©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation and Desensitization of the Human -Adrenergic Receptor
INVOLVEMENT OF G PROTEIN-COUPLED RECEPTOR KINASES AND cAMP-DEPENDENT PROTEIN KINASE (*)

(Received for publication, February 15, 1995; and in revised form, May 11, 1995)

Neil J. Freedman (1)(§) Stephen B. Liggett (¶) Douglas E. Drachman (1) Gang Pei (1) Marc G. Caron (3) Robert J. Lefkowitz (1) (2)

From the  (1)Howard Hughes Medical Institute, Departments of Medicine (Cardiology), (2)Biochemistry, and (3)Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Persistent stimulation of the -adrenergic receptor (AR) engenders, within minutes, diminished responsiveness of the AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the AR (like that of the related AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the -adrenergic receptor kinase (ARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the AR within 3-5 min. Both cell types also express ARK1 and the associated inhibitory proteins -arrestin-1 and -arrestin-2, as assessed by immunoblotting. Agonist-induced AR desensitization in 293 cells correlates with a 2 ± 0.3-fold increase in phosphorylation of the AR, determined by immunoprecipitation of the AR from cells metabolically labeled with P. This agonist-induced AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, ARK1, ARK2, or GRK5 significantly increases agonist-induced AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified ARK1, ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the AR. Consistent with a GRK mechanism, receptor-specific desensitization of the AR was enhanced by overexpression of -arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the AR involves phosphorylation of the receptor by both PKA and at least ARK1 in intact cells. Like the AR, the AR appears to bind either -arrestin-1 or -arrestin-2 and to react with rhodopsin kinase, ARK1, ARK2, and GRK5.


INTRODUCTION

The mammalian AR()is coupled through G to the activation of adenylyl cyclase and is important in the regulation of heart rate and contractility(1) , lipolysis by adipose tissue(2) , and blood pressure homeostasis(3) , among other vital functions. Like many G-coupled receptors (4) , the AR demonstrates receptor-specific or homologous desensitization(2, 5) ; persistent or repetitive stimulation decreases the receptor's ability to activate adenylyl cyclase. In contrast to desensitization of the AR, however, little is known about desensitization of the AR at the molecular level.

Because it shares physiologic agonists, G protein coupling, and a 54% amino acid homology with the AR(6) , the AR might be expected to share regulatory mechanisms with the AR as well. Occurring within seconds to minutes of agonist exposure, short term desensitization of the AR/G/adenylyl cyclase system involves phosphorylation of the receptor by two classes of serine/threonine kinases (for review, see (4) ). The second messenger-dependent kinases PKA and protein kinase C phosphorylate and desensitize the AR, but because they phosphorylate multiple proteins, they mediate a generalized cellular hyporesponsiveness known as heterologous desensitization(4) . Second messenger-independent G protein-coupled receptor kinases (GRKs)(7) , by contrast, initiate a two-step process of homologous desensitization by phosphorylating only activated receptors. Once phosphorylated, the receptors bind to inhibitory proteins, the -arrestin isoforms(8, 9) , which uncouple the receptors from G. Like the AR, the AR possesses both a canonical site for PKA phosphorylation in the carboxyl-terminal portion of its third intracellular loop and a serine/threonine-rich cytoplasmic tail (6) , the region of the AR phosphorylated by GRKs(4, 10) .

That the AR may be desensitized by a GRK-initiated mechanism is suggested by the examination of myocardium from failing human hearts. In the lethal condition of human chronic heart failure, depressed -adrenergic responsiveness accompanies a 2-3-fold up-regulation in the expression of the -adrenergic receptor kinase (ARK1 or GRK2)(11) . Despite this provocative association, however, the only mechanistic investigation of short term AR desensitization published thus far suggests that only a PKA-dependent mechanism, and not a GRK-dependent mechanism, effects AR desensitization(5) . We therefore utilized a combination of in vitro reconstitution and intact cell model systems to assess directly the role of GRKs and -arrestin isoforms in both the agonist-induced phosphorylation and desensitization of the AR.


EXPERIMENTAL PROCEDURES

Materials

All cell culture reagents were procured from Life Technologies. Human embryonal kidney (293) cells and Spodoptera frugiperda (Sf9) cells were obtained from American Type Culture Collection. H-89 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Nonidet P-40 and okadaic acid were obtained from Calbiochem, and staurosporine was from Boehringer Mannheim. Ascites containing the monoclonal antibody 12CA5 was obtained from Berkeley Antibody Co. 3-Isobutyl-1-methylxanthine (IBMX), forskolin, (-)-isoproterenol bitartrate, and the AR/AR antagonists nadolol and (-)-alprenolol were obtained from Sigma. The AR antagonist ICI-118,551HCl and dopamineHCl were procured from Research Biochemicals International. [P]Orthophosphate (carrier-free), [I]iodocyanopindolol, [2,8-H]adenine, [-P]ATP, [-P]ATP, [H]cAMP, L-[S]methionine/L-[S]cyteine (EXPRESS protein labeling mix), and [8-C]cAMP came from DuPont NEN. Restriction enzymes were obtained from Promega.

