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In many cells catecholamines induce a translocation of beta-adrenergic receptors from the cell surface to intracellular vesicular sites. We have postulated that the translocation event is the result of ligand-induced endocytosis of the receptor, probably via clathrin-coated pits. Previously, we demonstrated that reduction of cellular ATP content with antimycin A completely blocked endocytosis of epidermal growth factor and translocation of beta-adrenergic receptors in 1321N1 astrocytoma cells. However, the effect of reduction in ATP content on endocytosis remains controversial. In the present report, we demonstrate that reduction of ATP content to a level5% of that in control cells is sufficient to prevent endocytosis of [125I]iodotransferrin and translocation of beta-adrenergic receptors. The further demonstration that reactions leading to the return of internalized transferrin or beta-adrenergic receptors to the cell surface are blocked after relatively modest reductions in ATP content provides further evidence of the similarity in the processes subserving diacytosis of beta-adrenergic receptors and transferrin. The differential requirement for ATP of the two arms of diacytosis provides the basis for an explanation of the controversy regarding a requirement for ATP in endocytosis via clathrin-coated pits.

The kinetics of the return of internalized beta-adrenergic receptors to the plasma membrane were measured in human astrocytoma cells. The movement of [125I]iodopindolol-labeled receptors back to the plasma membrane was measured directly and was shown to occur with a t1+2 of 3-4 min. Unlabeled receptors appeared to exhibit the same kinetics of externalization. The process was not inhibited by low concentrations (1-10 microM) of propranolol or even high concentrations of isoproterenol (0.1-1.0 mM). Higher concentrations of propranolol (0.1-1.0 mM) and other lipophilic amines inhibited externalization. The results are consistent with the proposal that catecholamine-induced beta-adrenergic receptor internalization and externalization (diacytosis) occur via the clathrin-coated pit/endosome pathway.

Recombinant turkey erythrocyte beta-adrenergic receptors expressed in murine L cells exhibited characteristic avian subtype selectivity for agonists and antagonists. In 10 of the 11 clones studied, no agonist-induced internalization of receptor was observed, although agonist-induced uncoupling of receptor and adenylyl cyclase occurred rapidly. GTP caused little or no decrease in affinity for beta-adrenergic agonists. Such behavior is commonly observed in avian erythrocytes. In contrast, one clone was susceptible to agonist-induced receptor internalization and down-regulation even though it exhibited characteristic avian beta-adrenergic ligand-binding properties. The affinity of this variant receptor for agonists was also notably reduced by GTP. Electrophoresis of affinity-labeled receptor from this clone indicated an apparent size of about 33 kDa, about 12 kDa less than that of the native or recombinant turkey beta-adrenergic receptor. Genomic DNA from this cell line that encodes the receptor was cloned and partially sequenced. The coding region of the original receptor cDNA was interrupted after codon 412 (out of 483) and was followed by 36 base pairs of novel sequence prior to the first in-frame stop codon. These results suggest that the lack of both hormone-induced internalization and GTP-sensitive, high affinity binding of agonists that is characteristic of the beta-adrenergic receptor in avian erythrocytes is due to intrinsic properties of the receptor. The restoration of these phenomena in a C-terminally truncated mutant receptor suggests the importance of the C-terminal domain in determining these processes.

The redistribution of beta-adrenergic receptors (beta-AR) during agonist-induced desensitization has been compared to the process of receptor-mediated endocytosis of epidermal growth factor (EGF) in human astrocytoma cells (1321N1). [125I]EGF exhibited saturable binding to high affinity (KD = 1-2 nM) receptor sites on intact 1321N1 cells. [125I]EGF was found to internalize rapidly using an acid wash technique to remove surface bound hormone. Sucrose density gradient fractionation following exposure to EGF revealed a redistribution of EGF binding sites from high density (heavy peak) to low density (light peak) regions of the gradient. The light peak binding probably represents EGF in internalized vesicles formed during endocytosis. Low temperature (4 degrees C) or the presence of the lectin concanavalin A (con A) inhibited this ligand-induced movement of EGF receptors. When cells were incubated simultaneously with EGF and the beta-AR agonist isoproterenol, both receptors were found to co-migrate in the low density regions of sucrose gradients. No evidence of heterologous ligand-induced receptor endocytosis was found. These results suggest that the EGF receptors and beta-AR are processed in parallel by 1321N1 cells.

