©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Agonist Regulation of -Adrenergic Receptor Subcellular Distribution and Function (*)

Maria I. Fonseca , Donald C. Button (1), R. Dale Brown (§)

From the (1) Department of Pharmacology m/c 868, University of Illinois at Chicago, Chicago, Illinois 60612 Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have monitored agonist-induced -adrenergic receptor (AR) redistribution by immunocytochemical procedures in concert with functional measurements of agonist-elicited [H]inositol phosphate (InsP) production in human embryonal kidney 293 cells stably expressing AR cDNA (HEK293/). Anti-peptide antibodies directed against the carboxyl-terminal decapeptide of the AR were prepared and shown to react specifically with AR on immunoblots and in situ in HEK293/transfectants. Treatment of HEK293/cells with norepinephrine (10 µ M) results in a rapid (5-15 min) and striking internalization of cell surface receptor as visualized by confocal immunofluorescence microscopy. Receptor redistribution is sustained in the presence of agonist, rapidly reversed upon agonist removal, and prevented by the antagonist prazosin. Receptor internalizes to endosomes, as shown by colocalization with transferrin receptor, an endosomal marker. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (50 n M) causes receptor endocytosis similar to agonist; agonist-induced internalization is blocked by the PKC inhibitor staurosporine (0.5 µ M). In parallel experiments, agonist-induced [H]InsP production is abolished by phorbol 12-myristate 13-acetate but potentiated by staurosporine. Inhibition of receptor internalization with hypertonic sucrose attenuates agonist-induced [H]InsP formation; this effect is reversed by concomitant inhibition of PKC with staurosporine. These results suggest that PKC-dependent phosphorylation occurring as a consequence of AR stimulation induces receptor desensitization and internalization. Internalized receptor is reactivated and continuously recycled to the cell surface during agonist exposure.


INTRODUCTION

The -adrenergic receptors (AR)() have long been recognized to play a key role in regulation of blood pressure by the sympathetic nervous system (1) . Considerable progress has been made toward a molecular description of the structures and signal transduction mechanisms of -adrenergic receptors (2) . The primary structure of the -adrenergic receptors identified by molecular cloning corresponds to the predicted topographic model of the superfamily of G protein-coupled receptors, which features a motif of seven transmembrane domains (3) . Substantial evidence indicates the importance of agonist and G protein-regulated phospholipase C to generate phosphoinositide-derived second messengers for Casignaling in response to -adrenergic receptor activation (2, 4) . The convergence of recent pharmacological (5, 6, 7, 8) and molecular cloning (9, 10, 11, 12) studies have revealed the presence of isoforms of -adrenergic receptors. The different structural subtypes of -adrenergic receptors have been proposed to couple to distinct pathways of phosphoinositide signaling and cellular Caregulation (4, 13) .

Despite these important advances, significant gaps remain in our understanding of receptor signaling. One area of key interest concerns the disposition of the receptor in the cell membrane, and the influence of agonist on receptor distribution, responsiveness, and metabolism. Desensitization of AR responses by agonist has been reported although the kinetics of these processes vary among different systems (14, 15, 16) . Controversy also exists over whether agonists cause sequestration of the receptor from the extracellular surface (16, 17, 18) . Furthermore, prolonged agonist exposure has been reported to decrease total receptor number in some (19, 20) but not all systems (14, 21) .

Attempts to study receptor distribution at the subcellular level have been limited by the lack of specific structural probes of the receptor. In other systems, immunological approaches have provided powerful tools for studying receptor localization and organization at the cellular level, as well as revealing important insights into the mechanisms of receptor regulation (22, 23, 24) . In the present study we have developed a novel anti-peptide antibody directed against a unique decapeptide sequence corresponding to the carboxyl terminus of the -adrenergic receptor. The antibody specifically recognizes the receptor on immunoblots and in situ in human embryonal kidney 293 (HEK 293) cells transfected and selected for stable expression of the cDNA for the receptor (HEK293/). Using this experimental system we have combined immunocytochemical localization of the receptor with functional measurements of [H]inositol phosphate production to examine the mechanisms of agonist-induced receptor redistribution and modulation of receptor responsiveness. The results provide evidence for a cyclic mechanism of receptor activation, desensitization, internalization, and recovery upon agonist stimulation.