Plasmid Constructs

Recombinant DNA manipulations were carried out by standard techniques(12) . The influenza hemagglutinin nonapeptide epitope (YPYDVPDYA) for the monoclonal antibody 12CA5 (13) was added to the amino terminus of the human AR cDNA (6) with the polymerase chain reaction. The 5` (sense strand) primer was 5`-CGCGGGGGATCCATGTACCCATACGACGTCCCAGACTACGCCGGCGCGGGGGTGCTCGTCCTG-3`, with the start codon and nucleotides 4-24 of the native sequence underlined; the 3` (template strand) primer was 5`-GGTCGGCGCTGGCCAGGGAC-3` (nucleotides 399-380). The resultant polymerase chain reaction-generated 438-base pair fragment and the expression vector pcDNA I (Invitrogen) were cut with BamHI and PstI and ligated to generate the construct pcDNA I/125`. A 1.2-kilobase pair AR cDNA fragment produced by digestion with Bgl2 (Klenow-blunted) and PstI was then subcloned into pcDNA I/125`, which had been prepared by digestion with XhoI (Klenow-blunted) and PstI. The resulting construct was designated 12AR. A SacI (T polymerase-blunted)/BglII fragment of the human AR cDNA was subcloned into the baculovirus shuttle vector pVL1393 (Invitrogen) to create pBac. After digestion with NcoI and SalI, the 12CA5 epitope-tagged AR cDNA (14) was Klenow-blunted and subcloned into pcDNA I to create ``12AR.'' The cDNAs for bovine ARK1(15) , bovine ARK2(16) , and bovine rhodopsin kinase (17) were subcloned into pcDNA I using HindIII, EcoRI/NdeI (Klenow-blunted), and HindIII/BamHI sites, respectively. The bovine GRK5 construct in pcDNA I (10) and the human D-dopamine receptor cDNA in pCMV5 (18) have been described previously. Rat -arrestin-1 and -arrestin-2 (9) were subcloned into pcDNA I by blunting with T polymerase after digesting with SacII/SalI and KpnI/SalI, respectively. To create the K220R mutant of bovine ARK1, we amplified a 327-base pair AccI fragment with the the following 5` (sense strand) primer: 5`-CGCGGGGTCTACGGCTGCCGGAAGGCCGACACGGGCAAGATGTACGCCATGAGGTGTCTGGAC-3` (the mutagenic codon is underlined). Cut with AccI, this fragment and the (dephosphorylated) ARK1/pBC12BI construct (15) were ligated. The ARK1 K220R cDNA was then subcloned into pcDNA I, as above. Dideoxy sequencing was used to confirm the specificity of mutagenesis in all polymerase chain reaction-derived fragments.

Insect Cell Culture and Infections

All recombinant baculovirus operations were conducted according to standard protocols(19) . Sf9 cells were co-transfected in monolayer culture with pBac and BaculoGold® (PharMingen) DNA, according to the manufacturer's instructions. The resultant recombinant baculovirus (AcAR) was plaque-purified and used to infect log-phase cultures of Sf9 cells in 100-ml spinner flasks with a multiplicity of infection about 0.5. Forty-eight hours into infection, the Sf9 cells expressed 5 pmol of AR/mg of cell protein.

Mammalian Cell Culture and Transfections

Chinese hamster fibroblast (CHW) cells were grown in 90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 µg/ml streptomycin and 100 units/ml penicillin (medium A), as described previously(20) . Cells were transfected with either the human AR (20) or the human AR (6) cDNAs in the expression vector pBC12BI (20) , and stably transfected clones were selected as described previously(20) . Cells used for these experiments expressed either the AR or AR at 300 fmol/mg of membrane protein.

293 cells were incubated at 37 °C in 95% air, 5% CO and grown in medium B (90% minimal essential medium with Earle's salts, 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 units/ml penicillin). The evening before transfection, 4 10 cells were plated per 90-mm dish. These cells were transfected on day 1 by calcium phosphate co-precipitation(21) . Each plate received 10 µg of DNA total, comprising 0.25 µg of 12AR along with either a 4-fold molar excess of D-dopamine receptor plasmid, a 7-fold molar excess of GRK plasmid, a 10-fold molar excess of -arrestin plasmid, a 16-fold molar excess of ARK1 K220R plasmid, or a 3-fold molar excess of both ARK1 and -arrestin-2 plasmids; the balance of DNA comprised the empty vector pcDNA I. For 12AR experiments, each plate received 10-15 µg of DNA comprising 5-10 µg of 12AR and 2.5-5 µg of either ARK1 or -arrestin-2 plasmids, with the balance comprising just pcDNA I. Cells were split on day 2 into assay dishes as follows: for phosphorylation assays, 1 10 cells/9.6 cm well of six-well dishes; for cAMP accumulation assays, 2 10cells/3.8 cm well of 12-well dishes. Assays were performed on day 4. AR expression ranged from 0.8 to 2.5 pmol/mg of cell protein (see below); within a single experiment, however, AR expression among various co-transfected cell types varied from control cells by <30%, except where noted in the legend of Fig. 8.


Figure 8: Overexpression of -arrestins augments desensitization of the AR in a manner additive to that of ARK1. 293 cells were co-transfected with the 12AR (stripedbars) and an expression plasmid for either no cDNA (control), -arrestin-1 (arr1, n = 3), -arrestin-2 (arr2, n = 3), ARK1 (n = 4), or ARK1 along with -arrestin-2 (n = 4). ISO-stimulated cAMP production in intact cells was assayed as in Fig. 6C. 293 cells co-transfected with the 12AR (blackbars) and either control (empty vector), ARK1, or -arrestin-2 plasmids were also assayed (n = 3) for ISO-stimulated cAMP production as above, except that cells were stimulated with 50 µM ISO (EC in this AR system). Conversion of H into cAMP in this figure is expressed as a percentage of the cognate control cell value; mean ± S.E. is plotted. In AR control cells, the values (mean ± S.D.) for percent conversion of H into cAMP were 0.04 ± 0.03 (basal) and 1.8 ± 1 (ISO-stimulated); untransfected cells gave 3 ± 2% of AR control cell ISO responses. In AR control cells, basal values for percent conversion of H into cAMP were 0.12 ± 0.01, and ISO-stimulated values were 1.3 ± 0.3. Untransfected cells gave 19 ± 4% of AR control cell responses. By immunoblotting, overexpression levels of ARK1 and -arrestin-1 or -2 were 20 times endogenous levels in AR experiments, and 10 times endogenous levels in AR experiments. In AR/ARK1/-arrestin2 experiments, cell lines expressed the AR at the following levels (pmol/mg): 1.2 ± 0.2 (AR control cells), 2.1 ± .02 (AR/ARK1 cells), and 1.6 ± 0.3 (AR/ARK1/-arrestin2 cells). AR density over this range of AR expression levels showed no effect on ISO-stimulated cAMP accumulation (data not shown). *, p < 0.01 (-arrestin-1), p < 0.001 (-arrestin-2) compared with control cells; , p < 0.001 compared with control, and p < 0.05 compared with -arrestin-1 or -2 cells; , p < 0.001 compared with control and p < 0.05 compared with ARK1 cells.




