A Comparison of Catecholamine-induced Internalization of @-Adrenergic Receptors and Receptor-mediated Endocytosis of Epidermal Growth Factor in Human Astrocytoma Cells INHIBITION BY PHENYLARSINE OXIDE*

The ligand-induced internalization of @-adrenergic receptors and the receptor-mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not pre- vented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; how- ever, the inhibitory action of PA0 was blocked and reversed by bifunction~ thiols such as 2,3-dimercap- toethanol or di~othreitol. The results are consistent with the interaction of PA0 with vicinal sulfhydryl groups to form a stabile ring structure. PA0 did not prevent isoproterenol-induced uncoupling (desensiti- zation) of @-adrenergic receptors even though receptor internalization was completely blocked. The effects of PA0 on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not de-tectably altered by PA0 under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of @-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups. The activation by catecholamines of the @-adrenergic receptor (BAR1)-linked adenylate cyclase

The activation by catecholamines of the @-adrenergic receptor (BAR1)-linked adenylate cyclase is a transient effect in most cells. Following rapidly after the activation process, a set of temporally related events occur that lead to loss of response of the cell to catecholamines. As a part of this desensitization process, a portion of the cell surface BAR was shown to change form in a manner consistent with receptor internalization (1)(2)(3)(4). Thus, BAR can be isolated from desensitized cells in a membrane form depleted of other plasma membrane marker proteins. Such receptors are poorly accessible to hydrophilic ligands such as isoproterenol ( 3 ) and CGP-1217'7 (4); whereas, BAR on the surface of intact cells * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertiemnt" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Fellow of the Deutsche Forschungsgemeinschaft. lj To whom correspondence should be addressed Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.
' The abbreviations used are: BAR, @-adrenergic receptor; BAL, 2,3-dimercaptopropanol; CGP-12177,4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-%one hydrochloride; CYP, cyanopindolol; DTT, dithiothreitol; EGF, epidermai growth factor: PAO, phenylarsine oxide; Hepes, ~-(Z-hydroxyethyI)-l-piper~neeth~esu~on~c acid. or in lysates from control cells are readily accessible to these hydrophilic ligands. These observations have been i n t e~~~d as consistent with the proposal. that the ca~~ho~amine-induced form of BAR represents BAR with the ligand binding site on the inside of endocytotic vesicles. It follows that BAR might be internalized and processed in a manner similar to receptor-mediated endocytosis of certain polypeptides (asialoglycoprotein, insulin, EGF, low density lipoprotein, etc; for review, see Ref. 5). Internalization of protein-receptor complexes appears to involve sequestration in clathrin-coated pits and internalization by endocytosis in vesicles which simultaneously can contain different protein-receptor complexes (6,7).
The processing of polypeptide ligand and receptor appears to vary. In some cases the receptor is efficiently shuttled back to the cell surface 1) upon diisociation of the ligand and its degradation such as for asialoglycoprotein (8) and low density lipoprotein (9), or 2) with the protein ligand intact such as for transferrin (10). In some cases both ligand and receptor are degraded (11). There is evidence that separation of a ligand destined for degradation (asialoglycoprotein) from the receptor destined to recycle to the cell surface occurs in tubular organelles termed "compartments of uncoupling of receptor and ligand" (8).
In this report we compare the effects of the trivalent arsenical, phenylarsine oxide (PAO) on catecholamine-induced in-ternaIization of BAR and receptor mediated i n t e~a i i~t i o n of EGF in the human astrocytoma cell line 1321N1. PA0 previously had been shown to inhibit internalization of a variety of proteins (12-14) including EGF (15). We demonstrate that 1) PA0 inhibits the internalization of both BAR and EGF, 2) the effect of PA0 is reversed or prevented by bifunctional but not monofunctional sulfplydryi agents, and 3) the effect of PA0 cannot be explained on the basis of inhibition of energy metabolism.