N 6, O2'-dibutyryl cyclic AMP (DBcAMP) (3 x 10-4 M) causes reproducible changes in the morphology, growth rate and terminal cell density of 3 different types of human tumour cells grown in culture. Human tumour astrocyte lines 1181N1 and EH-118MG show generalized ablation of membrane systems when grown in the presence of this cyclic AMP analogue. This is not consistent with differentiation; it may be a toxic effect but possibly represents the response of these cell types to DBcAMP. These cells also grow more slowly and to lower terminal cell densities as a function of the concentration of DBcAMP. Although the growth kinetics demonstrate a return of these malignant cells to a state of density-dependent growth, micrographic analyses of inter-cellular relationships reveal that the cells still grow in overlapping patterns. Thus as judged by the commonly accepted criteria, DBcAMP does not induce contact inhibition of growth. All observed changes induced by the presence of DBcAMP are rapidly and completely reversed upon its removal from the growth medium. Human neuroblasts (NB1) grown in the presence of DBcAMP show irreversible morphological changes and the rate and extent of cell division is reduced. There is marked hyperplasia of intracytoplasmic microfilaments, which is considered consistent with differentiation.

As a part of the process of agonist-induced desensitization, 1321N1 human astrocytoma cells lose up to 95% of their beta-adrenergic receptors, as detected by 125I-hydroxybenzylpindolol (125IHYP) binding, after 12-24 h of exposure to isoproterenol. In preconfluent cultures the loss of beta-receptors is completely reversible upon removal of isoproterenol, with receptor levels reaching 100% of control levels within 48-72 h. Addition of cycloheximide (5 micrograms/ml) upon removal of agonist does not prevent the recovery of receptors. After an initial 4-h lag, receptors accumulate in the presence of cycloheximide until the same receptor level is reached that was present at the onset of desensitization. Confluent cultures, which have a reduced number of receptors per cell, recover beta-receptors to only 60 to 70% of control levels following removal of isoproterenol. In addition, cycloheximide blocks the recovery of receptors in these cultures. The effects of cycloheximide on the accumulation of receptors during cell growth suggest that receptors are stable in preconfluent cultures and that turnover only occurs later when cultures are confluent. The data also indicate that long term exposure of cells to catecholamine results in a form of the beta-adrenergic receptor that is undetectable by 125IHYP binding but, nonetheless, retains its primary amino acid structure. The undetectable receptors appear to be retained until agonist is removed, whereupon they become detectable by 125IHYP binding with a t1/2 of about 36 h in the presence of cycloheximide.

In conclusion, our data indicate that exposure of 1321N1 astrocytoma cells to catecholamines initiates a series of reactions that decreases cellular responsiveness to catecholamines and eventually results in a loss of functional beta AR from the cell. Whether the series of reactions depicted in Fig. 1 indeed represents a sequential process is not yet known. The molecular nature of the beta AR modifications responsible for uncoupling, internalization, and loss of binding also remains unknown. Nonetheless, these reactions are not restricted to the beta AR/AC system of 1321N1 astrocytoma or C62B glioma cells, since a number of investigators working with a variety of homogeneous cell systems have reported the occurrence of similar phenomena (2,7,8,13-16,19,21,28). Thus, the basic framework of catecholamine-induced modification of the properties of beta AR in astrocytoma cells is likely representative of the general phenomenon of agonist-specific desensitization of the beta AR-linked AC of mammalian cells.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.

