[CANCER RESEARCH 41, 2175-2181, June 1981]0008-5472/81 /0041-OOOOS02.00

Characterization of Phorbol Ester Receptors and Their Down-Modulation

in GH4Ci Rat Pituitary CellsSusan Jaken,1 Armen H. Tashjian, Jr.,2 and Peter M. Blumberg3

Interdisciplinary Programs in Health [S. J.] and Laboratory of Toxicology [A. H. T.], Harvard School of Public Health, and Department of Pharmacology, HarvardMedical School [A. H. T., P. M B.I, Boston, Massachusetts 02115

ABSTRACT

Phorbol ester binding to intact GH4d cells, a continuousstrain of rat pituitary cells, was measured using [3H]phorbol

12,13-dibutyrate. The binding was saturable; Scatchard analysis indicated one class of high-affinity binding sites (Kd, 11nM) with 6.4 pmol [3H]phorbol 12,13-dibutyrate bound per mg

cell protein at saturation. The relative binding affinities for otherphorbol esters and analogs were similar to those demonstratedfor binding to homogenates of chick embryo fibroblasts andmouse skin, as well as for tumor promotion in vivo. A closecorrelation was shown to exist between the binding affinities ofthese derivatives and their potencies for inducing biologicalresponses in GH4d cells, such as decreases in binding ofepidermal growth factor and thyrotropin-releasing hormone. A

distinctive new finding is the down modulation of phorbol esterbinding sites on GH4Ci cells by both homologous and heterol-

ogous ligands. Prolonged exposure to phorbol esters or thyrotropin-releasing hormone produced a loss of available [3H]-

phorbol 12,13-dibutyrate binding sites with a maximal de

crease to about 20% of control after 24 hr of treatment.Scatchard analysis indicated that the decrease in binding wasdue to a loss of receptors with no change in affinity. Thebiological significance of phorbol ester receptor down modulation is not yet known; however, it may represent a mechanismfor attenuating cellular responsiveness to phorbol esters intheir continued presence.

INTRODUCTION

Tumor promoters are compounds which are not carcinogenicbut enhance the probability of tumor formation following exposure to a subthreshold dose of a carcinogen. The process oftumor promotion has been studied in detail in mouse skin withthe potent tumor promoters, the phorbol esters (1 -4, 9, 37).

Tumor promotion, however, is not unique to mouse skin. Themultistep nature of carcinogenesis has been demonstrated inliver, bladder, and mammary gland (for review, see Ref. 17). Inaddition to their action as tumor promoters, the phorbol estersmediate a variety of biological responses in cells grown inculture. These include changes in membrane activity such asincreased uptake of nutrients, increased phospholipid biosynthesis, increased membrane fluidity, increased release of ar-achidonic acid, and uncoupling of /3-adrenergic receptors (for

1Fellow of the Interdisciplinary Programs in Health. Harvard School of Public

Health.2 Recipient of a research grant from the National Institute of Arthritis. Metab

olism, and Digestive Diseases (AM 1101).3 Recipient of a Research Career Development Award from NIH and a research

grant from the National Cancer Institute (CA 22895). To whom requests forreprints should be addressed.

Received November 24, 1980; accepted February 19. 1981.

reviews, see Refs. 2, 3, and 12). The phorbol esters also alterthe binding of several peptide hormones (EGF,4 TRH, and

somatostatin) to their cell surface receptors (5, 22, 28, 34).Exposure to phorbol esters also results in long-term responses

which probably involve transcriptional and translational eventssuch as increases in plasminogen activator, inhibition or stimulation of DMA synthesis, and inhibition or stimulation of theprocess of differentiation (for reviews, see Refs. 2, 3, and 12).In general, the phorbol esters which are most potent in mediating biological responses in cells in culture are also thosewhich are most potent as tumor promoters in mouse skin. Itthus seems likely that the phorbol esters are acting at homologous targets in vitro and in vivo.