Inhibition of Receptor Endocytosis by Phenylarsine Oxide
92% air and 8% COZ at 37 "C in a humidified incubator. Cells were seeded at 1.5 X lo5 cells/cm2 and were subcultured 4 days prior to the experiment.
Binding of Cyanopindolol-To determine the total number of BAR, lysates or membranes were incubated with 50 p~ (10 X KD) "I-CYP in 154 mM Nac1, 20 mM Tris, 5 mM &f&12, pH 7.4, in a total volume of 0.25 ml for 2 h at 30 "C. Nonspecific binding was determined in the presence of 1 p M propranoiol. intenalizution of BAR-Cells were incubated with 1 p~ (-)isoproterenol for 20 min at 37 "C to induce receptor internalization. The internalized form of BAR was detected by two different methods. 1) Sucrose density gradient centrifugation, in which desensitized cells were washed with Hepes-Eagle's medium incubated with 0.25 mg/ml concanavalin A (19) for 20 min at 4 "C then lysed by hypotonic shock in 1 mM Tris, 2 mM EDTA, pH 7.4, for 20 min at 4 "C. Lysates were layered on top of a step gradient consisting of 3.2 ml of 15%, 4 ml of 38%, and 4 ml of 60% sucrose (w/v in 20 mM Tris HCI, pH 7.4) and centrifuged for 30 min at 35,000 rpm in a Beckman SW 40 rotor. The interfaces were collected and diluted k1 with 154 mM NaCl, 20 mM Tris, 5 mM MgClz, pH 7.4, and BAR was determined by binding of ' 25]1-CYP. Internalized BAR migrated preferentially at the 15-38% interface and BAR on the plasma membrane at the 3840% interface.
2) Competition binding, in which desensitized cells were washed and lysed by hypotonic shock in 1 mM Tris, 2 mM EDTA, pH 7.4, for 20 min at 4 "C. Competition binding was carried out with diluted lysates using 10 pM '251-CYP and a range of concentrations of CGP-12177 (incubation was carried out at 30 'C for 2 h (20)). Internalized BAR (vesicles in the lysate) exhibited a marked inaccessibility to CGP-12177. The increase in '251-CYP binding in the presence of 10 nM CGP-12177 provided a antit tat ion of internalized BAR (see Fig. 5).
Epidermal Growth Factor Binding--Intact 1321N1 cells were washed once with Hepes-Eagle's medium containing 0.1% bovine serum albumin and incubated with 4 ng/ml (lo5 cpmlml) EGF. The incubation was either performed at 37 "C for up to 120 min or, to determine a single cycle of internalization, cells were incubated with '=I-EGF for 2 h at 4 "C, washed, and then incubated at 37 "C with nonlabeled EGF in the same buffer. After incubation at 37 "C, cells were chilled and washed five times with assay buffer. To determine total binding, cells were solubilized in 0.1 N NaOH, and cell-associated radioactivity was determined in a y-counter. To determine internalized EGF, cells were washed for 5 min with 40 mM sodium acetate, pH 4.5, to remove cell-surface-bound EGF and solubilized afknvards.
To separate internalized EGF from plasma membrane EGF on a sucrose step gradient, cells were lysed by hypotonic shock after washing them. When cells had been treated with sodium acetate, they were washed afterwards thoroughly with assay buffer to readjust to pH 7.4 before lysis. Centrifugation was as described above for BAR.
