©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Phosphorylation in Agonist-promoted (2)-Adrenergic Receptor Sequestration
RESCUE OF A SEQUESTRATION-DEFECTIVE MUTANT RECEPTOR BY betaARK1 (*)

(Received for publication, June 13, 1995; and in revised form, August 18, 1995)

Stephen S. G. Ferguson(§)(¶) Luc Ménard (§) Larry S. Barak (**) Walter J. Koch Anne-Marie Colapietro Marc G. Caron (§§)

From the Howard Hughes Medical Institute Laboratories and Department of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The beta(2)-adrenergic receptor (beta(2)AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta(2)AR mutant (beta(2)AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta(2)AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta(2)AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta(2)AR-Y326A mutants, demonstrated that the beta(2)AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (betaARK1). However, overexpression of betaARK1 led to the agonist-dependent phosphorylation of the beta(2)AR-Y326A mutant and rescue of its sequestration. betaARK1-mediated rescue of beta(2)AR-Y326A sequestration could be prevented by mutating putative betaARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta(2)AR could be attenuated by overexpressing a dominant-negative mutant of betaARK1 (C betaARK1-K220M). These findings implicate a role for betaARK1-mediated phosphorylation in facilitating wild-type beta(2)AR sequestration.


INTRODUCTION

The exposure of the beta(2)-adrenergic receptor (beta(2)AR) (^1)to catecholamines initiates its biological response via coupling to the stimulatory G protein (G(s)), which then mediates the stimulation of adenylyl cyclase(1, 2) . However, this receptor-mediated adenylyl cyclase response to agonist is followed by a rapid uncoupling of the receptor from its effector system, termed desensitization. The mechanisms of desensitization have been particularly well studied using the beta(2)AR as a model system(2) . Several studies have demonstrated that the functional uncoupling of the beta(2)AR from G(s) is the consequence of its phosphorylation by one of two types of kinases(3, 4, 5, 6, 7, 8, 9) . Desensitization of agonist-occupied or activated beta(2)AR involves phosphorylation by a growing family of G protein-coupled receptor kinases, of which beta-adrenergic receptor kinase (betaARK1) is a member(3, 5, 9) . This phosphorylation serves to promote the binding of beta-arrestin to the receptor, which when bound further uncouples the receptor(10, 11, 12) . Moreover, cAMP-dependent protein kinase (PKA) phosphorylation can desensitize the beta(2)AR in response to elevated intracellular cAMP levels(6, 7, 8, 9) .

In addition to functional uncoupling of the beta(2)AR and G(s), agonist-mediated receptor internalization (sequestration) results in spatial uncoupling, such that in response to agonist plasma membrane receptors are removed to an intracellular compartment, probably into endosomes(13) . This has led to speculation that sequestration might represent a major mechanism of beta(2)AR desensitization. However, a large body of experimental evidence suggests that this is not the case, as both pharmacological manipulations and mutant receptors have been used to demonstrate that the beta(2)AR desensitizes in the absence of receptor sequestration(4, 14, 15, 16, 17) . In addition, receptor desensitization proceeds much faster than sequestration(18, 19) . Thus, sequestration mostly affects receptors that have already been uncoupled from G(s). This has led to the suggestion that receptor sequestration, rather than playing a role in receptor desensitization, might play a more important role in mediating the resensitization of desensitized receptors (14, 15, 16, 20) .

Sequestered beta(2)ARs are phosphorylated to a lesser extent than plasma membrane-associated receptors, which prompted the proposal that beta(2)AR sequestration might be triggered by phosphorylation(20) . However, further investigation of this hypothesis using phosphorylation site-deficient beta(2)AR mutants, as well as various truncated beta(2)ARs, led to the conclusion that phosphorylation was not a prerequisite of beta(2)AR sequestration(3, 9, 21, 22) . Nonetheless, recent data have renewed interest in the role of betaARK phosphorylation in receptor sequestration. Tsuga et al.(23) demonstrated that overexpression of betaARK1 could facilitate m2 muscarinic acetylcholine receptor sequestration, whereas overexpression of a dominant-negative betaARK1 could attenuate it.