In contrast to the wealth of information on biological responses to phorbol esters, much less is known about theirmechanism of action. Recently, Driedger and Blumberg (15)demonstrated the existence of specific cellular binding sites forphorbol esters. Using [3H]PDBU as the radioactive probe, the

binding of phorbol esters has been characterized in homogenates of chick embryo fibroblasts (15), mouse skin (11), andmouse brain (16). In the case of chick embryo fibroblasts, thebinding affinities for a series of phorbol esters and relatedditerpenes agreed quantitatively with their biological potenciesfor inducing loss of fibronectin, a transformation-sensitive pro

tein (15). The binding affinities to mouse skin preparationswere similar to those of the chick embryo fibroblasts; moreover,they were consistent with the tumor-promoting activity of the

derivatives. This indicates that the binding site being measuredin vitro is homologous to the binding site relevant to tumorpromotion in vivo.

In this report, we demonstrate that the binding assay caneasily be adapted to intact cells, in particular, GH4Ci rat pituitary cells. This approach allows an investigation of the regulation of the receptor activity with changes in the cellular environment. GH4Ci cells are a continuous strain of rat pituitarycells originally derived from an X-ray-induced pituitary tumor

(36). The advantage to using GH4C, cells for a study of themechanism of action of phorbol esters is that they have well-

characterized functional receptors for several growth factorsand hormones. Specific high-affinity receptors for TRH, EGF,

and somatostatin have been identified in these cells (18, 32,33). These receptors mediate the biological responses ofGH4Ci cells to these regulatory peptides, including the synthesis and release of prolactin and growth hormone.

Treatment of GH4Ci cells with phorbol esters results inincreased release and synthesis of prolactin and decreased

4 The abbreviations used are: EGF, epidermal growth factor; TRH. thyrotropin-

releasing hormone; PDBU, phorbol 12,13-dibutyrate; ED50, halt-maximally effective dose; F10', Ham's Medium F-10 supplemented with 15% horse serum and

2.5% fetal bovine serum without antibiotics; PMA, phorbol 12-myristate 13-acetate.

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production of growth hormone (28). In addition, phorbol esterscause a decrease in the binding of TRH, EGF, and somatostatinto GH4C, cells (27). Thus, GH4Ci cells provide a convenientsystem for studying phorbol ester-mediated changes in membrane activity, long-term biosynthetic responses, and peptide

hormone binding. In this study, we have characterized thebinding of phorbol esters to GH4Ci cells and have examinedthe relationship between binding affinities of selected phorbolesters and analogs and their ED50's for several biological

responses. In addition, we describe the novel finding thatphorbol esters induced a loss of their own binding sites intarget cells.

MATERIALS AND METHODS

Cell Culture. GH4Ci rat pituitary cells were grown in F10*

as described (36). Cells were grown in 16-mm multiwell dishes(Falcon Plastics, Oxnard, Calif.) in 0.5 ml F10+ for 1 to 2

weeks, at which time they had reached a plateau density ofabout 7 x 105 cells/well. Compounds to be added to thecultures were prepared in 100-fold excess in F10+. Cultures

which had received fresh medium 48 to 72 hr previously wereused in most experiments.

Binding of [3H]PDBU to Intact Cells. The assay for [-'H]-

PDBU binding to cell homogenates as developed by Driedgerand Blumberg (15) was adapted for measuring binding to intactcells. Cell monolayers were incubated in binding medium consisting of 200/il F10+, 50/il 0.75% dimethyl sulfoxide in F10+,

and other compounds as indicated. The assay was begun byaddition of 50 /il [3H]PDBU in F10*. The cultures were thenreturned to the incubator (37°; 5% CO2-95% air). Equilibriumbinding was reached within 15 min with 20 nM [3H]PDBU, and

the amount of specific binding remained constant for 60 min.Thus, there was no indication of modulation of the bindingduring the assay period. A 30-min incubation period was used

in most experiments after which the multiwell dishes wereremoved from the incubator and placed on ice. Aliquots of themedium were removed to measure directly the concentrationof free [3H]PDBU. The remaining medium was aspirated, and

the monolayers were rinsed quickly 3 times with 0.2 ml of ice-cold F10+. The importance of maintaining low temperatures

during the washing can be recognized from studies of phorbolester binding to mouse brain homogenates. The Kd for [3H]-

PDBU binding to brain tissue is similar to that in GH.,C, cells(~8 nw). The dissociation rate constant in mouse brain wasshown to have a fi/2 of 62.1 min at 4°and 1.75 min at 30°

(16). Cell lysates were collected in 0.6 ml of 0.1 N NaOH, andaliquots were prepared for scintillation counting in Aquasol.Specific binding was calculated from the difference in radioactivity in the absence or presence of excess PDBU (20 UM).