EXPERIMENTAL PROCEDURES

Materials

Cell culture media, reagents, and normal goat and rabbit sera were from Life Technologies, Inc. Fetal bovine serum was from Irvine Scientific (Irvine, CA), Bioproducts Source (Indianapolis, IN), or Atlanta Biologicals (Norcross, GA). [H]Inositol (20 Ci/mmol) and [H]prazosin (70-80 Ci/mmol) were from DuPont NEN. [H]Phenoxybenzamine (20 Ci/mmol) was synthesized according to Kan et al. (25) by Amersham Int. (Buckinghamshire, United Kingdom). It was stored at -20 °C under nitrogen. Purity was assessed by thin layer chromatography on silica gel developed with toluene:methanol (85:15). Sephadex G-50, (-)-norepinephrine-(+)-bitartrate, L-phenylephrine-HCl, phorbol 12-myristate 13-acetate, staurosporine, calphostin C, poly- D-lysine, bovine serum albumin, gelatin, keyhole limpet hemocyanin, superoxide dismutase, and catalase were from Sigma. Prazosin-HCl was a gift from Pfizer (Groton, CT). Phentolamine-HCl was a gift from Ciba-Geigy (Summit, NJ). Quantitative protein assay reagents were from Pierce. Electrophoresis reagents, goat anti-rabbit secondary antibodies, and other immunochemical supplies were from Bio-Rad or Jackson Immunoresearch. Mouse monoclonal anti-transferrin receptor antibodies were from Amersham or Boehringer. Nitrocellulose (0.45 µm) was from Schleicher and Schuell. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide was from Calbiochem. Digitonin (lot 1191) was from Gallard-Schlesinger (Carle Place, NY). Wheat germ agglutinin-Sepharose was from E-Y Labs (San Mateo, CA). A cDNA clone of the -adrenergic receptor (pBC12BI/, Ref. 9) was generously provided by Dr. Robert J. Lefkowitz, Duke University Medical School. Additional reagents and supplies were obtained from conventional sources and were the highest purity available.

Generation of Anti-peptide Antibodies

A peptide with the sequence (C)-SNMPLAPGHF corresponding to residues 506-515 at the carboxyl terminus of the hamster -adrenergic receptor (9) was synthesized commercially (Multiple Peptide Systems, La Jolla, CA). Purity was higher than 90% as assessed by reverse phase high performance liquid chromatography. Identity of the peptide was confirmed by mass spectroscopy. The peptide was coupled to keyhole limpet hemocyanin with m-maleimidobenzoyl- N-hydroxysuccinimide through the terminal cysteine residue by Multiple Peptide Systems and rabbit immunizations (two rabbits with boosts at 2-week intervals) were performed by Bethyl Laboratories (Montgomery, TX). Antisera were screened by standard enzyme-linked immunosorbent assay procedures (26) against immobilized peptides, and gave titers up to 1:10,000. [H]Phenoxybenzamine Labeling and Partial Purification of -Adrenergic Receptors-Reaction of -adrenergic receptors with [H]phenoxybenzamine was performed essentially as described previously (27) . Briefly, DDTMF-2 cells (0.5 10cells/ml, 3 10cells/preparation) were harvested, washed, and resuspended in 1% of the original volume of phosphate-buffered saline (PBS, 140 m M NaCl, 5 m M KCl, 10 m M NaPO, pH 7.4). [H]Phenoxybenzamine (70-100 n M) was added for 60 min at 30 °C with gentle agitation. The reaction was quenched by adding 0.1% -mercaptoethanol and incubating an additional 30 min. Cells were diluted with 2-3 volumes of PBS, washed by centrifugation, resuspended in one-tenth volume of PBS, and sonicated on ice for 2 min. Membranes were collected by centrifugation (20,000 g, 60 min, 4 °C). Solubilization of membranes with digitonin and wheat germ agglutinin chromatography of [H]phenoxybenzamine-labeled receptor were performed as described previously (28) . Receptor samples were desalted on Sephadex G-75 columns equilibrated with 50 m M NH-HCO, concentrated, and stored at -70 °C for use in immunoblotting or autoradiography protocols.