Human astrocytoma cells (1321N1) in culture respond to pharmacological concentrations of prostaglandins and catecholamines with a marked increase in the accumulation of cyclic AMP. However, growth of 1321N1 cells in the presence of low concentrations (0.003 to 0.1 muM) of prostaglandin E1 (PGE1) results in a marked reduction in the responsiveness of the cells-even to concentrations of PGE1 that normally stimulate maximal accumulation of cyclic AMP. Occasionally, a partial reduction in the responsiveness to catecholamines was observed in cells grown in the presence of PGE1. When it occurred this effect could be correlated with an increase in the cyclic AMP-degradation capacity of the cells. This loss of responsiveness to catecholamines could be completely reversed by 1-methyl-3-isobutylxanthine, a potent inhibitor of phosphodiesterase activity in 1321N1 cells. The consistently observed and more profound desensitization to the effects of PGE1 could not be correlated with an increase in cyclic AM-degradative capacity. Accordingly, 1-methyl-3-isobutylxanthine was only minimally effective in reversing desensitization to PGE1. It is concluded that the inability of 1321N1 cells grown in the presence of PGE1 to accumulate cyclic AMP upon subsequent challenge with PGE1 is primarily due to a selective desensitization of the PGE1-activated adenylate cyclase.

Exposure of postconfluent 1321N1 human astrocytoma cells to 1.0 microM isoproterenol for 12-24 hr results in a 90% loss of beta-adrenergic receptors. Upon removal of agonist, recovery of beta-receptors to control levels occurs within 72 hr. The recovery of receptors is completely blocked by cycloheximide [R. C. Doss, J. P. Perkins, and T. K. Harden, J. Biol. Chem. 256:12281-12286 (1981)]. In contrast cycloheximide does not block recovery of beta-receptors after down-regulation in preconfluent cultures. To determine unambiguously if beta-receptor synthesis accounts for the recovery of receptors after down-regulation, post confluent cultures were incubated with isoproterenol and then transferred to agonist-free medium containing either normal or "heavy" (2H, 13C, 15N) amino acids. The rate and extent of beta-receptor recovery were similar in both normal and heavy amino acid-containing medium. When beta-receptors that had recovered in the heavy amino acid-containing medium were labeled with 125I-cyanopindolol, solubilized in Lubrol PX, and subjected to centrifugation on a 5-15% sucrose density gradient, they exhibited an increased mass compared to beta-receptors that recovered in the presence of normal amino acids. These results confirm that the density shift method is a useful approach for the study of beta-receptor synthesis and that new receptor synthesis occurs during recovery of beta-receptors from catecholamine-induced down-regulation in postconfluent cultures.

Human astrocytoma cells (EH118MG) respond to catecholamines and prostaglandins with a marked increase in the rate of formation of cyclic AMP. Treatment of EH118MG cells with cholera toxin (10 to 100 ng/ml) for 45 to 60 min caused an increase in cellular cyclic AMP content (5- to 10-fold over basal). Cholera toxin also decreased the K0.5 for isoproterenol 10- to 50-fold and decreased the K0.5 for prostaglandin E1 (PGE1)30- to 100-fold, while increasing the maximal response to PGE1 by 1.5- to 3-fold. Treatment with cholera toxin did not change the K1 values for beta-adrenergic receptor antagonists such as propranolol, alprenolol, and sotalol. Direct binding studies using [125I]iodohydroxybenzylpindolol indicated no significant changes in the number of beta-receptors or in the kinetics of the interaction of the radioligand with receptors after treatment of cells with the toxin. Competition binding studies with propranolol and sotalol revealed no toxin-induced change in Kd values for these antagonists. Treatment with cholera toxin caused only small decreases (2- to 3-fold) in the Kd values for binding of isoproterenol and norepinephrine. It is concluded that cholera toxin has little direct effect on the binding of agonists or antagonists to beta-receptors, but instead increases the efficiency of coupling of receptor and catalytic moieties of adenylate cyclase.