The equilibrium dissociation constants for other phorbol esters and analogs were estimated from dose-dependent inhibition of [3H]PDBU binding. The values for K¡were corrected forthe concentration of [3H]PDBU using the equation

EDsoK,

t+i.Ka

with 30 or 300 nM [3H]PDBU for 24 hr. At this time, the

conditioned media were collected, and the cells were removedwith Viokase (Viobin Corp., Monticello, III.). Both the media andthe cell pellets were extracted 3 times with equal volumes ofethyl acetate containing 1 jug PDBU per ml. The recovery ofradioactivity in the ethyl acetate phase was >95%. The extractswere dried under a stream of nitrogen and analyzed by thin-

layer chromatography as described by Driedger and Blumberg(15). Greater than 98% of the radioactivity in the cell extractsand >90% in the conditioned medium extracts migrated with[3H]PDBU.

Other Methods. 3H-TRH binding was measured as described

by Hinkle and Tashjian (18). EGF was iodinated according tothe method of Carpenter and Cohen (6) to a specific activity of1300Ci/mmol. Binding was measured during a 1-hr incubationperiod in F10+. Nonspecific binding was estimated in the

presence of 33 nM unlabeled EGF. Protein was measured bythe method of Lowry ef al. (24) or by the Bio-Rad protein assay

in lysates prepared from cell monolayers which had beenrinsed twice with 0.01 M phosphate-0.15 M NaCI (pH 7.4).

Materials. Materials were obtained from the followingsources: 3H-TRH (100 Ci/mmol), New England Nuclear (Bos

ton, Mass.); EGF, Collaborative Research, Inc. (Waltham,Mass.); PMA, Chemical Carcinogenesis (Eden Prairie, Minn.);mezerein, Chemical Carcinogenesis and John Douros, NationalCancer Institute (Bethesda, Md.); and somatostatin, BeckmanInstruments, Inc. (Palo Alto, Calif.). All others were from SigmaChemical Co. (St. Louis, Mo.). [3H]PDBU (3.42 Ci/mmol) was

synthesized according to the method of Kreibich and Hecker(21).

RESULTS

Characterization of [3H]PDBU Binding. The binding of[3H]PDBU to GH4C, cells was saturable (Chart i A). Scatchard

analysis (31 ) indicated one class of high-affinity binding sites

(Chart IB). From 3 separate experiments, the dissociationconstant (Ka) was calculated to be 11.3 ±1.3 nM (S.D.) with6.41 ±0.23 pmol [3H]PDBU bound per mg cell protein (equiv

alent to approximately 200,000 binding sites per cell). Atsaturating concentrations, approximately 65% of the cell-as

sociated radioactivity represented specific binding.

5 06c\o*

I 0.4

20 40 60[3H] PDBu, nM

2468

] PDBu Bound, pmol/mg

where L is the concentration of [3H]PDBU (8).

Identity of Bound and Free Ligand. Cells were incubated

Chart 1. Specific binding of [3H]PDBU to GH„C,cells. GH..C, cells wereincubated with increasing concentrations of |'H|PDBU for 60 min. Specific

binding was calculated from the difference in cell-associated radioactivity measured in the absence and presence of excess nonradioactive PDBU at eachconcentration of [3H]PDBU. In A. the curve was drawn according to the binding

parameters determined in Chart 1B Each point represents the mean of triplicatecultures: bars, range, ß, Scatchard analysis. The binding parameters weredetermined from linear regression of the data shown in Chart 1/V In this experiment, the Ka was 11.7 nM. with 6.3 pmol [3H]PDBU bound per mg cell protein at

saturation.