Figure 6: Overexpression of GRKs augments both agonist-induced phosphorylation and desensitization of the AR. 293 cells transiently co-transfected with the 12AR and either the empty vector (Empty, control) or an expression plasmid for one of 4 GRKs (ARK1, ARK2, GRK5, or rhodopsin kinase, RK) were metabolically labeled with P and treated as in Fig. 5, except that 100 nM ISO was used to stimulate the cells. A, an autoradiogram of immunoprecipitated ARs is representative of four performed. Sample 11 was from untransfected cells. B, the radioactivity in the immunoprecipitated AR bands was quantitated, and AR phosphorylation in ISO-treated cells (stimulated) was normalized to AR phosphorylation in unstimulated control cells (basal) as follows: ((stimulated - basal)/(basal)). The resulting values for AR phosphorylation as ``-fold above control basal'' are plotted as the mean ± S.E. from n = 4 (ARK1, ARK2, and GRK5) or n = 2 (RK) independent experiments. C, 293 cells from the same transfected cell pools used in B were labeled with [H]adenine and stimulated with 100 nM ISO, 50 µM forskolin, or vehicle (for basal values) for 6 min as described under ``Experimental Procedures.'' ISO-stimulated cAMP production is normalized to forskolin-stimulated cAMP production for each cell line, and plotted as a percentage of the forskolin-normalized ISO response in control cells. The values for percent conversion of H into cAMP in control cells were 0.06 ± 0.02 (basal), 1.8 ± 0.3 (ISO-stimulated), and 2.8 ± 0.1 (forskolin-stimulated); forskolin-stimulated values for GRK-overexpressing cells ranged from 95 to 103% of control. Untransfected cells gave 6 ± 2% of 12AR-transfected cell responses. Shown are results (mean ± S.E.) from two independent experiments performed in triplicate. Compared with control cells, *, p < 0.02, and , p < 0.05.




Figure 5: Stimulus-induced phosphorylation of the AR: (effects of GRK inhibition). A, 293 cells were co-transfected with the 12AR, and one of three expression plasmids: one without a cDNA insert (empty), one for ARK1, or one for a dominant negative mutant (K220R) of ARK1. After metabolic labeling with P, cells were challenged with control (-) or 10 µM ISO-containing (+) medium for 3 min and then washed and solubilized. The autoradiogram was produced as in Fig. 4. B, 293 cells were co-transfected with the 12AR and either the empty vector (control) or the K220R ARK1 mutant plasmid. Experiments (n = 4 for ISO, n = 2 for TPA and forskolin) were carried out as in Fig. 4, except that cells were exposed to 1 µM okadaic acid immediately before the stimulation with forskolin or TPA. The difference between stimulated and basal AR phosphorylation is plotted as a percentage of this difference observed in control cells (mean ± S.E.). *, p < 0.001 for the comparison with control cells.




Figure 4: Stimulus-induced phosphorylation of the AR: (effects of PKA inhibition). 293 cells transiently transfected with the 12AR were metabolically labeled with P in the absence (control) or presence (+H-89) of 20 µM H-89. Cells were then challenged with control (None) or stimulus-containing medium for varying times: 3 min with ISO (10 µM ISO); 10 min with TPA (3 µM TPA), cAMP (3 mM dibutyryl cAMP), and Forsk (50 µM forskolin with 0.5 mM IBMX). Receptors were immunoprecipitated and resolved by SDS-polyacrylamide gel electrophoresis. A, the autoradiogram from a single experiment is shown. Samples 6 and 12 derive from untransfected cells, and the positions of molecular weight markers are shown by arrows. B, three replications of this experiment are summarized, with AR phosphorylation (mean ± S.E.) expressed as (((stimulated/basal) - 1) 100) for control (darkbars) and H-89-treated (stripedbars) cells. With or without H-89 preincubation, basal levels of cellular AR phosphorylation were indistinguishable.



Desensitization and Adenylyl Cyclase Assays

CHW cells at 90% confluence were incubated for 2 h in medium A without serum, and then exposed at 37 °C to this medium containing 100 µM ascorbate with (desensitized cells) or without (control cells) 1 µM(-)-isoproterenol (ISO) for the indicated times. Cell dishes were then transferred to ice and washed 4 times with 20 ml of ice-cold phosphate-buffered saline (PBS). Cells were next scraped into 5 mM TrisCl, pH 7.4 (25 °C), 2 mM EDTA, and membranes were prepared as described previously(20) . Adenylyl cyclase activity of membrane preparations from control and desensitized cells was assayed, and data analysis was conducted as described previously (20) .

To assay the accumulation of cAMP in intact cells, transiently transfected 293 cells in 12-well dishes were labeled by incubation in 0.5 ml/well of 30% medium B, 70% minimal essential medium with 2 µCi of [H]adenine/ml overnight or 4 µCi of [H]adenine/ml for 3 h. Assays were performed at 37 °C in room air, in medium C (1 mM IBMX, 100 nM AR antagonist ICI-118,551 in minimal essential medium with Earle's salts). Paired 12-well dishes (ISO-pretreated and control) were handled together, and each comprised four sets of triplicate wells. Labeling medium was aspirated, and cells were exposed to medium C (0.5 ml/well) with or without 10 µM ISO or 2 mM dibutyryl cAMP for the indicated times (exposure 1). Afterward, media were aspirated, and the cells were washed with 1 ml/well of medium C. Cells were then challenged (or rechallenged) with 0.5 ml/well medium C with or without 10 µM ISO, 10 µM dopamine, or 50 µM forskolin for 4 min (exposure 2). Subsequently, dishes were transferred to ice, and cells were lysed by the addition of 0.5 ml/well of 2 stop solution (0.2 mM cAMP and 9 nCi/ml [C]cAMP in 5% (w/v) perchloric acid). The cAMP accumulated in the cells and media was quantitated chromatographically by the method of Salomon (22) and expressed as the percent incorporation of H into cAMP (H in cAMP per well / total uptake of H per well). [C]cAMP counts were used to normalize results for column yield, which averaged 50%. The intra-assay coefficient of variation on triplicate determinations was 10%. The cAMP produced in response to stimulation during exposure 2 (see above) was calculated as the cAMP accumulation in stimulated cells minus the cAMP accumulation in unstimulated cells from the same 12-well dish. Data were normalized to values obtained on the control dish of each pair (i.e. the dish treated with control medium during exposure 1).