The characteristics of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase system is compared in C62B and C6BD12 rat glioma cells and 1321N1 human astrocytoma cells. The process of receptor-specific (or agonist-specific) desensitization is demonstrated in the C62B subline. Thus, upon exposure of C62B cells to catecholamine a rapid decline (t 1/2 = 6-8 min) in isoproterenol-stimulated adenylate cyclase activity occurs with minimal loss in basal or NaF-stimulated activity. With the same time course an uncoupled population of beta-receptors is formed and can be isolated as a low density, particulate subfraction of cell lysates. These early desensitization processes are not blocked by cycloheximide. With extended exposure to catecholamines beta-receptors are lost from detection with a t 1/2 of 150-180 min. This process in C62B cells exhibits the same characteristics observed by others for other sublines of C6 cells (Homburger, et al. J. Biol. Chem. 255: 10436, 1980; and Fishman, et al. Mol. Pharmacol. 20: 310, 1981) and by us for human astrocytoma cells (Su et al. J. Biol. Chem. 255: 7410, 1980). In addition, the existence in C62B cells of a cyclic AMP-induced, cycloheximide-sensitive desensitization process, such as that reported by others (Terasaki, et al. Adv. Cyclic Nuc. Res. 9: 33, 1978) is confirmed. Thus, it appears that C62B cells exhibit all of the characteristics typically attributed to receptor-specific desensitization in addition to the cyclic AMP-mediated heterologous process described previously.

Incubation of human astrocytoma cells (1321N1) with low concentrations of isoproterenol results in a specific loss of responsiveness to catecholamines as evidenced by a decreased accumulation of cAMP in intact cells, a reduction in isoproterenol-stimulated adenylate cyclase activity, and a decrease in beta-adrenergic receptor density, as measured by the specific binding of 125I-hydroxybenzylpindolol. The kinetics of desensitization suggest the involvement of two different reactions. The initial reaction involves a rapid loss of adenylate cyclase activity with little loss of beta-adrenergic receptors. Subsequently, a slower reaction results in the loss of measurable beta-adrenergic receptors. The degree of loss of both parameters was similar after 24 h of desensitization. It is concluded that the loss of beta-adrenergic receptors is an event that occurs as a result of the initial uncoupling of the beta-receptor-linked adenylate cyclase.

The interactions of agonists and antagonists with beta-adrenergic receptors on intact 1321N1 human astrocytoma and C62B rat glioma cells were studied by using the radioligand (-)-[125I]iodopindolol. Competition binding assays were performed at 37 degrees C under equilibrium conditions and in short-time nonequilibrium assays that approximated initial velocity conditions for binding of the radioligand. The theoretical basis and experimental validation of the initial velocity approach for determining binding affinities of rapidly equilibrating ligands are presented. For the agonists isoproterenol and epinephrine, high binding affinities that approximated their apparent affinities for binding in membranes and for increase of cyclic AMP concentrations in intact cells could be demonstrated only in short-time assays; in contrast, much lower affinities were observed with equilibrium (60-min) assays as reported previously for various cell lines. High-affinity binding of isoproterenol to 1321N1 cells also was observed in equilibrium (6-hr) binding assays carried out on ice. These results indicate that in the native state the intact cell beta-adrenergic receptor has a high binding affinity for agonists and suggest that incubation at 37 degrees C in the presence of an agonist converts the receptors to a form with low affinity for agonists.