Immunoblotting

Proteins were resolved by electrophoresis through 10% SDS-PAGE minigels and electrotransferred overnight (300 mA) to nitrocellulose in transfer buffer composed of 0.7 M glycine, 25 m M Tris-HCl, pH 7.7 (29) . Immunoblots were processed according to the manufacturer's procedure (Bio-Rad). Briefly, nitrocellulose strips corresponding to individual lanes from the gel were cut and incubated in blocking buffer (500 m M NaCl, 20 m M Tris-HCl, pH 7.5) containing 3% gelatin for 1 h, followed by anti-peptide antisera (1:500-1:1000 dilution) in blocking buffer containing 1% gelatin and 0.05% Tween 20 for 2 h. The strips were washed with blocking buffer and incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase as specified by the manufacturer (Bio-Rad). Immunoblots were developed with 5-bromo-4-chloro-3-indoylphosphate and nitro blue tetrazolium. In some experiments, parallel immunoblotting and autoradiography were performed on [H]phenoxybenzamine-labeled receptor preparations. In this case replicate nitrocellulose strips from the electrotransfer procedure either were immunoblotted as described above, or were subjected to autoradiography by exposure with Kodak X-Omat film with intensifying screens for 2 weeks at -70 °C.

Immunofluorescence Staining

HEK 293 cells stably expressing receptor were subcultured in 35-mm dishes containing poly- D-lysine-coated coverslips. At subconfluence (2-4 days), cells were processed for immunostaining. Treatments of cultures with experimental agents were performed in growth medium for the indicated times before processing. Treatment with norepinephrine (NE) was always accompanied with propranolol (1 µ M) to prevent activation of endogenous -adrenergic receptors, and with catalase (10 µg/ml) and superoxide dismutase (10 µg/ml) to prevent catecholamine oxidation. Cells were fixed in 3.7% formaldehyde in PBS for 15 min. Fixed specimens were blocked for 2 h at room temperature with PBS containing 10% normal goat serum, 1% bovine serum albumin, 0.05-0.1% Triton. For single antibody labeling, specimens were incubated with receptor antibody (1:500-1:1000 dilutions in blocking solution) at 4 °C overnight, followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson) at a dilution of 1:400 in the blocking buffer (1 h incubation at room temperature). In double immunofluorescence labeling experiments, mouse monoclonal anti-transferrin antibody (1:100 dilution) was incubated with the fixed cells for 3 h at room temperature followed by addition of anti-receptor antibody and overnight incubation with both antibodies at 4 °C. Respective antibody labelings were visualized with Lissamine rhodamine-goat anti-mouse and fluorescein isothiocyanate-goat anti-rabbit IgGs. Coverslips were mounted in Slow Fade mounting solution (Molecular Probes). Immunofluorescence staining was examined by conventional inverted fluorescence microscopy (Zeiss or Nikon) or by confocal fluorescence microscopy with 1-µm optical sections. Confocal fluorescence microscopy was performed at the Electron Microscopy and Imaging Facility, Northwestern University School of Medicine, Chicago.

Stable Expression of -Adrenergic Receptors in HEK 293 Cells

A 2.1-kilobase EcoRI fragment containing the coding sequence of the hamster -adrenergic receptor (pBC12BI/, kindly provided by Dr. Robert J. Lefkowitz, Duke University Medical School) was subcloned into pBluescript SK(+) (Stratagene, La Jolla, CA). The pBluescript/plasmid was used as the source of a 2.1-kilobase HindIII- XbaI fragment that was inserted at the HindIII and XbaI sites of the eukaryotic expression vector pRc/CMV (Invitrogen). The construct, pCMV/, is capable of high levels of receptor expression in mammalian cells.

pCMV/was used to transfect cultures of HEK 293 cells (adenovirus-transformed human embryonal kidney cells, Ref. 30), seeded at 1-2 10cells/10-cm dish by the calcium phosphate-mediated gene transfer method of Chen and Okayama (31) . One day following exposure of cells to the DNA precipitate, cultures were re-plated at a 5-10-fold lower density in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 400 µg/ml G418. Cultures were maintained in selection medium for 2-4 weeks with fresh medium changes twice per week. Resistant colonies were pooled and stock cultures of pooled transfectants stably expressing receptors (10-20 pmol/mg membrane protein by Scatchard analysis) were propagated in growth medium containing G-418. Measurement of Agonist-elicited [H]Inositol Phosphate Production-Protocols for equilibration of HEK 293 cell cultures with [H]inositol, experimental exposure to agonist, and analysis of [H]inositol phosphate production were performed essentially as described previously (32) . Briefly, cultures equilibrated 24-48 h with [H]inositol (2-5 µCi/ml) were exposed to physiological salt solution supplemented with LiCl (Li-PSS, composition in millimolar: NaCl, 130; LiCl, 10; KCl, 5.4; CaCl, 1.8; MgCl, 1.6; NaHPO, 1.0; D-glucose, 5.5; HEPES, 10; pH 7.4) and specified agonist additions for indicated times at 37 °C. Addition of the -adrenergic agonist norepinephrine was routinely accompanied by 1 µ M propranolol and 10 µg/ml each of superoxide dismutase and catalase. Reactions were terminated with ice-cold 10% trichloroacetic acid. Samples were extracted with ethyl ether, neutralized, and fractionated by anion exchange chromatography (Dowex 18-formate). The columns were eluted with water to remove [H]inositol and then total [H]inositol phosphates were collected by eluting with 1.2 M ammonium formate in 0.1 M formic acid according to published procedures (32) .