To assess ISO-stimulated cAMP accumulation in 293 cells co-transfected with the 12AR and either a GRK or a -arrestin, each distinct co-transfected cell type was plated in a single row of triplicate wells in 12-well dishes. Cells were labeled as described above, but after aspiration of labeling medium and before stimulation, cells were washed with 1 ml/well of 37 °C minimal essential medium. Subsequently, cells were treated with 0.5 ml/well of medium C for 7 min at 37 °C and then challenged with an additional 0.5 ml/well of medium C with or without various concentrations of ISO or 100 µM forskolin for 6 min. Reactions were terminated by the addition of 1 ml/well of 2 stop solution, and cAMP was assayed as described above. Pilot ISO concentration/response experiments with these conditions demonstrated the EC to be 100 nM and the maximally effective concentration to be 1 µM ISO. AR experiments were conducted in a similar manner, except that endogenous 293 cell ARs were antagonized with 20 µM nadolol instead of 100 nM ICI-188,551.

Immunoblotting

CHW cells were washed twice in PBS, and scraped into 0 °C lysis buffer (10 mM TrisCl, pH 7.4 (25 °C), 2 mM EDTA with the following protease inhibitors: 0.1 mM PMSF; 10 µg/ml benzamidine, leupeptin, and soybean trypsin inhibitor; 5 µg/ml aprotinin; 1 µg/ml pepstatin A). Following cell disruption (20) , the supernatant fraction taken after a 15-min, 100,000 g spin was designated as the cytosolic fraction. Whole cell extracts of 293 cells were prepared in a similar fashion, without centrifugation. Immunoblotting was performed with previously characterized antisera(9, 23) , essentially as described previously (9) except that chemiluminescent detection of immune complexes was performed with ECL reagents and Hyperfilm-ECL (Amersham Corp.), according to the manufacturer's instructions. -Fold overexpression of proteins in transfected cells was quantitated by end point dilution of transfected cell homogenates.

Intact Cell Phosphorylation

Assays were performed at 37 °C, in phosphate-free Dulbecco's modified Eagle's medium, 20 mM HEPES, pH 7.4, with antibiotics as above (medium D). Cells were washed with 2 ml of medium D/well. Labeling was then conducted for 40 min in a CO incubator with 0.5 ml of medium D/well containing 100 µCi of P/ml. In PKA inhibition experiments, this labeling medium contained 0.04% dimethyl sulfoxide with or without 20 µM H-89. Next, to each well was added 0.5 ml of medium D containing vehicle with or without the indicated stimulating compound, and incubation continued for 3 min (with ISO) or 10 min (with forskolin, dibutyryl cAMP, and TPA). In ARK1 K220R experiments, 0.25 ml of medium D containing 3 µM okadaic acid (1 µM final concentration) was added to labeled cells immediately before forskolin or TPA stimulation (and 7 min before ISO stimulation) to inhibit cellular phosphatases. In experiments designed to inhibit both PKA and protein kinase C, 0.5 ml of medium D containing 0.1% dimethyl sulfoxide with or without 1 µM staurosporine was added to each well during the terminal 10 min of labeling. Stimulations were terminated by transferring dishes to ice, adding 2 ml of ice-cold PBS/well, washing once with PBS, and adding 0.5 ml/well of RIPA+ buffer (150 mM NaCl, 50 mM TrisCl, pH 8.0 (25 °C), 5 mM EDTA, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, with 10 mM NaF and 10 mM disodium pyrophosphate as phosphatase inhibitors, and protease inhibitors as above, in lysis buffer). Solubilized cells and debris were transferred to tubes in a total volume of 0.8 ml. After 1 h on ice, debris was separated from soluble material at 200,000 g for 15 min at 4 °C in a TLA 100.2 rotor (Beckman). From the supernatant fraction, 730 µl were transferred to 1.5-ml conical tubes. Two 15-µl aliquots were removed for protein assay (Bio-Rad DC protein assay kit). The remaining volume was processed for immunoprecipitation, each step of which was conducted at 0-4 °C. Pre-clearing was effected by adding to each tube 100 µl of 10% (v/v) protein A-Sepharose beads (Pharmacia Biotech Inc.) in 3% (w/v) bovine serum albumin/RIPA+ and rotating for 1 h. After the beads were pelleted at 10,000 g for 15 s, the supernatant fluid was transferred to a new tube containing protein A beads (as above) and 12 µg of the monoclonal IgG 12CA5. After 2 h on an inversion wheel, beads were pelleted, and the supernatant fluid was removed. The beads were next washed 3 times with 1 ml of RIPA+ and aspirated to dryness with a 28-gauge needle. Subsequently, 60 µl of 1 Laemmli sample buffer (24) was added to each sample. Immune complexes were dissociated by heating to 65 °C for 10 min and resolved by SDS-polyacrylamide gel electrophoresis in 10% gels. Gel lanes were loaded with the volume of sample buffer necessary to give equivalent amounts of AR from each sample, as follows. The AR density (pmol/mg of whole cell protein) of each transfected cell line was determined on a nonradioactive aliquot of each transfected cell population and was multiplied by the value for RIPA+-solubilized protein in each immunoprecipitation tube; the resultant receptors/tube values were then normalized and used to adust gel loading volumes. The gels were stained, dried, and subjected to autoradiography on DuPont Reflection film with an intensifying screen at -80 °C for 48-72 h. The same gels were analyzed quantitatively with a Molecular Dynamics PhosphorImager. Identical rectangles were drawn about each receptor band, and the signal intensity within each rectangle was integrated. Specific radioactivity in the receptor bands was determined as the difference between radioactivity in the receptor rectangle and radioactivity in an identical rectangle drawn in lanes of untransfected parental cells. Automatic background subtraction was not used.

Stoichiometry of AR Phosphorylation

Biosynthetic labeling and immunoprecipitation (25) were employed to determine the stoichiometry of AR phosphorylation in intact 293 cells. Briefly, transfected cells were labeled in medium B containing dialyzed fetal bovine serum, with either L-[S]methionine/L-[S]cyteine (50 µCi/ml met) for 48 h or with P (0.5 mCi/ml) for 7 h. Cells were stimulated (or not) with 10 µM ISO, washed twice with PBS, and then processed for immunoprecipitation as above, except that fluorography was used to image the S-labeled proteins(24) . Gel bands corresponding to the AR were cut, processed, and counted in a liquid scintillation counter as described previously(25) . The total counts contained in the AR bands were corrected by subtracting nonspecific counts obtained from the cognate bands of gel lanes derived from untransfected cells. The specific activities of S-labeled proteins and cellular [P]ATP were determined as described previously(25) .