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The radioactivity was reversibly associated with the cells.Following equilibration with 20 nM [3H]PDBU, the cell-associ

ated radioactivity could be displaced by incubation with 20¡IMPDBU at 37°.Within 5 min after addition of excess nonra-dioactive PDBU, 92% of the specifically bound [3H]PDBU was

displaced; within 10 min, 100% of the specifically bound[3H]PDBU was displaced (data not shown).

The binding affinity of the receptor for other phorbol estersand mezerein was estimated by dose-dependent inhibition of[3H]PDBU binding. The calculated K¡'sare listed in Table 1.

The biologically inactive parent alcohol, phorbol, did not compete for [3H]PDBU binding. In general, the binding affinities

were of the same rank order as has been demonstrated forchick embryo fibroblasts, mouse skin, and mouse brain (11,15, 16).

Decrease in Specific [3H]PDBU Binding with Time of Incubation. When GHid cells were incubated with 16 nM [3H]-

PDBU for 6 and 24 hr, there was a decrease in cell-associated

radioactivity when compared to cultures exposed for 15 min(Chart 2, open bars). The cell-associated radioactivity still

remaining after 6 or 24 hr was reversibly associated with thecells. The radioactivity could be removed by either dilution (2incubations in F10+ without PDBU for 5 min at 37°, hatched

bars) or exchange (addition of unlabeled PDBU to a finalconcentration of 20 p.Mfor 7 min at 37°,stippled bars). Similar

results were obtained when cells were incubated for 6 or 24 hrwith 160 nM [3H]PDBU. The decrease in binding was not dueto degradation of [3H]PDBU. After a 24-hr incubation with either30 or 300 nM [3H]PDBU, greater than 98% of the ethyl acetate-

extractable radioactivity from the cell lysate and 90% from theconditioned media migrated on thin-layer chromatography withauthentic [3H]PDBU (see "Materials and Methods" for details).

Characterization of Homologous Down Modulation. Thedata in Chart 2 demonstrate that cell-associated [3H]PDBU wasremoved from the cells with thorough washing at 37°. By

analogy, following incubation with unlabeled PDBU, the PDBUcould be washed out of the cells, and the available bindingsites could be measured as usual with [3H]PDBU. This protocol,

which yields results quantitatively similar to those obtained withcontinual incubation with [3H]PDBU, was used to characterize

the PDBU-induced loss of phorbol ester binding sites, i.e..

homologous "down modulation," a term used here to refer to

a loss of binding activity without implying any mechanismmediating this loss.

The dose-response curves for PDBU-induced loss of [3H]-

PDBU binding are shown in Chart 3. Cells were incubated for6 or 24 hr with PDBU prior to measuring the binding with[3H]PDBU. The ED5o's for PDBU at 6 and 24 hr were about 5

and 1.7 nM, respectively. The decrease in the ED50 with timewas due to an increase in the potency of each dose withincreasing time; e.g., at 6 hr, 0.3 ng/ml resulted in no significant loss of binding, and at 24 hr, a 32% decrease wasobserved. The maximal response was achieved with 30 ng/mlat 6 hr and 10 ng/ml at 24 hr. This difference in ED5o's with

time of incubation was found in each of 3 experiments. Treatment with other phorbol esters and analogs also resulted inloss of [3H]PDBU binding (Table 1). The maximal response was

the same for all 4 derivatives (54 ±6% of control at 6 hr, 19±3% of control at 24 hr).

Effects of Heterologous Compounds on [3H]PDBU Binding.

Several compounds which have been reported to alter or mimicthe biological actions of phorbol esters in vivo were tested fortheir effects on [3H]PDBU binding. Dexamethasone, retinoleacid, and EGF have been shown not to compete for [3H]PDBU

binding in homogenates of chick embryo fibroblasts or mouseskin (11,15); however, those results did not eliminate thepossibility of an indirect modulation of phorbol ester bindingwhich could occur in intact cells.