Determination of the Intracellular ATP Concentration-Cells were s o~u b~~z e d by addition of 1.5 ml of 70 "C 0.14 N HNO, to each dish. Dishes were incubated for an additional 20 min at 37 "C to allow complete solubilization. The supernatant was collected and centrifuged for 10 min at 4000 X g. The pH of the s u~r n a t~t was adjusted to 7.0 by adding 0.6 N NaOH in 20 mM Tris. ATP content was determined using a bioluminescence assay. Light emission was detected with a Beckman scintillation counter using a single photomultiplier. 50 pl of the CLS reagent was diluted in 3.5 ml of reaction buffer (20 mM Hepes, 10 mM MgC12, 2 m M EDTA, 0.18 mM DTT, and 0.15 mM AMP, pH 7.0). Samples (100 p1) were added to 1 ml of this reaction mixture and light emission was measured for 1 min. The signal was constant for at least 5 min due to the in~bition of the initial rate by excess AMP. Under these conditions, a range of 5 (IO5 cpm)-40 pmol (lo6 cpm) of ATP could be determined. ATP content is expressed as nmol/lo6 cells.
Determination of Adenylate Cyclase Activity-Adenylate cyclase activity was determined in cell lysates by the method of Salomon et ai. (21), which involves the separation of [32P]cAMP from r3'P]ATP on sequential columns of Dowex 50 and Alumina.

RESULTS
Incubation of 1321N1 cells with 1251-EGF at 4 "C leads to selective binding to cell surface receptors (22). When such labeled cells are washed and the incubation is continued at 37 "C, in the presence or absence of labeled EGF, a rapid translocation of Iz5I-EGF occurs from sites on the cell surface to sites within the cell (15). This tr~slocation, which presum-

RG. 1. ~t e r n~a t i o n
of epidermal growth factor. Cells were incubated with 4 ng/ml(105 cpm/ml) of lBI-EGF for 2 h at 4 "C. The medium was changed, nonlabeled EGF (4 ng/ml) was added in Hepes-Eagle's medium supplemented with 0.1% bovine serum albumin (37 "C), and cells were incubated for the time indicated. A, cells were washed and lysed, and plasma membrane (0) was separated from the vesicular fraction (0) on a sucrose step gradient. B, cells were incubated with sodium acetate, pH 4.5, to remove cell-surfacebound EGF, washed, lysed, and separated on a sucrose step gradient.
ably occurs by receptor-mediated endocytosis, can be followed in either of two ways. Upon incubation at 37 "C of cells prelabeled at 4 "C with IZ5I-EGF, the label rapidly moves from sites where the label is dissociated by acidic conditions (presumably cell surface sites) to sites where it is not dissociated (15). We also have found that, conco~itantly, acid-stabile 1251-EGF appears in low density fractions of cell lysates upon centrifugation over sucrose gradients; whereas all acid-labile '%EGF is associated with higher density fractions. The effect of PA0 on this translocation has been investigated by combining both techniques. The centr~ugation procedure was modified' to substitute a step gradient for a continuous gradient of sucrose, the internalized (acid-stabile) lz5I-EGF is collected at a 15/38% interface, while the cell surface (acidlabile) label accumulates at a 38/60% interface. Fig. 1 shows the time course of translocation of previously bound (4 "C) '251-EGF during a subsequent 37 "C incubation. Clearly, EGF bound at 4 "C is almost exclusively associated with the plasma membrane fraction (Fig. lA), but rapidly moves into the low density fraction with time at 37 "C.
In a related experiment, the cells were washed at low pH to remove membrane-bound lZ5I-EGF prior to lysis and centrifugation over sucrose gradients. The results (Fig. 1B) indicate that very little acid-stable l2'1-EGF is present immediately after binding at 4 "C. When the incuba~on was continued at 37 "C, acid-stable lz51-EGF first appeared in association with the heavy fractions and shortly thereafter in the light fractions of the gradient.
In Fig. 2 the effect of PA0 on binding and internalization of 'T-EGF is ~u s t r a~. The protocol used is somewhat

FIG. 2. Effect of P A 0 on EGF internalization.