Membrane Phosphorylation Assays

Sf9 cell plasma membrane fragments were prepared as described previously (26) from AcAR-infected cells, yielding 50-80 pmol of AR/mg of protein. GRKs were purified as described previously (10, 16, 26) from Sf9 cells infected with recombinant baculoviruses for ARK1, ARK2, and GRK5(10, 26) . Phosphorylation reactions were conducted at 30 °C for 15 min, with 1 pmol of AR in Sf9 membranes/tube, exactly as described in (26) .

Radioligand Binding and Immunofluorescence

To determine the AR or AR expression, transfected 293 cell homogenates, Sf9 cells or membranes, or CHW cell membranes (20) were subjected to saturation binding with 0.7 nM [I]iodocyanopindolol, as described previously(20) . The protein concentration of cell homogenates was estimated by the method of Bradford(24) , using bovine -globulin as a standard. Immunofluorescence and flow cytometry were used to quantitate 12AR expression and 12AR sequestration and were performed essentially as described previously(27) . Cells with fluorescence 2 standard deviations above the mean of untransfected cells were considered positive. By this criterion, 12AR transfection efficiencies in these experiments ranged from 35 to 50%.

Statistical Analysis

ISO concentration/response curves for adenylyl cyclase activity were generated by an iterative nonlinear least squares technique(14) . The t test for the comparison of two independent means was used, with pooled estimates of common variances(28) . Throughout the text, one-tailed p values are given.


RESULTS

To test the possibility that the AR is desensitized by a GRK-initiated mechanism, we first sought to compare the pattern of short term AR desensitization with that of the AR, which is known to be regulated by at least a ARK1-initiated mechanism(4, 29, 30, 31) . In CHW cells stably transfected with either the AR or the AR, a 5-min exposure to a saturating dose of the AR agonist ISO engenders substantial desensitization of the ISO-stimulated adenylyl cyclase response (Fig. 1). Under assay conditions favoring the detection of ARK1-initiated desensitization (29) , the maximum adenylyl cyclase response in membranes from ISO-pretreated cells was 69 ± 8% and 72 ± 10% of control values for the AR and AR, respectively; EC remained essentially unchanged for the AR and increased 2.7 ± 0.7-fold for the AR. With a 30-min ISO pretreatment, desensitization was even more pronounced; the maximum adenylyl cyclase response in membranes from ISO-pretreated cells was 57 ± 5 and 60 ± 10% of control values for the AR and AR, respectively. EC increased insignificantly for the AR and 3.2 ± 0.8-fold for the AR.


Figure 1: Short term desensitization of the AR resembles that of the AR. CHW cells stably transfected with either the AR (top) or the AR (bottom) were exposed to control or 1 µM ISO-containing medium for 5 or 30 min at 37 °C. Cells were then washed, and membranes were prepared for ISO-stimulated adenylyl cyclase assay. Shown are the mean values from six separate experiments performed in triplicate. For each experiment, the difference between the ISO-induced maximal cyclase activity and the basal cyclase activity seen in control cells was designated 100%, and all other cyclase activities in that experiment were normalized to this value. Basal cyclase activities were 3.5 ± 0.8 and 4.9 ± 1 pmol cAMP/min/mg of protein; maximal ISO-stimulated values were 12.2 ± 1.8 and 17.2 ± 2.4 pmol cAMP/min/mg of protein for the AR and the AR membranes, respectively.



To the extent that the AR and AR desensitization observed in CHW cells derives from a GRK-initiated mechanism, we would expect the desensitization to be homologous or receptor-specific. To assess the homologous nature of short term AR desensitization, we used 293 cells transiently co-transfected with the AR and the D-dopamine receptor plasmids. When these cells were prestimulated with a receptor-saturating dose of ISO for 3 min, their accumulation of cAMP in response to a subsequent 4-min, maximal stimulation with ISO or dopamine differed significantly from that observed in nonprestimulated control cells. The response to ISO was reduced by 51 ± 12%, and the response to dopamine was reduced by only 12 ± 3% (Fig. 2). The disparity between reductions in responses to the two agonists may derive from homologous desensitization of the AR on the one hand and heterologous desensitization of the D-dopamine receptor on the other hand. Alternatively, the disparity between ISO-induced reductions in agonist response may derive from other receptor-specific differences in regulation. To test these possibilities, we prestimulated the cells with dibutyryl cAMP in order to activate exclusively PKA and engender only heterologous desensitization of both the AR and the D-receptor (Fig. 2). In contrast to the ISO-prestimulated cells, the dibutyryl cAMP-prestimulated cells showed equivalent desensitization of ISO- and dopamine-induced responses; a 17 ± 1% reduction in response to ISO and a 16 ± 6% reduction in response to dopamine was seen. PKA-induced desensitization thus appears equivalent for the AR and D-dopamine receptor. Moreover, for the D-dopamine receptor, dibutyryl cAMP-induced desensitization is indistinguishable from ISO-induced desensitization. Together, these observations suggest that, with receptor-saturating concentrations, agonist-induced desensitization of the AR in transfected 293 cells is largely homologous. To estimate the contribution of PKA to this homologous desensitization, we used H-89 (32) to inhibit PKA in these intact cells. Prestimulation with ISO under these conditions failed to affect the response to subsequent stimulation with dopamine, but it still resulted in AR desensitization, manifested by a 23 ± 6% (p < 0.025) reduction in the response to subsequent stimulation with ISO (Fig. 2).