Additional Procedures

Culture of DDTMF-2 (28) and HEK 293 (24, 30) cell lines were performed as described previously. Receptor density was assessed by [H]prazosin binding to intact cells or membrane preparations by established procedures (16) . Protein determinations were performed by Bradford (33) or bicinchoninic acid assay (Pierce).


RESULTS

-Adrenergic Receptor Recognition by Immunoblotting Using Anti-peptide Antibodies

Anti-peptide antibodies were prepared in rabbits against a unique amino acid sequence (amino acids 506-515) that corresponds to the cytoplasmic carboxyl terminus of the cloned hamster -adrenergic receptor. The antiserum reacted with high specificity and sensitivity to the cognate peptide by enzyme-linked immunosorbent assay (data not shown).

Specific immunoreactivity of this antibody toward the receptor polypeptide was tested on immunoblots by two approaches, as shown in Fig. 1. First, receptors from DDTMF-2 cells were covalently radiolabeled with [H]phenoxybenzamine, solubilized with digitonin, and partially purified by wheat germ agglutinin chromatography (27) . Replicate samples of this preparation were subjected to SDS-PAGE and examined by autoradiography to visualize the radiolabeled receptor, or by immunoblotting. As shown in Fig. 1 A, Lane 1, [H]phenoxybenzamine labeled polypeptides of approximate molecular mass = 80 kDa. The antiserum immunoreacted with a band that co-migrated with the radiolabeled polypeptide (Fig. 1 A, Lane 2). Previous studies have identified this 80-kDa peptide as the -adrenergic receptor. Labeling was specifically blocked by the -adrenergic antagonist prazosin (Ref. 27, and confirmed in these experiments, data not shown). The diffuse labeling pattern arises from the glycosylation of the receptor protein (28) .


Figure 1:In vitro recognition of -adrenergic receptor by immunoblotting with anti-peptide antibodies. A, [H]phenoxybenzamine-labeled DDTMF2 cell membranes, partially purified on wheat germ agglutinin-Sepharose, were subjected to SDS-PAGE (40-50 fmol of receptor/lane), electrotransferred to nitrocellulose, and visualized by autoradiography ( Lane 1) and immunoblotting with antiserum directed against the carboxyl-terminal decapeptide sequence from the receptor ( Lane 2), as indicated under ``Experimental Procedures.'' Positions and apparent masses (in kDa) of molecular weight standards are indicated. B, crude membrane extracts from wild type HEK 293 cells ( Lane 1), or cells transfected and stably expressing the receptor ( Lane 2) were immunoblotted with the antiserum.



Specific immunoreactivity of the antiserum was further demonstrated by immunoblotting against crude membrane fractions prepared from HEK 293 cells transfected to stably express the receptor. The antibody reacted with polypeptides of approximate molecular mass = 80 kDa present in membrane extracts from transfected cells (Fig. 1 B, Lane 2), but absent in membranes from untransfected cells (Fig. 1 B, Lane 1). Furthermore, the immunoreactivity of antibody 506-515 was completely abolished by prior absorption with its corresponding peptide (data not shown).

Agonist-induced Redistribution of -Adrenergic Receptors

The capability of the antibodies to recognize the receptor in situ was tested by immunofluorescence staining of pooled populations of HEK 293 cells selected for stable AR expression. The pattern of immunostaining obtained with this antibody appears diffusely and uniformly distributed over the cell surface (Fig. 2 A). Specificity of the immunostaining was demonstrated by the following criteria (data not shown). First, preabsorption of the antiserum with the corresponding peptide abolished the specific staining. Second, no positive immunofluorescence was observed when the anti-peptide antiserum was substituted with preimmune serum. Third, no immunofluorescence was observed in untransfected HEK 293 cells. Furthermore, no immunostaining was observed if the cells were not first permeabilized with detergent allowing access of the antibody to the cell interior. This latter result is consistent with the proposed cytoplasmic location of the peptide epitope recognized by the antibody.