Upper panel, cells were preincubated for 5 min with 100 p~ PAO. EGF (4 ng/ml, IO5 cpm/ml) was added to the medium, the cells were incubated at 37 "C for the time indicated, washed; and lysed, and plasma membrane (0) was separated from cytosolic vesicles (0) on a sucrose step gradient. Lower panel, cells were incubated with 4 ng/ml (10' cpm/ ml) of IZ5I-EGF for 2 h at 4 "C. Medium was changed and 4 ng/ml EGF plus 100 p~ PA0 were added. The incubation was carried out at 37 "C for the time indicated. Cells were washed, incubated at pH 4.5, washed, lysed and plasma membrane (0) was separated from cytosolic vesicles (0) on a sucrose step gradient. different than that described above for Fig. 1; namely, cells were exposed to lZ5I-EGF at 37 "C and the appearance of label in light (internalized) and heavy (cell surface) fractions of sucrose gradients was measured. Pretreatment of the cells for 5 min at 37 "C with 100 pM PA0 (IDso = 30 phi) completely inhibited appearance of '=I-EGF in the light fraction, whereas binding of lZ51-EGF to the cell surface appeared unaffected. Not only was no translocation of cell surface EGF to internalized EGF detected, but incubation of labeled cells at low pH prior to lysis revealed that PA0 also prevents the formation of acid-stable 1251-EGF associated with the heavy fractions of the gradient (Fig. 2, lower panel, and compare Fig.  1B). The difference in the scales of the y axes of Fig. 2, upper and lower panels, should be noted.
In the following experiments, cells were preincubated at 37 "C for 5 min with PA0 at a concentration of 100 PM. These conditions result in complete inhibition of receptor-mediated endocytosis even after extensive washing of the cells postexposure. This is operationally important since, under the exposure conditions chosen, the complicating secondary actions of PA0 are readily reversed by washing as discussed below.
P A 0 had been used by others to inhibit internalization of EGF (15) and other polypeptides (13, 14). PA0 is a trivalent arsenical and as such can form readily reversible bonds with single sulfhydryl groups, or quite stable ring structures upon reaction with vicinal sulfhydryl groups such as those in lipoic acid (23). In an attempt to characterize the chemical basis of inhibition of EGF endocytosis by PAO, the effects ofa number TABLE I Reversibility of the inhibition by PA0 of internalization of EGF Cells were pretreated for 5 min with PA0 or 0.1% dimethyl sulfoxide in Hepes-Eagle's medium supplemented with 0.1% bovine serum albumin. 4 ng/ml (10' cpm/ml) of lZ5I-EGF was added and the cells were incubated either for 2 h at 4 "C or for 30 min at 37 "C. The cells were transferred on ice, washed, lysed, and layered on top of a sucrose step gradient. The 15/38% sucrose interface was collected and filtered. and radioactivitv was determined. of sulfhydryl compounds were tested for their capacity to prevent or reverse its effects. The protocol was to incubate 1321N1 cells for 5 min at 37 "C with PA0 plus or minus a sulfhydryl compound, then lZ5I-EGF was added for an additional 30 min and internalization was measured as the increase in radioactivity in the light fraction of sucrose gradients. Monofunctional sulfhydryl compounds (glutathione, 2-mercaptoethanol) did not prevent the inhibitory action of PA0 while bifunctional reagents (DTT and BAL) prevented the inhibition almost completely (Table I). The results in Fig.  3 demonstrate that not only can DTT and BAL prevent the action of PA0 but they also reverse the inhibition. Addition of 10 X molar excess of DTT or BAL caused an immediate increase in internalized lZ5I-EGF and a complementary decline in '=I-EGF associated with the cell surface.