Figure 2: Homologous desensitization of the AR. 293 cells transiently co-transfected with the 12AR and human D-dopamine receptor were metabolically labeled with [H]adenine and challenged with control medium (control cells) or stimulus-containing medium at 37 °C for varying times: 3 min with ISO (10 µM ISO); 10 min with dBcAMP (2 mM dibutyryl cAMP); 3 min with 10 µM ISO preceded by a 40-min incubation with 20 µM H-89 (ISO/H-89). Cells were then washed and rechallenged for 4 min at 37 °C with control medium (for basal activity) or medium containing 10 µM of either ISO or dopamine. Plotted is the difference between the stimulated and cognate basal values for cAMP production, as a percent of the appropriate control cell value. Control cell values for percent conversion of H into cAMP were 0.20 ± 0.08 (basal), 2.4 ± 0.9 (ISO-stimulated), and 2.4 ± 0.1 (dopamine-stimulated). For ISO-prestimulated cells, these values were 2.5 ± 0.6 (basal), 3.6 ± 0.6 (ISO-stimulated), and 4.3 ± 0.2 (dopamine-stimulated). Untransfected cells demonstrated <3% of transfected cell responses. The means ± S.D. of two independent experiments performed in triplicate are shown. Compared with naive cells, *, p < 0.05;**, p < 0.025;***, p < 0.005. Compared with cells prestimulated and restimulated with ISO, , p < 0.05; , p < 0.025. Responses elicited after H-89 pretreatment differed from those elicited without such pretreatment (p < 0.05).



Agonist-induced receptor sequestration cannot explain the desensitization findings. Cells treated identically to those assayed for desensitization were processed for immunofluorescence with the monoclonal antibody 12CA5, which is specific for the amino-terminal epitope-tagged AR. By flow cytometry, the desensitized cells appear to lose only 13 ± 1% of their plasma membrane receptors by the time of restimulation with agonist. Over the range of receptor expression used in these experiments, such a small change in receptor number has no effect on ISO-stimulated cAMP production (data not shown).

If homologous desensitization of the AR in CHW or 293 cells is to be attributed to a GRK-initiated mechanism, one should be able to demonstrate the expression of both GRK(s) and the associated inhibitory -arrestin(s) in these cells, as well as agonist-induced phosphorylation of the AR. Fig. 3shows that both CHW and 293 cells express ARK1 and -arrestin-1 (M 50,000). Additionally, the CHW membrane fraction (data not shown) and 293 cells (Fig. 3) express -arrestin-2 (M 45,000). Similar immunoblots (data not shown) demonstrate the expression of GRK5 by 293 cells.


Figure 3: Immunoblots for ARK and -arrestin isoform expression in 293 and CHW cells. Top panel, 30 µg of 293 cell homogenate, 50 µg of CHW cell cytosolic fraction, and 25 ng of purified ARK1 were loaded separately onto a 10% SDS-polyacrylamide gel and subjected to electrophoresis and immunoblotting for ARK isoforms as described under ``Experimental Procedures.'' Bottom panel, 50 µg of 293 cell homogenate, 50 µg of CHW cell cytosolic fraction, and 5 µg of homogenate from 293 cells transfected with expression plasmids for either -arrestin-1 (arr1) or -arrestin-2 (arr2) were loaded separately onto a 12% SDS-polyacrylamide gel and treated as above, but with antiserum specific for -arrestin isoforms. The positions of biotinylated molecular weight markers (Amersham Corp.) are indicated with arrows.



Agonist-induced phosphorylation of the AR in intact 293 cells is demonstrated in Fig. 4. In unstimulated cells (lane1), the AR exists as a phosphoprotein migrating with a M of 69-79 10. Upon stimulation with a receptor-saturating dose of ISO (10 µM) for 3 min or with 3 µM TPA for 10 min, the 293 cells increase AR phosphorylation approximately 2-fold above basal levels. This phosphorylation corresponds to 2.4 ± 0.9 mol of phosphate incorporated/mol of receptor (n = 3 experiments). AR phosphorylation increases only approximately 50% above basal levels when 293 cells are stimulated for 10 min with 3 mM dibutyryl cAMP or 50 µM forskolin, 0.5 mM IBMX, even though such forskolin stimulation is sufficient to increase cellular cAMP at least 2.2-fold more than the 3-min ISO stimulation (assuming a 35% transfection efficiency, data not shown). Used at a concentration that inhibited heterologous desensitization (Fig. 2), H-89 pretreatment of cells abolished forskolin- and dibutyryl cAMP-induced AR phosphorylation but inhibited ISO- and TPA-induced AR phosphorylation by only approximately 60 and 40%, respectively (Fig. 4). When 1 µM staurosporine is used to abolish forskolin-, dibutyryl cAMP-, and TPA-stimulated AR phosphorylation, ISO-stimulated AR phosphorylation is reduced by 56 ± 16% (data not shown, n = 3). However, these cell-permeant kinase inhibitors may overestimate the relative activity of PKA in agonist-stimulated AR phosphorylation. At the concentrations used in these experiments, H-89 or staurosporine inhibits purified ARK1 activity by about 15% (data not shown), and H-89 appears to inhibit partially the protein kinase C-mediated AR phosphorylation demonstrated above. The results from these experiments, in aggregate, indicate that a kinase or kinases other than PKA or protein kinase C must be involved in agonist-induced phosphorylation of the AR.

To establish a role for one or more GRKs in agonist-induced phosphorylation of the AR, we sought to inhibit this agonist-induced phosphorylation by overexpression of a dominant-negative mutant (K220R) of ARK1. Kong et al.(30) have shown this mutant to be a competitive inhibitor of wild-type ARK1 with regard to phosphorylation of the purified AR. As shown in Fig. 5A, agonist-induced AR phosphorylation is increased 40% in ARK1-overexpressing cells and decreased 50% in ARK1 K220R-overexpressing cells, compared with control cells. By immunoblotting, we found overexpression of both ARK1 and ARK1 K220R in these 293 cells to be 20-fold over endogenous levels (data not shown). Fig. 5B shows that whereas overexpression of ARK1 K220R reduces agonist-induced AR phosphorylation by 45 ± 8%, it has essentially no effect on TPA- or forskolin-induced AR phosphorylation. Thus, a GRK in 293 cells seems to effect about 50% of the rapid, agonist-induced phosphorylation of the AR.

