors (Kd -0.13 riM) with a low number of receptors per cell (-600 per cell). This receptor was found to be specific as FSH, LH, GnRH, and TGF-[31 do not compete for binding. This is the first description o f binding sites for this protein hormone on K562 cells. © 19aa Acaaemic Press, Inc.

Activin A, or FSH Releasing Protein, is a protein hormone originally isolated from gonadal fluids of several species (I-6).

The biological response first

associated with activin A was the stimulation of FSH secretion from cultured rat pituitaries (1,2).

Sequence analysis showed that activin A is a member of the TGF-[3

gene family (2), and similar to TGF-[~, is only active as a homodimer (~A~A). Subsequently, a factor was isolated from the conditioned media of a leukemia cell line which causes the differentiation of mouse Friend cells and which shares sequence identity with activin (7,8).

More recently, activin A was shown to

stimulate the differeniiation of the human erythroleukemic cell line K562 (9).

In

the present study, we have investigated how the biological activity to activin A is mediated at the receptor level by developing a radioreceptor assay using iodinated recombinant activin A as ligand and K562 cells as a receptor source. MATERIALS AND METHODS Recombinant activin A was a generous gift from Anthony Mason, Genentech (San Francisco, CA). [125I]Bolton-Hunter reagent was obtained from New England Nuclear (Boston, MA). K562 cells were obtained from American Type Culture Collection (Rockville, MD). TGF-[31 was obtained from R&D Systems, Inc. (Minneapolis, MN). FSH and LH were obtained from SIGMA Chemical Co. (St. Louis, Me). Abbreviations: FSH, follicle stimulating hormone; LH, luteinizing hormone; TGF]31, transforming growth factor-beta one; FCS, fetal calf serum; BH, Bolton-Hunter; SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; TCA, trichloroacetic acid; HEPES, [ 4 - ( 2 - h y d r o x y e t h y l ) - l - p i p e r a z i n e e t h a n e s u l f o n i c acid; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; KIU, Kallikrein Inactivator Units. 0o06-291X/88 $1.5o Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Radioiodination of activin A: Activin A was iodinated using the BoltonHunter method (10). Briefly, recombinant activin A (2 ~tg) was radioiodinated with 1 mCi Bolton-Hunter reagent (NEN) at 4 o c . The final reaction volume was 36 ~tL in 140 mM sodium phosphate buffer, pH 8.5. The reaction vial was vortexed every 10 min. for 1 hr and then stored at 4 ° overnight. Separation of iodinated activin ([125I]BH-activin) from unreacted Bolton-Hunter reagent was accomplished on a G-25 column (0.7 X 20 cm) equilibrated with 0.22 M sodium phosphate buffer, pH 8.5, 0.2% fish gelatin (equilibration buffer) and pretreated twice with 10 mg of fish gelatin. Fractions (0.5 mL) were collected and assayed for [125I]BH-activin by electrophoresing peaks of radioactivity on 15% SDS-PAGE followed by autoradigraphy. [125I]BH-activin eluted in the void volume. Generally, greater than 90% of the cpms in this peak were TCA precipitable. Cell line: The human erythroleukemic cell line K562 was maintained in suspension culture in RPMI 1640 nutrient media supplemented with 12% FCS. B i n d i n g o f [ 1 2 5 I ] B H - a c t i v i n to K562 c e l l s : Cells were harvested by centrifugation and rinsed twice in HEPES-EDTA buffer (137 mM NaCI, 5 mM KCI, 0.7 mM Na2HPO4, 2 mM EDTA, 25 mM HEPES, pH 7.4) and once in binding buffer (RPMI 1640, 0.1.% BSA, 1000 KIU/mL, 25 mM HEPES, pH 7.4. Cells were then suspended in binding buffer and used within 15 rain. Cells (4 xl06 in 400 ltL) were then incubated with 300,000 epm [125I]BH-aetivin at 2 5 o c (unless otherwise stated) and kept in suspension by constant rotation. The reaction was terminated by centrifuging 0.35 mL of the incubation over 50 IxL dibutyl phthalate oil (Kodak) in a 0.4 mL Sarstedt polypropylene mierofuge tube. After centrifugation (Beckman microfuge) for 4 rain at 25°C, the tip of the tube was cut through the oil layer and counted in a y c o u n t e r . Results are the average of duplicate determinations. Nonspecific binding was determined in the presence of 13.4 nM unlabelled activin A. In a typical experiment, between 5 - 7% of the total radioactivity added was bound to the cells and the background (incubations with no cells) was < 0.1%. In the presence of excess activin A, 50 - 78% of the total bound cpms were displaceable. There was no specific binding to the polypropylene tubes.