Since PA0 reacts with sulfhydryl groups and since other sulfhydryl-reactive compounds inhibit mitochondrial function and/or glycolysis, it was possible that the inhibitory effects of PA0 on endocytosis were caused by lowering cellular ATP. We3 and others (10,(24)(25)(26)(27), have shown that agents that lower ATP levels inhibit receptor-mediated endocytosis. How-C. Hertel, S. J. Coulter, and J. P. Perkins, manuscript submitted for publication. rnhi~ition of Receptor ~n~c y~o s~ by P~n y~p s i~ Oxide ever, based ob the results shown in Fig. 4, this mechanism seems not to offer a viable explanation for the actions of PAO. PA0 has no effect on ATP levels in 1321N1 cells under conditions (Fig. 4B) where it completely prevents internalization of '251-EGF (or BAR, see below). Treatment of cells in a nutrient-depleted medium with PA0 caused a modest decline in ATP levels, but this was prevented by the monofunctional sulfhydryl compound glutathione (Fig. 4A) which does not prevent the inhibition of endocytosis by PA0 (Table I}. One of the purposes of this study was to extend our comparison of the mechanisms subserving ligand-induced BAR internalization and receptor-mediated EGF internalization. To this end, the effects of PA0 on BAR internalization have been determined. Numerous previous experiments (1)(2)(3)(4) have been consistent with the idea that exposure of 1321N1 cells to isoproterenol results in the rapid translocation of cell surface BAR to a location within cytosolic vesicles. Such vesicles can be isolated in low density fractions of sucrose gradients. In addition, the BAR in such vesicles appears to be inaccessible to hydrophilic ligands such as CGP-12177 (4,28). These two properties have been used to quantitate endocytosis of BAR. Thus, we have measured the appearance of BAR in low density fractions of cell lysates after centrifugation over sucrose gradients, or we have measured changes in the apparent affinity of the hydrophilic ligand CGP-12177 as a competitor of 1251-CYP binding to cell lysates. PA0 (100 PM) was added to cells 5 min prior to a 20-min incubation with 1 p~ isoproterenol. Complete inhibition of the formation of BAR with low affinity for CGP-12177 or of f o~a t i o n of the low density form of BAR was observed (compare Fig. 5). The apparent IDso for PA0 for a 5-min incubation at 37 "C was 30 y~ for inhibition of BAR internalization as well as for EGF internalization. As was the case for EGF internalization, bifunctional but not monofunctional sulfhydryl compounds inhibited or reversed the effect of PA0 on BAR internalization (Table 11).
Initially our experiments with BAR were complicated by an effect of PA0 on BAR function per se; that is to say, a n t~o n i s t binding was reduced in agonist-pretrea~ cells (Table 11). However, this effect of PA0 was found to be reversed by extensive washing of the cells after the standard 5-min exposure to PAO. Such washing (5 x 10 ml over 5 min) did not reverse inhibition of BAR internalization. Also, ad- dition of glutathione or mercaptoethanol simultaneously with PA0 prevented loss of antagonist binding, but had no effect on the inhibition by PA0 of BAR internalization.
The KO and Bmx values for 1251-CYP binding were determined by Scatchard plot analysis after pretreatment of 1321N1 cells under various conditions (Table 111). PA0 alone had no detectable effect on either KB or B,, for 1251-CYP. However, treatment of cells with PA0 in the presence of isoproterenol resulted in a 35% decrease in the Bmx and a 35% increase in KO (Table 111). Washing the cells before adding isoproterenol, or including glutathione in the mixture, prevented both effects.
The effect of PA0 on BAR binding properties also was determined by directly exposing cell lysates to PAO. Neither the KD nor the Bmax for 12sI-CYP were altered by the presence of P A 0 (100 PM) in a standard binding assay (Fig. 6). However, PA0 had a marked effect on the apparent affinity of BAR for the agonist isopro~renol (Fig. 7). The typical shallow or biphasic competition curve for isoproterenol in the absence of GTP (29) was observed in lysates from control cells. PA0 caused a significant increase in the apparent affinity for isoproterenol. The inclusion of either GTP (500 PM) or glutathione (1 mM) prevented or reversed this effect of PAO;

Inhibition of Receptor Endocytosis by
Phenylarsine Oxide

Inhibition of formation of low affinity for CGP-12177
Cells were pretreated for 5 min at 37 "C with 100 pM PAO. 1 pM isoproterenol together with the thiol reagents was added for an additional 20 min. Cells were lysed and binding of 10 pM '251-CYP was determined in the presence or absence of 10 nM CGP-12177. Nonspecific binding was determined in the presence of 1 p M propranolol. The right column shows specific binding of lBI-CYP. This value was defined as 100%. The left column shows binding of '2sI-CYP in the presence of CGP-12177. This is expressed as per cent of binding in the absence of CGP-12177 (right column). LOW percentage lZ5I-CYP binding is therefore due to high accessibility for CGP-12177, which means less receptor internalization.