GH4Ci cells were pretreated for 24 hr with retinoic acid (10~7M), leupeptin (100/¿g/ml), dexamethasone (10~7 M), EGF(10~7M), or somatostatin (10~7 M). [3H]PDBU binding was measured

in the continued presence of these compounds. None of thesecompounds decreased [3H]PDBU binding. Therefore, at least

in GH4Ci cells, it would appear that any modulation of phorbolester-mediated responses by these compounds occurs at astep subsequent to the phorbol ester-receptor interaction.

Effect of TRH on [3H]PDBU Binding. GH,C, cells have

specific receptors for the tripeptide, TRH (Kd, 10 nM). WhenGH4Ci cells were incubated with increasing concentrations ofTRH, there was a dose-dependent loss of [3H]PDBU binding

(Chart 4). TRH treatment also results in a decreased number ofreceptors for TRH (19).

Table 1

Comparison of binding affinities with potencies for biological responsesKi's for the compounds tested were determined by dose-dependent inhibition of [3H]PDBU binding.

Inhibition of '25I-EGF binding was measured by preincubating cultures with various concentrations of thetest compounds for 5 min and then adding 125I-EGF (-100,000 cpm/well) for 60 min in the continuedpresence of the phorbol ester or mezerein. Inhibition of 3H-TRH binding was measured by preincubatingcultures with the compounds for 3 hr and then adding 3H-TRH (2 nM) for 60 min in the continued presence

of the phorbol ester or mezerein. These preincubation periods have been shown to be the minimal timesnecessary for observing maximal phorbol ester effects on EGF and TRH binding (27). Inhibition of [3H]PDBU

binding (homologous down modulation) was measured in cultures treated for 24 hr with the compoundsindicated. The compounds were removed from the cells by washing twice with F10* and then incubatingtwice with 0.5 ml F10* at 37°for 10 min. The amount of phorbol ester binding activity remaining was thendetermined during a 30-min incubation with [3H]PDBU.

CompoundPMA

PDBUPhorbol-1 2,1 3-dibenzoateMezereinPhorbolK

or K(nM)2.6± 1.3a

11.3± 1.0617.0± 3.0a64.0 ± 17.0"

>1600EDso

for homologous down mod

ulation(HM)1.9±0.7a

1.6 ±0.75b9.0 ±1.0a

31.0 ±2.0aEDM

for loss of'"I-EGF bind

ing(nM)6.9±0.9a

14.0 ±7.0629.0 ±3.0a38.0 ±1.0aEDso

for loss of3H-TRH bind

ing(nM)4.1±0.9a

13.0 ±5.0o

Mean ±range of duplicate independent experiments.' Mean ±S.D. of triplicate independent experiments.

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CLo 1500

o 1000

500

15min-1-Jl

.Incubation!6hr24hrIncubation

Incubation

Chart 2. Decrease in and reversibility of specific [3H]PDBU binding with timeof incubation. GH4C, cells were incubated with 16 nwi [3H]PDBU for 15 min, 6 hr,

or 24 hr. At each time, the amount of total cell-associated radioactivity followingwashing with ice-cold F10* was determined O. Two approaches were used to

determine if the cell-associated radioactivity was reversibly associated with thecells, (a) The medium containing [3H]PDBU was removed from some cultures andreplaced with F10* (37°). The cultures were then incubated for 5 min at 37°.

This washing procedure was repeated once. The medium was removed, and thecell monolayers were dissolved in 0.1 N NaOH W).(b) Nonradioactive PDBU wasadded to the cultures (without rinsing) to a final concentration of 20 /IM. Thecultures were returned to the incubator for 7 min. The medium was then removed,and the cell monolayers were dissolved in 0.1 N NaOH (H). Nonspecific bindingdenotes binding during a 15-min incubation with 16 nw [3H]PDBU and 20 /IM

PDBU. Each value represents the mean of duplicate cultures; bars, range. Similarresults were obtained in 2 other experiments.

moo.

O.I I.O IO 100

PDBu, ng/ml

Chart 3. Dose-dependent decrease in pHJPDBU binding with phorbol esterpretreatment. GH4C, cells were pretreated with the indicated concentrations ofPDBU for 6 to 24 hr. At these times, the monolayers were washed as describedin the legend to Table 1. The amount of phorbol ester binding activity remainingwas then determined during a 30-min incubation with [3H]PDBU. Each point

represents the mean of triplicate cultures; bars, range. Similar results wereobtained in 2 separate experiments. The arrows indicate the ED50's. •,6-hr

treatment; O, 24-hr treatment.