These antibodies were used to study the influence of agonist exposure on receptor distribution. As shown in Fig. 2 B, exposure of HEK 293/cells for 3 h to experimental growth medium containing 10 µ M norepinephrine produced a striking redistribution of the staining pattern of the receptor and the appearance of highly non-uniform aggregates of intense immunostaining. The receptor antagonist prazosin (1 µ M) blocked agonist-induced redistribution of immunostaining (Fig. 2 C), confirming that the redistribution was specifically mediated by agonist occupancy of the receptor.


Figure 2: Agonist induced redistribution of the -adrenergic receptor. HEK 293 cells stably expressing the receptor were treated with growth medium containing specified additions for 3 h, and processed for immunofluorescence microscopy using antiserum directed against the receptor as described under ``Experimental Procedures.'' A, control, no additions; B, 10 µ M norepinephrine; C, 10 µ M norepinephrine plus 1 µ M prazosin. Scale bar, 10 µm.



The kinetics of agonist-induced redistribution are indicated in Fig. 3. Redistribution was evident within 5 min of agonist exposure, the earliest interval examined, essentially complete by 15 min, and sustained during agonist exposure intervals up to 24 h.

The agonist-induced redistribution was reversible. Exposure of cells to norepinephrine for 1 h followed by removal of agonist and addition of fresh growth medium containing prazosin restored the original diffuse pattern of immunostaining within 15-30 min (Fig. 4). Similar reversal of receptor redistribution was observed upon removal of agonist from cultures treated with norepinephrine for 24 h (data not shown).

Localization of Internalized Receptors

In order to visualize receptor localization at higher spatial resolution, the staining of control and agonist-treated cells was examined by confocal immunofluorescence microscopy. Scanning of sequential optical sections (1-µm thickness) of individual cells was performed. Fig. 5 shows representative control and agonist-treated images obtained at comparable focal planes relative to the attachment surface. Control cells (Fig. 5 A) displayed immunostaining on the cell periphery, consistent with localization of the receptor at the cell surface. NE-treated cells (Fig. 5 B) showed non-uniform and bright patches of immunofluorescence localized internally throughout the cytoplasm; some aggregates also appeared to be associated with the cell surface. These images clearly demonstrate that the -adrenergic agonist elicits a profound redistribution of receptor within these cells, and they strongly suggest that a significant fraction of receptors translocate from the cell surface to an intracellular location.


Figure 5: Agonist-induced -adrenergic receptor redistribution observed with confocal microscopy. Confocal microscopy images (optical section at z = 2 µm above attachment surface) are shown of: A control cell, or B, NE-treated cell (10 µ M). Treatment interval was 3 h. Scale bar, 10 µm.



Internalization of many receptors and integral membrane proteins occurs by endocytosis to a common endosomal compartment from which further intracellular sorting or recycling to the cell surface proceeds (34) . To determine whether receptor trafficking occurs by a similar pathway we performed immunofluorescence colocalization of the internalized receptors with transferrin receptor, a classic endosomal marker. Each antigen was labeled with its corresponding primary antibody and visualized with fluorescein isothiocyanate-() or Lissamine rhodamine-(transferrin) conjugated secondary antibodies, respectively. Confocal images obtained from control cells showed that receptor is mainly localized to the cell surface while transferrin receptors reside in internal vesicles (Fig. 6, A and B). After 1 h NE exposure, the internalized receptor colocalizes with transferrin receptor in endosomes (Fig. 6, C and D). This result indicates that receptor translocates to endosomes during agonist exposure. In addition, agonist-induced receptor internalization was completely prevented by pretreatment of cells with hyperosmotic sucrose solutions (0.45 M), a procedure which has been shown previously to inhibit receptor-mediated endocytosis (Ref. 35, data not shown). In sum, these results are consistent with an endocytic pathway of receptor internalization.


Figure 6: Co-localization of -adrenergic receptor and transferrin receptor. Cells were treated (1 h) with growth medium containing indicated norepinephrine additions and then were fixed, permeabilized, and labeled simultaneously with rabbit polyclonal antiserum directed against the receptor (1:1000 dilution) and a mouse monoclonal antibody specific for transferrin receptor (1:100 dilution). Immunofluorescence detection was achieved with fluorescein isothiocyanate-goat anti-rabbit IgG (receptor) and Lissamine rhodamine-goat anti-mouse IgG (transferrin receptor) secondary antibodies. Confocal images are shown of a control cell ( A and B) and a NE (10 µ M) treated cell ( C and D) labeled with AR ( A and C) and transferrin receptor ( B and D) antibodies.