Beta-Adrenergic receptors and epidermal growth factor receptors are both expressed on the cell surface of human astrocytoma cells. Incubation with a catecholamine or epidermal growth factor results in rapid internalization of the respective receptor. The internalized receptors co-migrate in light fractions on sucrose gradients. Astrocytoma cells maintain a constant ATP concentration by either glycolytic or mitochondrial ATP production. When cells are incubated in a medium depleted of substrates for glycolysis and gluconeogenesis, addition of inhibitors of mitochondrial ATP synthesis causes a rapid reduction in cellular ATP content. An immediate return to control ATP levels occurs upon addition of an appropriate nutrient, such as glucose. Decreasing the cellular ATP content to less than 10% of control markedly inhibits internalization of beta-adrenergic receptors and epidermal growth factor. The inhibition of endocytosis is reversed as soon as the intracellular ATP content is restored. Previous work by others (Clarke, B.L., and Weigel, P.H. (1985) J. Biol. Chem. 260, 128-133) suggested that ATP is not required for internalization (per se) of asialoglycoprotein in hepatocytes but was required for recycling of the asialoglycoprotein receptor. In contrast, our results indicate that in astrocytoma cells the process of internalization of epidermal growth factor and beta-adrenergic receptors, per se, is highly ATP dependent.

Receptor-mediated internalization of epidermal growth factor (EGF) occurs by a process involving initially clathrin-coated pits on the cell surface and the subsequent formation of ligand-containing endosomes. Using a modified acid wash technique, cell surface-bound EGF was removed. Utilizing sucrose density centrifugation, the residual cell-associated EGF was separated into plasma membrane-associated and intracellular vesicle-associated forms. Using these procedures we have identified a transient form of cell-associated EGF that is still attached to the plasma membrane but not accessible to the extracellular fluid. This form of EGF appears to be the precursor for endosomic EGF. We suggest that this intermediate form represents the receptor-ligand complex shown by electronmicroscopy to be located in narrow-necked plasma membrane invaginations (Willingham, M. C., and Pastan, I. (1980) Cell 21, 67-77).

Human astrocytoma cells (1321N1) exhibit adenylate cyclase activities coupled to independent receptors for catecholamines and prostaglandins of the E-series. Exposure of the cells to either norepinephrine or prostaglandin E1 (PGE1) results in an initial rapid accumulation of cyclic AMP but also results in a progressive loss of responsiveness of the cells to agonists. Initially, the desensitization is in large part agonist-specific. However, with continued exposure to high concentrations of norepinephrine, partial loss of responsiveness to PGE1 occurs, and vice versa. The mechanism underlying this phenomenon does not appear to involve inactivation of the effectors, formation of an inhibitory substance in the culture medium or an increase in the rate of excretion of cyclic AMP from the cell. Blockade of protein synthesis (85%) by 5 mug/ml cycloheximide did not change the rate or extent of desensitization. When desensitized cells were incubated in the presence of the effectors, responsiveness was essentially completely recovered with a t1/2 of 5-7 hr. Cycloheximide recovery reduced. Norepinephrine-induced desensitization to either norepinephrine or PGE1 was blocked by sotalol, a beta receptor blocking agent. Incubation of the cells with dibutyryl cyclic AMP caused desensitization to both norepinephrine and PGE). The results suggest that catecholamine-induced desensitization occurs as a result of interaction of the agonist with the same receptor that is linked to activation of adenylate cyclase. Cyclic AMP appears to mediate at least the non-specific aspect of agonist-induced desensitization.

Exposure of 1321N1 human astrocytoma cells to the protein kinase C (PKC) activator phorbol 12-myristate, 13-acetate (PMA) led to a rapid and concentration-dependent decrease in isoproterenol (ISO)-stimulated adenylate cyclase (AC) activity in cell lysates. This desensitization of beta-adrenergic receptor (BAR) function was mimicked by mezerein, which also activates PKC, but not by 4-O-methyl-PMA, which is a very weak activator of PKC. Pretreatment with PMA led to desensitization of AC activity stimulated by ISO and by prostaglandin E1, in contrast to the beta-receptor-specific desensitization induced by ISO. Stimulation of AC activity by forskolin and by fluoride remained unaltered. The extent of desensitization observed with PMA plus ISO was greater than with either agent alone. Desensitization with PMA did not result in internalization of BAR, as assessed by sucrose density gradient centrifugation assays and by assays of competition by the hydrophilic ligand ISO for radioligand binding to intact cell receptors. PMA pretreatment did not alter the apparent affinity of the agonist ISO for intact cell BAR, nor was the potency of ISO for stimulation of AC activity altered. The protein kinase inhibitor H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] inhibited the desensitization induced by PMA but not that induced by ISO. These results indicate that activation of PKC can lead to desensitization of receptor-stimulated AC activity but that agonist-induced desensitization of BAR-stimulated AC activity occurs by a different mechanism.