If a GRK-initiated mechanism is important for desensitization of the AR, we should expect that overexpression of one or more GRKs would not only increase agonist-induced AR phosphorylation but also decrease agonist-induced cAMP signaling by virtue of more rapid or enhanced desensitization. When cells were co-transfected with the AR and either ARK1, ARK2, GRK5, or rhodopsin kinase, AR phosphorylation increased 3.2-4.5-fold over that seen in the absence of GRK overexpression (Fig. 6, A and B). When 293 cells from the same transfected populations used in phosphorylation assays were used in intact cell cyclase assays, we found that this augmented AR phosphorylation correlated with a significant 27-41% reduction in ISO-stimulated cAMP production (Fig. 6C). By contrast, 50 µM forskolin-stimulated cAMP production was not affected by GRK overexpression; GRK-overexpressing cells showed 98 ± 3% of control cell responses.

To corroborate the transfected cell findings of GRK-mediated AR phosphorylation, we used purified GRKs to phosphorylate the AR in purified plasma membrane fragments. Fig. 7shows that phosphorylation of the AR by ARK1 was agonist-dependent. The phosphorylated AR (indicated by the arrow) co-migrates with the AR from photoaffinity-labeled AcAR-infected Sf9 cell membranes (Fig. 7) and is absent from samples of uninfected Sf9 cell membranes subjected to ISO-stimulated phosphorylation by ARK1 (data not shown). Similar results were obtained when phosphorylation reactions were conducted with ARK2 or GRK5 (data not shown).


Figure 7: Phosphorylation of the AR in Sf9 cell membranes. AcAR-infected Sf9 cell membranes were incubated with [-P]ATP with or without 100 µM ISO and in the absence or presence of purified ARK1, as described under ``Experimental Procedures.'' Samples were then subjected to SDS-polyacrylamide gel electrophoresis in 10% gels. Shown (at left) is an autoradiogram of one such gel, representative of three performed. The position of the phosphorylated AR is indicated by the arrow, and it co-migrates with the photoaffinity-labeled AR (at right). For photoaffinity labeling, membranes from Sf9 cells were resuspended in 75 mM TrisCl, pH 8 (25 °C), 12.5 mM MgCl, 2 mM EDTA. Approximately 2 pmol of AR in these membranes were incubated with 2 nM [I]iodocyanopindololdiazirine (I-CYP)(Amersham Corp.), with or without 20 µM of the AR antagonist(-)-alprenolol, as described previously(46) . Photolysis was performed at 0 °C for 10 min with a 254-nm ultraviolet lamp suspended 15 cm above the samples. Samples were then processed as described previously (46) and 0.1 pmol of receptor was loaded/lane.



The paradigm for GRK-initiated receptor desensitization predicts that an arrestin homologue should be important to the short term desensitization of a GRK-regulated receptor(7) . To test this prediction, we overexpressed -arrestin-1 or -arrestin-2 along with the AR in 293 cells and assessed the effect of this overexpression on ISO-stimulated cellular cAMP accumulation (Fig. 8). Each -arrestin isoform was expressed to levels 20-fold above endogenous levels ( Fig. 3and data not shown). Under these conditions, -arrestin isoforms reduce ISO-stimulated cAMP production by 20% (p < 0.01). Similar levels of ARK1 overexpression reduce ISO-stimulated cAMP production by 33%, significantly more (p < 0.05) than the -arrestin isoform effect (Fig. 8). ISO-stimulated cAMP production was diminished even further, by 49%, when we co-expressed the AR with ARK1 and -arrestin-2 (p < 0.05 compared with the ARK1 effect). Thus, the effects of ARK1 and -arrestin isoform overexpression on AR-mediated signaling appear to be additive in this system.

To demonstrate that ARK1 and -arrestin isoforms diminish ISO-induced cAMP responses by interacting specifically with the AR, and not with some downstream component of the signaling pathway, we took two approaches. First, we stimulated the cells used in Fig. 8with 25-50 µM forskolin and found cellular cAMP responses all to be within 5% of control cell values. Second, we overexpressed either ARK1 or -arrestin-2 with the AR and replicated the AR experiments described above. Although coupled to G like the AR, the AR fails to undergo short term desensitization(14) . In contrast to experiments in AR-expressing cells, overexpression of neither ARK1 nor -arrestin-2 affects ISO-stimulated cAMP production in AR-expressing cells (Fig. 8).


DISCUSSION

In this series of experiments, we have demonstrated for the first time that rapid desensitization of the AR in intact cells involves agonist-induced phosphorylation of the receptor itself. Our cell culture model for AR phosphorylation suggests that any of four GRKs, when overexpressed, can phosphorylate and initiate desensitization of the human AR. Furthermore, because we can immunologically identify at least ARK1 and GRK5 in untransfected 293 cells, our ARK1 dominant negative experiments suggest that either ARK1, GRK5, or both kinases actually do phosphorylate the AR in intact cells. Whereas the ability of GRK5 to phosphorylate activated receptors has been previously characterized, our work is the first to correlate this phosphorylation with functional receptor desensitization.

The importance of agonist-induced phosphorylation in the desensitization of the AR is well established(20, 29, 33) . For the AR, the actions of PKA and ARK1 appear to be independent events that contribute roughly equally to receptor phosphorylation and desensitization induced by receptor-saturating concentrations of agonist. Our results in transfected 293 cells suggest a similar scheme for the AR. Assessed with either inhibitors of PKA and GRKs or with specific activators of PKA, agonist-promoted phosphorylation and desensitization of the AR appear to be due to approximately equal contributions from GRKs and PKA.

With respect to rapid desensitization and susceptibility to GRK-mediated phosphorylation, the AR appears to be remarkably similar to the AR. Although certain intracellular domains of the AR may differentiate it from the AR at the level of G coupling (34, 35) and agonist-promoted receptor sequestration(36) , the primary structures relevant to rapid desensitization are indeed similar between the receptor subtypes. Both receptors possess PKA sites in their third intracellular loops(6) . While the AR cytoplasmic tail contains 11 serine and threonine residues thought to be candidates for GRK-mediated phosphorylation(10, 20) , the cognate AR region contains 10. This minor difference is almost certainly insignificant, however, since the actual stoichiometry of agonist-induced AR phosphorylation by intact cells (like that of the AR) approximates 1 mol of phosphate/mol of receptor(37) . Furthermore, regions of the activated AR other than the cytoplasmic tail substrate domain are known to be very important in ARK1 activation(38) . The receptor's first intracellular loop is one such region. Indeed, a peptide corresponding to amino acids 57-71 of the human AR has been shown to inhibit ARK1-mediated AR phosphorylation and ARK1-initiated AR desensitization(39) . The human AR shares 73% homology and 80% similarity with the AR in this domain(6) .