Role of Protein Kinase C in -Adrenergic Receptor Internalization

Recent studies suggest that protein kinase C-mediated phosphorylation may regulate receptor internalization (36) . Using immunocytochemical procedures we tested the involvement of protein kinase C in agonist-induced receptor internalization. The characteristic receptor internalization induced by norepinephrine is shown in Fig. 7 A. Inhibition of protein kinase C by staurosporine (0.5 µ M) completely blocks the NE-induced receptor redistribution (Fig. 7 B). Moreover, activation of protein kinase C by PMA (50 n M) induces receptor internalization to endosomes. Fig. 8, A and B, respectively, show colocalization of -adrenergic receptors and transferrin receptors in cells treated with PMA. The kinetics of PMA-induced receptor internalization are comparable to those observed with agonist, and the effects of PMA are blocked by staurosporine (data not shown). The possibility that these pharmacologic agents may exert actions in addition to their effects on protein kinase C should also be considered. However, the body of data outlined above which show the parallels between the actions of agonist and PMA, and the inhibition of their effects by staurosporine, strongly suggest that protein kinase C-dependent phosphorylation plays an important role in the mechanism of agonist-induced receptor internalization in this system.

Activation and Desensitization of Receptor Response

Experiments were next designed to investigate the relationship between receptor redistribution and functional responsiveness. Activation of -adrenergic receptors by norepinephrine elicits a sustained and robust accumulation of [H]inositol phosphates in cultures equilibrated with [H]inositol and stimulated in the presence of LiCl to prevent inositol phosphate degradation (Fig. 9 A). Under these conditions, accumulation of [H]inositol phosphates is linear over at least 60 min of agonist exposure.

The sustained and linear accumulation of inositol phosphates stimulated by agonist presents an apparent conflict with the rapid agonist-induced receptor internalization. To reconcile this conflict we reasoned that the observed inositol phosphate response might reflect multiple processes of receptor activation, desensitization, or internalization. To test this hypothesis cells were subjected to treatments which had been shown previously to alter the subcellular distribution of the receptor, and the effects on agonist-stimulated [H]inositol phosphate production were measured. Results are shown in Fig. 9 B. Activation of protein kinase C by PMA abolishes agonist-stimulated [H]inositol phosphate production. Conversely, inhibition of protein kinase C by staurosporine (2 µ M, Fig. 9B) or calphostin C (0.5 µ M, data not shown) enhances agonist-stimulated inositol phosphate production. However, the linear accumulation of [H]inositol phosphates suggests that receptor reactivation must also occur during prolonged agonist exposure. Recent studies on -adrenergic receptors suggest that receptor internalization is necessary for recovery following agonist-induced desensitization (37) . We therefore tested the role of receptor internalization as a determinant of receptor responsiveness by measuring agonist-stimulated [H]InsP production in solutions containing hyperosmotic sucrose to block receptor internalization. As further shown in Fig. 9 B, this treatment attenuates agonist-stimulated [H]InsP production. Moreover, concurrent inhibition of protein kinase C with staurosporine in the presence of sucrose reverses the attenuation so that agonist responses again are potentiated relative to naive cultures. The elevation of basal InsP production by sucrose treatment does not affect the calculated responses, since all data were normalized relative to basal and agonist-stimulated responses of control cultures. These results indicate that activation of PKC which is predicted to occur as a consequence of agonist stimulation inhibits receptor responsiveness. Receptor internalization during agonist stimulation is necessary to sustain the functional response, suggesting that dynamic cycling of receptors between cell surface and internal compartments is required for the regeneration of active receptor.


Figure 9: Activation and desensitization of agonist-elicited [H]inositol phosphate production in HEK 293 cells. A, time course of agonist-elicited [H]inositol phosphate production. Cultures were equilibrated with [H]inositol, stimulated in Li-PSS containing the indicated agonist additions for the specified times, and [H]inositol phosphates were measured as described under ``Experimental Procedures.'' , 10 µ M phenylephrine; , no added agonist. B, regulation of receptor responsiveness by protein kinase C or receptor internalization. Cultures equilibrated with [H]inositol were incubated with Li-PSS containing the indicated additions for 20 min pre-treatment interval (1-ml incubation volume). Replicate cultures were then stimulated for a further 20 min by addition of phenylephrine (10 µ M final concentration, hatched bars) or vehicle ( open bars) to the pre-treatment solution. Quantitation of [H]InsP production was performed as described under ``Experimental Procedures.'' Data are corrected for basal [H]InsP production and normalized to agonist-stimulated [H]InsP production obtained in control cultures incubated with Li-PSS containing no further additions during the pre-treatment interval. Treatments: PMA, 100 n M; STP, staurosporine, 2 µ M; SUC, 0.45 M sucrose.