The dose-response curves for TRH-mediated loss of 3H-TRHbinding and the heterologous loss of [3H]PDBU binding were

compared in parallel cultures (Chart 4). The ED50 for TRH was1 nM for both effects.

Phorbol Ester Binding in Down-modulated Cells. Cells were

treated for 6 hr with TRH or PDBU, and the remaining phorbolester binding activity was analyzed by the method of Scatchard(Chart 5). In 3 experiments, TRH treatment resulted in a 60 ±14% (S.D.) decrease, and PDBU treatment resulted in a 50 ±8% decrease in the number of [3H]PDBU binding sites. In those3 experiments, the Kd's for PDBU were as follows: control

cultures, 11.3 ±1.3 nw; TRH-treated cultures, 14.0 ±3.7 nM;and PDBU-treated cultures, 13.5 ±2.3 nM. Therefore, therewere no significant differences in the affinity constants, and thedecreases in binding accompanying TRH and PDBU treatment

were due to a decrease in the number of available bindingsites.

Time Course of Phorbol Ester Receptor Down-Modulationand Recovery. The time course of TRH- and PDBU-mediateddown-modulation of [3H]PDBU binding was examined using

concentrations of TRH and PDBU which produced maximaleffects (Chart 6). Both ligands caused a loss of binding whichwas apparent within 3 hr and which reached a plateau at 24hr. In a separate experiment in which the cells received freshmedium ± PDBU (10 ng/ml) every day, the extent of thedecrease observed at 24 hr was maintained for 6 days (datanot shown).

Following removal of either TRH or PDBU, the binding activityreturned (Chart 7). After a 24-hr recovery period, the bindinghad increased to 75% of control; after a 48-hr recovery period,

the binding had returned to control levels. Several compoundswhich have been reported to modify or mimic the effects ofphorbol esters on cells and which do not affect [3H]PDBU

binding in GH4C, cells (see above) were tested for their effectson TRH- or PDBU-induced down-modulation and recovery from

O

M

Chart 4. Dose-dependent decrease in [3H]PDBU binding with TRH pretreat

ment. GH«Ci cells were pretreated with increasing concentrations of TRH for 24

hr. which is the time necessary for maximal effects. For the determination ofphorbol ester binding, [3H)PDBU was added directly to the cultures (without

changing the medium) for a 30-min incubation period. For the determination ofTRH binding, the cultures were rinsed 2 times with F10* and then incubated for60 min at 37° in 0.5 ml F10* to remove any cell-associated TRH (19). Themedium was removed and replaced with F10* containing 3H-TRH for a 60-min

incubation period. Each point represents the mean of duplicate cultures; bars,range. The arrows indicate the ED-a's. O, 3H-TRH bound; ». [3H]PDBU bound.

06

E•50.4

ÃŽ 0.2-

Hj PDBu Bound, pmol/mg

2468[3HJPDBu Bound, pmol/mg

Chart 5. Scatchard analysis of binding of [3H]PDBU in cells pretreated with

TRH or PDBU. A, GH«C,cells were incubated with PDBU (10 ng/ml) for 6 hr.The cultures were washed to remove cell-associated PDBU as described in thelegend to Table 1. The remaining phorbol ester binding activity was then measured using a range of concentrations of [3H]PDBU from 2 to 60 nw. Each point

represents the mean of triplicate cultures. Similar results were obtained in 2separate experiments. •,binding to untreated control cultures; O, binding toPDBU-treated cultures. B, GH4Ci cells were incubated with 2 x 10"r M TRH for

6 hr. The remaining phorbol ester binding activity was then measured by adding[3H]PDBU to the cultures without changing the medium. Final concentrations of[3H]PDBU ranged from 2 to 60 nM. Each point represents the mean of duplicate

cultures. Similar results were obtained in 2 separate experiments. •.binding tountreated control cultures; O, binding to TRH-treated cultures.