RESULTS Characterization of [125I]BH-activin :

The radioligand ( [ 1 2 5 I ] B H - a c t i v i n

A) was characterized by SDS-PAGE as a single band of radiolabelled protein (Fig. I). The pooled fractions containing [125I]BH-activin than 90% TCA-preeipitable cpms.

generally

contained

greater

The specific activity of the radioligand was

estimated to be 25 - 109 ~tCi/~tg as judged be the ability of the radioligand to stimulate the differentiation (i.e. increased hemoglobin content) of K562 ceils (11,12). T e m p e r a t u r e d e p e n d e n c e o f [ 1 2 5 I ] B H - a c t i v i n A b i n d i n g to K 5 6 2 c e l l s : At 25oc, maximal specific binding occured as early as 30 min and remained stable for up to 90 rain after which the binding decreased slightly (Fig. 2).

At 4 ° C ,

specific binding at 60 min was only 60% that at 25°C and appeared to still be increasing after 3.5 h r .

All subsequent experiments were performed at 25°C for

70 - 80 min. S a t u r a t i o n and C o m p e t i t i o n A n a l y s i s o f [ 1 2 5 I ] B H - a c t i v i n A B i n d i n g : Analysis of both saturation curves and competition curves by the computer programs ALLFIT and LIGAND (13-15) allowed a determination of the dissociation constant (Kd) of 0.13 x 10 -9 M (0.1 - 0.2 x 10 -9 M) and an estimated receptor content 845

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Fi~. 1 SDS-PAGE profile of [125I]BH-activin A detected by autoradiography. An aliquot of [125I]BH-activin was run on a 15% polyacrylamide gel. The gel was dried and exposed to film for 4 hr. Fi~. 2 Kinetics of specific binding of [125I]BH-activin A to K562 cells at temperatures indicated. Cells were rinsed twice in HEPES-EDTA buffer and once in binding buffer (RPMI 1640, 0.1% BSA, 1000 KIU/mL, 25 mM HEPES, pH 7.4). Cells (4 xl06 in 400 ILL) were then incubated with approximately 75 fmol [t25I]BH-activin at 25°C (0--0) or 4oc ([]--[]) and kept in suspension by constant rotation. The reaction was terminated by centrifugation through oil in a microfuge tube. The tip of the tube was cut through the oil layer and counted in a ~ counter. Results are the average of duplicate determinations. Nonspecific binding was determined in the presence of 13.4 nM unlabeUed activin A.

of 550 - 620 receptors per cell.

(A representative association curve is shown in

Fig. 3 and representative competition curves are shown in Fig. 4). S p e c i f i c i t y of B i n d i n g : In order to demonstrate the pharmacological specificity of this receptor, several peptides and proteins were tested for their ability to displace bound [125I]BH-activin A.

FSH (25 nM), LH (6.25 nM), GnRH

(1.25 ttM), and increasing concentrations of TGF-131 (from 2 pM to 10 nM) did not have any detectable ability to displace the radioligand (Fig. 4). DISCUSSION A radioreceptor assay has been established for [125I]BH-activin A on the human erythroleukemic cell line K562.