DISCUSSION

The development of an immunological probe for the -adrenergic receptor has enabled us to visualize the receptor with unprecedented subcellular resolution, and to elucidate mechanisms of agonist-induced internalization. Alterations in receptor responsiveness were observed in parallel functional measurements of agonist-elicited [H]InsP production. Evidence is presented suggesting the involvement of protein kinase C in both receptor internalization and desensitization which occur during agonist exposure.

Taken together, these data lead to a working model relating receptor activation, desensitization, and recycling. Salient features of the model are summarized as follows. 1) Agonist activates the receptor, generating inositol phosphate and diacylglycerol second messengers. Robust agonist-elicited phosphoinositide metabolism is demonstrated in Fig. 9; we have found functional coupling of this response to the end point of elevated intracellular Causing fura 2 methodology.() 2) Protein kinase C-mediated phosphorylation consequently causes feedback inhibition of receptor response. Pharmacologic activation of PKC by PMA inhibits receptor response to agonist. Conversely, inhibition of PKC with staurosporine during agonist stimulation potentiates receptor response. 3) Agonist induces receptor endocytosis by a mechanism involving protein kinase C-mediated phosphorylation. Internalized receptor co-localizes with transferrin receptor, a classic marker for endosomes. Receptor internalization is prevented by treatment of cells with hyperosmotic sucrose solution, which has been shown to block receptor-mediated endocytosis in other systems. Furthermore, activation of protein kinase C by PMA causes receptor internalization to endosomes, mimicking the effects of agonist. Conversely, inhibition of PKC with staurosporine blocks agonist-induced receptor internalization. 4) Receptor reactivation and recycling to the cell surface occur continuously during agonist exposure. Interruption of receptor internalization with hyperosmotic sucrose attenuates the sustained agonist response, consistent with an accumulation of desensitized cell surface receptor resulting from protein kinase C activation (see above). Simultaneous inhibition of protein kinase C relieves this inhibition by reducing the extent of receptor desensitization, suggesting that desensitized receptor is regenerated during internalization and subsequently returned to the cell surface. This result argues that the effect of sucrose treatment is specific for blocking receptor internalization rather than nonspecific uncoupling of receptor responsiveness. Upon removal of agonist, the initial cell surface disposition of the receptor is rapidly restored, further supporting the reversible nature of this process.

These results offer insights into several aspects of the relationship between -adrenergic receptor activation and desensitization. First, our model suggests that steady state receptor response will reflect a balance between the rates of receptor desensitization and recovery. The reactivation component of the model provides a means to reconcile observations of agonist-induced alterations in receptor properties with the relatively sustained physiological responses to -adrenergic stimulation which are observed in vivo (14, 38, 39) . Variability in receptor responsiveness among different experimental systems could then result from alterations in this balance. Specifically, our data point to a role for protein kinase C-mediated phosphorylation in the process of receptor desensitization. Variations in protein kinase C activation (arising, for example, from heterogeneous expression of specific PKC isoenzymes, alterations in diacylglycerol metabolism, or availability of key PKC substrates) would in turn affect the rate or extent of receptor desensitization, and contribute to the observed discrepancies among different experimental systems. Consistent with the proposed role of PKC in AR regulation, previous studies have shown that PMA treatment causes AR phosphorylation, modulation of agonist affinity, and uncoupling of the receptor from cellular responses (16) . A recent report (40) demonstrates that staurosporine treatment to inhibit PKC potentiates AR-mediated InsP production and contractile responses in rat aorta smooth muscle, similar to the results in this study.

A related consideration concerns the relationship between receptor number and response. Our data suggest that at steady state response a fraction of receptors will be desensitized or inaccessible to agonist compared to naive cells. Thus one may predict an initial transient activation of response followed by a decline to the maintained steady state. Interestingly, Nahorski and co-workers (41) describe such a temporal sequence of activation and rapid partial desensitization ( t< 30 s) of recombinant M3 muscarinic receptor coupling to phosphoinositide metabolism in transfected Chinese hamster ovary cells; partial desensitization of this receptor is accompanied by its phosphorylation. Moreover, agonist-induced alteration in receptor accessibility or responsiveness further predicts that an apparent excess of total receptors will be measured relative to the magnitude of the sustained response, whereas the initial rate or latency of response activation will more closely reflect the total number of receptors. Parallel measurements of receptor number and agonist-stimulated intracellular Caelevations in individual astroglial cells are consistent with this prediction (42) . HEK 293 cells express receptors in excess over physiological systems, and caution is always advisable in extrapolating results from expression systems to intact tissue. However, apparent receptor reserves have been documented in a number of preparations (43) . Our results provide a rationale to understand their physiological function.