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5-

ö 4

a•oe.o 3m3mg 2

5-

0 8 16 24

Hours of Exposure

Chart 6. Time course of loss of phorbol ester binding induced by PDBU orTRH. GH4C, cells were treated with PDBU (10 ng/ml) or 2 x 10~' M TRH, and[3H]PDBU binding was measured at the indicated time intervals. In cultures

treated with PDBU. the cells were washed as described in the legend to Table 1before the [3H]PDBU was added. Each point represents the mean of duplicate

cultures; bars, range. Similar results were obtained in one other experiment. O,[3H]PDBU binding to TRH-treated cultures; »,[3H]PDBU binding to PDBU-treatedcultures; A, [3H]PDBU binding to untreated control cultures.

down modulation. None of the compounds tested (EGF, 5 x10~8 M; retinole acid, 10~7 M; dexamethasone, 10~7 M; or

leupeptin, 20 jug/ml) inhibited PDBU-induced down-modula

tion. None of these compounds substantially inhibited recoveryfrom TRH- or PDBU-mediated down-modulation, although EGFtreatment resulted in a small decrease in 24-hr recovery fromPDBU-mediated down-modulation (18 ±6%; n = 3).

Comparison of Binding Affinities and EDw's for Biological

Responses. The dissociation constants for several phorbolesters and for mezerein together with the ED50's of these

derivatives for 3 biological responses were determined (Table1). All binding studies and biological responses were studiedon cells incubated in F10* in order to permit the closest

possible comparison between binding and biological potency.The phorbol ester-mediated decreases in binding of 125I-EGFand 3H-TRH are rapid effects which can be measured 1 or 4

hr, respectively, following exposure to phorbol esters (27). TheEDso's for these responses for the phorbol esters listed in Table

1 were all within a factor of 1.9 of the dissociation constants.In contrast to the rapid decrease in EGF and TRH binding,homologous down modulation occurred over a longer timecourse. The ED50's for the phorbol esters for homologous

down-modulation were less than those for heterologous down

modulation and were less than or equal to the dissociationconstants (Table 1).

The potency of the weakly promoting, highly inflammatoryresiniferonol derivative, mezerein, was also studied. In GH4Cicells, mezerein was the least biologically active. Mezerein alsohad the lowest binding affinity. These results were confirmedwith mezerein from 2 separate sources. Both batches were atleast 98% pure as determined by high-pressure liquid chro-

matography. The result with mezerein is noteworthy, since inmany (22, 38) but not all (14, 20) in vitro systems mezerein

03Q 2Q.

I -

-ÃŽ

4 8 12 16 20 24"

Hours After Washing

48

Chart 7. Time course of recovery of [3H]PDBU binding from down modulation.GH4C, cells were treated with PDBU (10 ng/ml) or 2 X 10~7 M TRH for 24 hr.

Cell-associated TRH was then removed as described in the legend to Chart 4.Cell-associated PDBU was removed as described in the legend to Table 1 exceptthat two 30-min incubations were used to coordinate the TRH- and PDBU-treatedcultures. The end of this 1-hr wash period was taken as the beginning of therecovery period (zero time). For determination of [3H]PDBU binding, the mediumwas removed, and fresh medium containing [3H]PDBU was added for a 30-min

incubation period. Each point represents the mean of duplicate cultures; bars,range. Similar results were obtained in one other experiment. Symbols aredescribed in the legend to Chart 6.

has been reported to have similar potency to PMA. In vivo,mezerein is within 2-fold of PMA in inflammatory potency but

is only 1/50 as potent as a complete promoter (35).

DISCUSSION

The results presented in this report demonstrate that specificphorbol ester binding can be studied in living cells. The bindingin GH4Ci cells was shown to represent a single class of high-

affinity sites with a Kd of about 11 nw for PDBU. The rapideffects of phorbol esters on plasma membrane activities indicate that a plasma membrane location of the receptor is likely.In fact, in mouse brain homogenates, specific [3H]PDBU binding

activity was exclusively particulate and was predominantly inplasma membrane-enriched fractions.5 In broken cell preparations of the GH4Ci cells, all of the specific [3H]PDBU binding

activity was likewise particulate (data not shown). A criticalfeature of the investigation was the finding that the structuralrelationships for binding corresponded well to the biologicalactivities of the compounds on the same cells (Table 1), findingsthat are consistent with the hypothesis that the biologicaleffects are mediated via these binding sites.