Generation of a biologically active

radiolabeUed ligand was of primary importance.

Iodination of recombinant

activin A by the Bolton-Hunter method resulted in a ligand that is radiolabelled to a high specific activity (25 - 109 mCi/mg) with maintenance of biological activity. The use of the Bolton-Hunter reagent provides a mild iodination method that is non-oxidative, as opposed to the harsh oxidative conditions generated by the use of chloramine

T.

Several binding parameters were characterized in order to validate this radioreceptor assay.

First, radiolabelled activin A showed temperature-dependent

association kinetics, being maximally bound at 25°C in 30 min, but not after 210 846

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Fi~. 3 Binding of [125I]BH-activin A to K562 cells. Left panel: Saturation analysis of 1251-BH-activin A to K562 cells. Total binding (0--0); nonspecifie binding (&._A); specific binding ([]--[]). Right panel: Scatchard plot from a competition experiment (competition curve not shown). K562 cells from semiconfluent flasks were rinsed twice with HEPES-EDTA buffer and once with binding buffer. For the saturation analysis, 3.5 - 4 x 106 suspended cells and 0 - 75 fmol radioligand were incubated in a final volume of 0.4 mL binding buffer. For the competition analysis, a constant amount of 20 fmol radioligand per tube was used. Cells were kept in suspension with constant rotation for 80 rain.

min. at 4°C.

Binding of [125I]BH-activin A showed saturability at 25°C.

Scatchard

analysis of binding data revealed a single class of binding sites on K562 cells having a Kd = 130 pM. This K d is higher than the ECs0 of activin A on K562 cells

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Binding competition study comparing the ability of activin A, TGF-131 (left Binding experiments were carried out using standard conditions, incubating 3.5 x 106 K562 cells in the presence of a constant amount of radioligand and increasing amounts of either activin A ( ), TGF-131 ([]--[]), or single concentrations of FSH (11--II, 25 riM), LH ( -- , 6.25 nM), or GnRH ( A - - A , 1.25 lxM). The displacements curves were analyzed using the programs LIGAND and ALLFIT. Fig. 4

panel), FSH, LH, or GnRH (right panel) to displace [125I]BH-activin.

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(~30 pM) suggesting that the biological response triggering differentiation may occur at low receptor occupancy.

Finally, the pharmacological specificity of the

activin A receptor was demonstrated by the fact that unrelated proteins FSH, LH, GnRH, and the related protein TGFq31 do not displace [125I]BH-activin A from K562 cells. The content of activin A receptors on K562 cells was found to be only 600 receptors per cell.

This is low in comparison to receptors for the similar protein

TGF-13 in other cell types (e.g. 18,000 receptors/cell in NRK-49F and BALBc 3T3 cells (16)).

However, it is comparable to receptor contents for differentiation factors on

erythroleukemic cells or erythroid precursor cells.

For example, there are < 500

receptors/cell for erythropoietin on the mouse erythroleukemic cell line SKT6 (17) and only 50 - 500 G-CSF (granulocyte-colony stimulating factor) receptors on bone marrow granulocytes (18).

The physiological relevance of this disparity

between receptor levels for different types of growth factors on different cell types

remains

unclear.

We have described the initial characterization of the activin A receptor on the activin A-responsive erythroleukemic cell line K562. release of FSH from the pituitary (1,2).

Activin A also causes the

We have obtained preliminary evidence

demonstrating activin A binding to pituitary cells.

Further characterization of

both the pituitary activin receptor as well as the K562 activin receptor is currently being performed, and may help to understand part of the diverse physiological actions of activin.

ACKNOWLEDGEMENTS We would like to thank Dr. Louise Bilezikjian, Scott Struthers and Joan Vaughan for helpful discussion. This research was supported by NIH grants HD06877 (to CAC) and HD-13527 (to WV) and conducted in part by The Clayton Foundation for Research, California Division. WV is a Clayton Foundation Investigator.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

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