We have investigated the relationship between the catecholamine-induced occurrence in 1321N1 human astrocytoma cells of beta-adrenergic receptors that exhibit low apparent affinity for hydrophilic ligands in short-time assays with intact cells and a population of beta-adrenergic receptors that migrate in a light vesicle fraction on sucrose density gradients. Pretreatment of cells with concanavalin A prevents the generation of both of these forms of the receptor during incubation with agonists but does not prevent the agonist-induced decrease in isoproterenol-stimulated cyclic AMP production that also occurs during desensitization. Selective labeling of the low affinity beta-receptors with 125I-pindolol followed by centrifugation on sucrose density gradients revealed that all of the receptors in the light vesicle fraction from desensitized cells were of the low affinity type, but that a portion of the low affinity receptors also migrated in a heavier sucrose fraction together with the plasma membrane. In contrast, in control cells, no low affinity receptors were present in the heavy sucrose fractions. The agonist-induced occurrence of these various forms of the beta-adrenergic receptor can be explained on the basis of current models of desensitization involving agonist-induced internalization of beta-adrenergic receptors.

Binding of 125I-iodopindolol (IPIN) to intact 1321N1 human astrocytoma cell B-adrenergic receptors was measured at 37 degrees and on ice. Control cells showed a single component of IPIN binding on ice with the same total number of receptors as measured at 37 degrees. In desensitized cells (pretreated for 20 min with 1 microM isoproterenol) approximately 40% of IPIN binding on ice exhibited kinetics similar to those observed in control cells. The remaining 60% of receptors were labelled by IPIN at a much slower rate requiring the use of very high concentrations of IPIN. Sucrose density gradient fractionation was used to separately study the labelling of plasma membrane receptors and those associated with a light vesicle fraction. Labelling by IPIN on ice of the plasma membrane receptors of control cells was rapid, labelling of the light vesicle receptors of desensitized cells was slow, and labelling of the plasma membrane receptors of desensitized cells appeared to occur with both rapid and slow components. Selective labelling of the plasma membrane receptors of intact cells thus could be obtained by incubation with IPIN on ice under selected conditions. Similar results were obtained when broken cell preparations from control and desensitized cells were used. The decreased binding of IPIN on ice to B-adrenergic receptors in the light vesicle fraction not only provides further evidence consistent with sequestration of B-adrenergic receptors during desensitization, it also provides a convenient and inexpensive means to assay the sequestration reaction.

Competition by beta-adrenergic agonists and antagonists for 125I-pindolol binding sites on intact cells (1321N1 human astrocytoma and C62B rat glioma) was measured using short time binding assays as previously described (Toews, M. L., Harden, T. K., and Perkins, J. P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3553-3557). Preincubation of cells with agonists converted about half of the cellular beta-adrenergic receptors from a form exhibiting high affinity for the agonists isoproterenol and epinephrine and the antagonist sotalol to a form exhibiting much lower apparent affinity for these ligands in short time assays. Exposure to agonists did not alter the affinity of receptors for the antagonist metoprolol. This change in the ligand binding properties of the receptor was rapid (t1/2 = 1-2 min following a lag of about 0.5 min), reversible (t1/2 = 6-8 min), and dependent on the agonist concentration present during the preincubation (K0.5 = 15 nM for isoproterenol). Both isoproterenol and sotalol attained equilibrium with the high affinity receptors very rapidly but equilibrated only slowly with those receptors exhibiting low apparent affinity in short time assays. These results are interpreted in terms of a model which postulates that both the low apparent affinity in short time assays and the subsequent slow equilibration of hydrophilic ligands with these receptors result from agonist-induced internalization of a fraction of cell surface beta-adrenergic receptors. The relationship of this change in receptor binding properties to other aspects of agonist-induced desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system is discussed.