Our results also provide evidence for a new phorbol ester-mediated biological action, namely, homologous down-modu

lation of phorbol ester binding. This effect occurs slowly (Chart6) as compared to phorbol ester effects on heterologous pep-tide receptor binding which can be maximal within 1 hr ofexposure to phorbol ester (27). The slow time course is similarto that observed for dhronic effects of phorbol esters on thesecells, including increased prolactin synthesis and decreasedproduction of growth hormone (28). Each of the phorbol estersstudied was more potent at causing homologous down-modu-

5 W. G. Dunphy, R. J. Kochenburger, M. Castagna, and P. M. Blumberg,Kinetics and subcellular localization of specific [3H]phorbol 12,13-dibutyrate

binding by mouse brain, submitted for publication.

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lation as compared to heterologous down-modulation of TRH

and EGF binding sites.Down-modulation of phorbol ester binding was also induced

by TRH but not by EGF or somatostatin under the conditionsused. The TRH- and PDBU-mediated effects share a similartime course for loss of [3H]PDBU binding (Chart 6) and recovery

(Chart 7). Each caused a decrease in the number of receptorswith no change in Kd (Chart 4). TRH and phorbol esters sharecertain other effects on GH4Ci cells, such as increased prolac-tin release and synthesis and decreased growth hormone production (25, 28). Other effects are not common to both. Forexample, phorbol esters, but not TRH, cause a decrease inEGF binding. Phorbol esters cause a rapid (1-4 hr) decrease

in TRH binding; TRH causes a decrease in TRH binding whichoccurs more slowly (2-24 hr) (19).

One mechanism for homologous down-modulation of certainpeptide hormone receptors involves ligand-induced clustering

of receptors, followed by internalization and degradation of thehormone-receptor complex (6, 10). In the case of EGF, which

has been studied extensively, the time course for loss ofbinding is usually very rapid. In fact, in GH4C, cells, there is aloss of 125I-EGF binding within 1 hr of exposure to EGF.6 The

difference in time course of EGF and phorbol ester-mediated

homologous down modulation may indicate that they proceedby different mechanisms. On the other hand, such differencescould be explained by differences in the individual turnoverrates for the 2 binding sites.

Down-modulation of receptors has been correlated with de

creased responsiveness to the homologous ligand in severalsystems (7). In GH4Ci cells, down modulation of TRH receptorsby triiodothyronine results in a decrease in the maximal extentof TRH-stimulated prolactin release (29). The importance of

receptor modulation in disease states is suggested by studieson insulin binding in the diabetic db/db mouse. Decreasedinsulin binding in fibroblasts from these mice has been correlated with decreased sensitivity to insulin-induced increases indeoxyglucose uptake and induction of ornithine decarboxylase(30). It is possible that loss of phorbol ester binding sites alsorepresents a mechanism for escaping from some phorbol ester-mediated biological effects. In fact, refractoriness to phorbolester-mediated processes has been reported. Phorbol ester-

mediated decrease in EGF binding in HeLa cells was lost withprolonged exposure (23). Inhibition by phorbol esters of theconversion of 3T3 fibroblasts to adipocytes was lost withcontinued exposure to phorbol didecanoate and phorbol di-benzoate but not PMA (13). Similarly, the phorbol ester-mediated inhibition of melanogenesis in B-16 melanoma cells was

lost with prolonged exposure (26).Measurements of phorbol ester binding in intact cells provide

the techniques necessary to investigate modulation of bindingby various compounds. Furthermore, it will now be possible todetermine whether modulation of the binding influences phorbol ester-mediated biological effects.

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1981;41:2175-2181. Cancer Res   Susan Jaken, Armen H. Tashjian, Jr. and Peter M. Blumberg 

Rat Pituitary Cells1C4Down-Modulation in GHCharacterization of Phorbol Ester Receptors and Their

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