Previously, Zhou and Fishman (5) used SK-N-MC neuroblastoma cells to demonstrate short term (30 min) agonist-induced AR desensitization. Desensitization in their system, however, was evident only by an increase in the EC for ISO-stimulated adenylyl cyclase activity in membranes from ISO-pretreated cells. Unlike our studies in CHW and 293 cells, their studies showed no desensitization of the ISO-stimulated adenylyl cyclase maximal response. Their membrane cyclase assays were probably confounded, though, by the presence of ARs in the SK-N-MC cells(40) . The AR can mediate virtually all of the ISO-stimulated SK-N-MC cell adenylyl cyclase response when concentrations of ISO reach the high (1 µM) levels required to elicit the maximal response of adenylyl cyclase(40) . Since the AR fails to desensitize(14) , it is therefore not surprising that the maximal adenylyl cyclase response to ISO also fails to desensitize in SK-N-MC cells. This AR contribution to ISO-stimulated SK-N-MC adenylyl cyclase activity also complicates the interpretation of other observations made by Zhou and Fishman(5) . For example, when SK-N-MC cells are permeabilized and loaded with heparin, a ARK1 inhibitor, no effect on ISO-induced desensitization is seen in (5) . From these data, Zhou and Fishman (5) infer that GRK-initiated desensitization is unimportant for the AR. If AR-mediated signaling in SK-N-MC cell membranes obscures GRK-initiated AR desensitization, however, one might expect no effect of heparin on the residual apparent AR desensitization. Similarly, AR-mediated signaling may underlie the failure of PKA activators to engender desensitization of ISO-stimulated adenylyl cyclase in SK-N-MC cells(5) . In light of this failure to desensitize the AR with PKA activation, the mechanism by which the peptide inhibitor of PKA attenuates ISO-induced desensitization in permeabilized SK-N-MC cells (5) remains unclear. In our AR-transfected 293 cell model system, we were able to study the ISO-stimulated cAMP signal of specifically the AR by fully antagonizing the endogenous 293 cell AR with ICI-118,551.

The AR is linked to GRK5 and ARK1 circumstantially. The relative expression of GRK5 mRNA is highest in myocardium(10) , where the human AR is also highly expressed(1) . A coordinate 50% down-regulation of the AR and 2-3-fold up-regulation of ARK1 has been observed in human chronic heart failure(11) , in which decreased cardiac responsiveness to -adrenergic agonists is thought to exacerbate the disease (for review, see (1) ). Nonetheless, uncoupling of the diseased myocardial human AR from effectors remains to be demonstrated. Previous human studies may have had limited sensitivity to detect ARK1-mediated AR uncoupling, however, since they utilized adenylyl cyclase assays of washed (i.e. ARK1-depleted) membranes prepared without phosphatase inhibitors (41) . Porcine models of heart failure, by contrast, have succeeded in demonstrating AR uncoupling(42) . Recently, AR uncoupling (preceding down-regulation) in porcine heart failure has been correlated with significant increases in myocardial GRK activity(47) . Also linking ARK1 with the AR in the heart are recent data from transgenic mice, showing that AR-mediated events are attenuated by myocardial overexpression of ARK1 and potentiated by myocardial overexpression of a ARK1 antagonist(43) . By directly demonstrating ARK1-mediated phosphorylation of the human AR, our data considerably strengthen the credibility of ARK1 as a potential therapeutic target in chronic heart failure.

In contrast to rhodopsin, the m-muscarinic acetylcholine receptor(44) , and the -adrenergic receptor(26) , the AR, like the AR (26) , appears to be a relatively promiscuous GRK substrate. What factors, then, determine the relative importance of any particular GRK to desensitization of the AR expressed in any particular tissue or cell type? The anatomic distributions of GRK expression are distinct, but overlapping(7) , and few data exist comparing expression levels among individual GRKs (besides ARK1 and ARK2(16) ). Nonetheless, it seems reasonable to speculate that cell-specific differences in both GRK subtype and quantity primarily determine which GRK, if any, significantly affects the signaling through the ARs expressed in that cell. Correlating GRK-mediated receptor phosphorylation with receptor desensitization, as we have done with the AR, seems critical to the evaluation of receptor-GRK interaction. Because distinct peptide substrate specificities have been demonstrated for ARK1 and GRK5 (44, 45) , it might be that various GRKs phosphorylate the same receptor at distinct serine or threonine residues, thereby engendering variable effects on receptor desensitization. The transfected cell system we have developed for studying the AR should prove valuable in exploring these possibilities.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL03008-02 (to N. J. F.) and HL-16037 (to R. J. L.). 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.

§
Recipient of a Clinical Investigator Development Award from the NHLBI, National Institutes of Health. To whom correspondence should be addressed: Dept. of Medicine, Duke University Medical Center, Box 3821, Durham, NC 27710.

Present address: Pulmonary/Critical Care Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267.

The abbreviations used are: AR, -adrenergic receptor; PKA, cAMPdependent protein kinase; ARK, -adrenergic receptor kinase; G protein, guanine nucleotide-binding regulatory protein; GRK, G protein-coupled receptor kinase; G, stimulatory G protein; ISO, (-)-isoproterenol; CHW, Chinese hamster fibroblast; 293 cells, human embryonic kidney cells; IBMX, 3-isobutyl-1-methylxanthine; TPA, 12-O-tetradecanoylphorbol-13-acetate; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfon-amide2HCl; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. Madan Kwatra and Susan Trukawinski for help in preparing the AR recombinant baculovirus, Dr. Julie A. Pitcher for in vitro assays of H-89 and staurosporine effects on GRK-mediated phosphorylation, and Humphrey Kendall and Grace Irons for expert technical assistance.

Note Added in Proof-Desensitization of the AR in CHW cells has recently been documented by Zhou et al.(48) , and the ability of heparin to inhibit this desensitization in permeabilized cells suggests a GRK-dependent mechanism(48) .


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