Tunicamycin, which inhibits formation of asparagine-linked glycoproteins, caused a concentration-dependent blockade of beta-adrenergic receptor (beta-AR) accumulation in 1321N1 human astrocytoma cells during growth in culture. A concentration of tunicamycin (0.1 microgram/ml) that inhibited receptor accumulation and [3H]mannose or [3H]glucosamine incorporation into glycoproteins by 90% had only a small effect (10%) on [3H]leucine incorporation into protein, and reduced the rate of cell growth. Incubation in drug-free medium subsequent to treatment of 1321N1 cells with tunicamycin for 48 hr resulted in recovery of beta-AR to control levels within an additional 48 hr. Exposure of cultures to isoproterenol (0.1 microM, 12 hr) caused an 80-90% loss of beta-AR in both pre- and postconfluent cultures; beta-AR recovered to control levels upon removal of isoproterenol. Although both tunicamycin and the protein synthesis inhibitor cycloheximide blocked beta-AR accumulation during growth of 1321N1 cells, neither agent inhibited the appearance of beta-AR during recovery from the down-regulated state in preconfluent cultures. However, cycloheximide, but not tunicamycin, blocked recovery of beta-AR after isoproterenol-induced loss of receptors in postconfluent cultures. In a previous report (Mol. Pharmacol. 26:424-429, 1984), we provided direct evidence that recovery of beta-AR from down-regulation in postconfluent cultures requires de novo synthesis of receptor protein. Thus, the results with tunicamycin are consistent with the idea that recovery of beta-AR in postconfluent cultures requires the synthesis of new beta-AR molecules, but as aglycoproteins that exhibit radioligand-binding characteristics similar to those of native glycoprotein beta-AR.

The ligand-induced internalization of beta-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 prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO 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 detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups.

Incubation of 1321N1 human astrocytoma cells with 1 microM isoproterenol rapidly results in the conversion of a portion of the beta-adrenergic receptors to a membrane form that can be separated from markers for the plasma membrane by sucrose density gradient or differential centrifugation. This "light peak" form of the receptor reaches a maximal level within 10 min of incubation of cells with catecholamine. Two types of experiments suggest that the early phase of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase can be separated into at least two reactions. First, the agonist-induced loss of catecholamine-stimulated adenylate cyclase activity precedes the appearance of beta-adrenergic receptors in the light peak fraction by 1-2 min. Second, pretreatment of cells with concanavalin A prior to induction of desensitization blocks the formation of the light peak form of beta-adrenergic receptors without blocking the "uncoupling" reaction as measured by catecholamine-stimulated adenylate cyclase activity. Specificity for the reaction that converts beta-adrenergic receptors to the light peak form is indicated by the lack of a catecholamine-induced alteration in the sucrose density gradient distribution of muscarinic cholinergic receptors, adenylate cyclase or the guanine nucleotide-binding proteins, Ns and Ni. The light peak of beta-adrenergic receptors migrates at a density similar to that of at least a portion of the activity of galactosyltransferase, a marker for Golgi. Enzyme marker activities for lysosomes and endoplasmic reticulum are not associated with this population of beta-adrenergic receptors. Taken together, these and other data suggest that incubation of 1321N1 cells with isoproterenol results in a rapid uncoupling of beta-adrenergic receptors from adenylate cyclase which is followed by a change in the membrane form of the receptor. This latter step most likely represents internalization of receptors into a vesicular form which may then serve as the precursor state from which receptors are eventually lost from the cell.