TABLE I11 Effect of PAOpretreatment on antagonist binding
Cells were pretreated for 5 min with PAO, followed by a 20-min incubation with or without isoproterenol. In line 6, cells were washed extensively before isoproterenol was added. In the experiment described in line 7, l mM glutathione was added together with isoproterenol. Cells were washed and lysed and B,, and KD for '251-CYP were determined in a saturation binding experiment according to Scatchard (33).

B,,
KO fmollmg protein p~ 0.1% dimethyl sulfoxide 11.3 6.9 0.1% dimethyl sulfoxide, 1 p~ iso-10.2 6.9 100 p M PA0 proterenol 11.9 7.5 100 pM PAO, 1 p M isoproterenol 7.4 10.9 100 PM PAO, wash 12.5 7.3 100 pM PAO, wash, isoproterenol 11.7 6.6 100 pM PAO, 1 mM glutathione, 1 11.6 9.6 p~ isoproterenol DTT caused a marked further decline in the affinity of BAR for isoproterenol. These results are similar to those of The effect of PA0 on an additional aspect of agonistinduced modification of BAR function, the responsiveness of adenylate cyclase to isoproterenol, has been examined ( Table  IV). Responsiveness of lysates from control cells (not treated with either isoproterenol or PAO) is expressed as the ratio of enzyme activity in the presence of isoproterenol to activity in its absence; the numerical value 12.5 indicates the magnitude of stimulation of basal activity by the BAR agonist. When cells were treated with isoproterenol for 20 min, then thoroughly washed and the cell lysate exposed to 10 p~ isoproterenol, the magnitude of stimulation was found to have been reduced to 8.9. The difference represents a 29% desensitiza- Binding of 1251-CYP to cell lysates was determined in the presence (0) or absence (0) of 100 p~ PAO. Data were analyzed according to Scatchard (33). tion. The activity of adenylate cyclase in the presence of 1 mM NaF was the same (ratios of 9.3 and 10.5) in both instances, confirming the selective nature of the desensitization. The effect of PA0 on desensitization was determined by interposing a 5-min preincubation of the cells with 100 PM PA0 and 1.0 mM glutathione. This condition completely blocks BAR or EGF internalization (see above). The subsequent 20-min exposure to isoproterenol resulted in a 35% desensitization. Again enzyme activity in the presence of NaF was found to be unaltered. PA0 caused a modest decrease in basal activity (1.5 to 1.2 pmol/min/mg of protein) and a modest increase in NaF activity (14.0 to 17.4 pmol/min/mg of protein). Basal activity was decreased by more than 50% in the presence of DTT or BAL (Table IV). As might be expected, desensitization occurred normally after pretreatment of cells with PA0 plus a bifunctional sulfhydryl agent (DTT, BAL).

DISCUSSION
Previous investigators have reported that PA0 inhibits internalization of protein into oocytes (12) and receptormediated endocytosis of protein nexin (14), EGF (15), and insulin (13). However, the mechanism of action of this trivalent arsenical was not explored. Early studies (23) established that trivalent arsenicals form stabile ring structures with molecules having appropriately spaced vicinal sulfhydryl groups. Five-membered rings appear to be most stabile. The equilibrium of such a reaction strongly favors the complex, and monofunctional sulfhydryl agents are not effective in competing in such a reaction (23). Thus, the results of our studies tentatively point to the involvement of vicinal sulfhydryl groups in the inhibitory action of PA0 on receptormediated endocytosis.