At present the signals controlling receptor internalization are not well known. Using radioligand binding measurements to assess receptor redistribution in Chinese hamster ovary cells transfected with receptor cDNA, Zhu and Toews (36) report that agonist or PMA cause receptor internalization which is blocked by staurosporine. Our studies which monitor the receptor directly with an immunological probe demonstrate a role of PKC-mediated phosphorylation in receptor internalization in this system. However, the site(s) of PKC phosphorylation and the molecular interactions which control receptor internalization are unknown. Mutagenesis studies of adrenergic receptors have identified a tyrosine-containing sequence (NP XXY) which is conserved among AR and similar to the internalization signal (NP XY) of constitutively recycling receptors (44) . It is tempting to speculate that PKC-mediated phosphorylation triggers receptor internalization by facilitating the interaction of this NP XXY locus with the endocytic machinery of the cell.

Significant parallels exist between our data and extensive studies on agonist regulation of -adrenergic receptor responsiveness and disposition, as follows. 1) Agonist exposure in a variety of systems leads rapidly to receptor desensitization, characterized by reduced ability of the cell surface receptor to couple functionally to the stimulatory G protein, G, and to activation of adenylyl cyclase (45) . 2) Phosphorylation of -adrenergic receptors by the second messenger activated cAMP-dependent protein kinase, as well as by specific -adrenergic receptor kinases, appears important for receptor desensitization (45) . Although the corresponding receptor kinases have not been described, our data do not rule out this possibility. Moreover, we show here evidence supporting involvement of protein kinase C-mediated phosphorylation in AR receptor desensitization and internalization. 3) -Adrenergic receptor desensitization is accompanied by rapid and reversible sequestration of the receptor (46, 47) . Elegant immunocytochemical procedures of von Zastrow and Kobilka (24) have demonstrated that -adrenergic receptor sequestration occurs by an endocytic pathway. 4) -Adrenergic receptor sequestration is necessary for reactivation of desensitized receptor (37) , possibly by a dephosphorylation mechanism (48) . Significantly, recycling of reactivated receptors appears to occur continuously in the presence of agonist (49) . 5) -Adrenergic receptor desensitization, sequestration, and down-regulation are independently regulated processes rather than occurring as an obligatory sequence of events (44, 50) . In additional studies we find no decrease in AR immunoreactivity or [H]prazosin binding activity following 24 h agonist exposure, demonstrating that internalization occurs independently of down-regulation (data not shown). These overall similarities suggest that members of the family of G protein-coupled receptors may share comparable cycles of activation, desensitization, and internalization; yet exhibit significant disparities in their temporal responses.

In conclusion, we demonstrate that agonist activation of receptors leads to a continuous cycle of receptor desensitization, internalization, and recovery. The availability of this defined and functionally coupled expression system combined with novel immunochemical probes should allow further progress in understanding the molecular interactions underlying receptor cycling. It will be of interest to extend these approaches to examine the organization and regulation of -adrenergic receptors in vascular tissues under normal and pathophysiological conditions.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 41470 (to R. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Pharmacology Research Associate of the National Institute of General Medical Sciences.

§
To whom correspondence should be addressed. Tel.: 312-996-5664; Fax: 312-996-1225.

The abbreviations used are: AR, -adrenergic receptor; HEK cell, human embryonal kidney cell; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; NE, norepinephrine; PMA, phorbol 12-myristate 13-acetate; InsP, inositol phosphate; Li-PSS, physiological salt solution supplemented with LiCl; PKC, protein kinase C.

D. Siwik and R. D. Brown, unpublished data.


ACKNOWLEDGEMENTS

We are indebted to Dr. Robert J. Lefkowitz for the -adrenergic receptor cDNA clone used in this study, to Drs. Brenda Russell and Herb Proudfit for use of fluorescence microscopy instrumentation, to Drs. John Reece and Palmer Taylor for helpful discussions, to Dr. Kelly Ambler for critically reading the manuscript, and to Fran Fries for assistance with cell culture.


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