Wiley and ~~~n g h~ (15) stated that PA0 caused an irreversible inhibition of EGF internalization in cultured human fibroblasts. It is a consistent finding that extensive washing of PAO-treated cells does not reinstate endocytosis. Irreversible inhibition could result from nonspecific interactions and in such a case PA0 would be a less useful tool in the elucidation of the molecular mechanism of endocytosis. For example, trivalent arsenicals are known to interact with a large variety of enzymes including those involved in ATP generation. Thus, irreversible inhibition of ATP production could lead to general debilitation of cell integrity and a loss in the capacity to mount a complex event like endocytosis. Others have cautioned against the use of PAO-treated cells after 3 4 h (13).
Our studies provide two useful, probably generally applicable items of information. First, the effects of PA0 on. endocytosis can be prevented and, more importantly, reversed by bifunctional sulfhydryl compounds. Second, PA0 inhibits endocytosis under con~tions where cellular ATP levels are not changed. The stability of the ~~b i t e d state and its reversal by bifunetional, but not monofunctional, sulfhydryl agents suggests a somewhat selective interaction with a biological molecule containing vicinal sulfhydryl groups.
One purpose of our studies was to examine further the similarities in the endocytosis of BAR and EGF. Preliminary work from our laboratory had shown that the time courses of internalization of BAR and EGF were similar (but not identical): both processes were blocked by reduced temperature, both were blocked by treatment of cells with concanav~in A (22), and both were blocked by a reduction in cellular ATP3 (although not to the same extent). The present study demonstrates that endocytosis of both BAR and EGF are inhibited in similar fashion by PAO. Furthermore, it is demonstrated that PA0 blocks the appearance of acid-stabile lZ5I-EGF in a plasma membrane fraction, suggesting that this form of the EGF-receptor complex is a consequence of endocytosis. The time course of its appearance (about 5 min earlier than in the low density fraction) suggests further that it occurs at an early step during e n d o c~s i s .
A possibly related observation was recently made with BAR (3). In this case, isopro~renol induced a form of the BAR that was inaccessible to hydrophilic ligands, but that co-migrated with the heavy fraction on sucrose gradients. A possible explanation for both of these observations is the formation of invaginated segments of the plasma membrane that were yet to form as completed endo-somes, as has been described in other systems (32).
Since P A 0 had secondary effects on BAR, it was necessary to develop conditions to separate its actions on BAR propertiesper se and effects on endocytosis. Fortunately, the former effects of PA0 were completely prevented by glutathione or 2-mercaptoethanol. When these secondary effects of PA0 were prevented, internalization of BAR remained inhibited; however, uncoupling between BAR and adenylate cyclase still occurred. Therefore, consistent with previous observations (19) it is possible to distinguish reactions leading to uncoupling and those leading to receptor internalization. It follows that 1321N1 cells possess a mechanism for uncoupling the cellular response to the ligand even when receptor internalization is prevented.
Ligand-induced endocytosis of BAR occurs in 1321N1 cells by a process we have yet to be able to distinguish from receptor-mediated endocytosis of EGF. However, our results only address similarities in the early steps of the overall process; thus, we cannot comment on possible similarities in steps subsequent to internalization such as recycling of receptor or ligand to the cell surface, or degradative pathways for receptor or ligand. However, to the extent that subsequent steps in the pathways can be shown to be similar, studies of BAR processing may be generally enlightening of the mechanism of endocytosis, since, of the common receptors studied in this area, only the BAR can be measured directly after internalization has occurred. The availability of a number of high affinity, highly selective, membrane-permeable radioligands for this receptor provides unique tools with which to follow the dynamics of receptor internalization and processing.