Previously, we reported that mutation of tyrosine residue 326 to an alanine residue in the seventh transmembrane domain of the beta(2)AR resulted in a sequestration-defective receptor (beta(2)AR-Y326A)(15) . The beta(2)AR-Y326A mutant, while unable to sequester, could desensitize, down-regulate, and be phosphorylated in response to agonist and, although its coupling was impaired, was able to maximally stimulate adenylyl cyclase in membranes (15) . More importantly, this receptor mutant was impaired in its ability to resensitize. This suggested that the beta(2)AR-Y326A mutant might provide an excellent tool for directly testing if agonist-promoted receptor sequestration played a role in receptor dephosphorylation. However, further examination of the ability of the beta(2)AR-Y326A mutant to be phosphorylated revealed that the beta(2)AR-Y326A mutant could not be used to study receptor dephosphorylation since, while it was a substrate for PKA phosphorylation, it did not serve as a substrate for phosphorylation by G protein-coupled receptor kinases. Thus, the previously described desensitization of this mutant (15) was likely the consequence of PKA- rather than betaARK-dependent mechanisms of desensitization. Interestingly though, we have used the beta(2)AR-Y326A mutant in conjunction with betaARK1 overexpression, as well as a dominant-negative betaARK1, to demonstrate that betaARK phosphorylation can facilitate beta(2)AR sequestration.

Tsuga et al.(23) , in a recent report, suggested that the ability of betaARK1 to facilitate m2 muscarinic acetylcholine receptor sequestration implied a unique role of phosphorylation for this class of G(i)-coupled receptor distinct from the G(s)-coupled beta(2)AR. The present results demonstrate a clear role for betaARK phosphorylation in the facilitation of beta(2)AR sequestration, suggesting that phosphorylation plays a broader role in agonist-promoted G protein-coupled receptor sequestration than previously envisaged.


EXPERIMENTAL PROCEDURES

Materials

I-Pindolol and [P]orthophosphate were purchased from DuPont NEN. Isoproterenol, propranolol, forskolin, IBMX, Nonidet P-40, and bovine serum albumin were acquired from Sigma. CGP-12177 was obtained from Boehringer Mannheim. Protein A-Sepharose 4 fast flow was supplied by Pharmacia Biotech Inc. 12CA5 ascites were purchased from Babco. Human embryonic kidney cells (293 cells) were from the American Tissue Culture Collection. Gentamicin, minimal essential medium, phosphate-free Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), and fetal bovine serum were provided by Life Technologies, Inc. Restriction enzymes were obtained from Promega. Hot Tub DNA polymerase and ECL Western blotting analysis system were supplied by Amersham Corp. All other chemicals and reagents were purchased from Fisher or VWR.

Plasmid Constructions

The putative sites of PKA and betaARK phosphorylation (9, 24, 25) were created as follows. The PKA and betaARK phosphorylation site-deficient human beta(2)AR mutants described by Hausdorff et al.(3) and the 12CA5 epitope-tagged beta(2)AR and beta(2)AR-Y326A in pBC (15) were used as templates for construction of PKA and betaARK phosphorylation site-deficient beta(2)AR-Y326A mutants. In brief, as described previously(3) , Ser, Ser, Ser, and Ser were replaced by an alanine residue to create the PKA phosphorylation site-deficient beta(2)AR mutant; whereas Ser, Thr, Thr, Thr, Ser, Thr, and Ser were replaced with an alanine residue and Ser, Ser, Ser, and Ser were replaced with a glycine residue to create the betaARK phosphorylation site-deficient beta(2)AR mutant. The first mutated PKA site in the third intracellular loop of the PKA site-deficient beta(2)AR was excised with NcoI-HpaI and used to replace the identical cassette of the 12CA5 epitope tagged beta(2)AR-Y326A cloned into pBC. The second mutated PKA site was created by replacing the NcoI-FspI cassette of the PKA phosphorylation site-deficient beta(2)AR in pBC with the identical cassette from beta(2)AR-Y326A containing the first mutated PKA site. A betaARK phosphorylation site-deficient 12CA5 epitope-tagged beta(2)AR-Y326A mutant in pBC was obtained by replacing the StuI-AccI cassette containing all the potential betaARK phosphorylation sites with the same cassette from the betaARK site-deficient beta(2)AR in pBC. pcDNA1/Amp constructs were obtained by excising the entire cDNA for each of the mutants from pBC using NcoI-SalI, blunting and subcloning them into the EcoRV site of pcDNA1/Amp.

A point mutation in bovine betaARK1 (K220M) was generated by polymerase chain reaction using a 5` primer encompassing an AccI (position 614) 5` of the lysine to be mutated. The codon at positions 658-660, AAG (lysine 220), was mutated to ATG (methionine). A 3` primer located 3` to a second AccI site (position 941) was used in conjunction with the 5` primer to generate the reading frame containing both AccI sites. The DNA was cloned into pCR(TM)II (Invitrogen) according to the specifications of the manufacturer. Positive clones were isolated, and the mutation, as well as the integrity of the coding sequence between both AccI sites, was confirmed by dideoxy DNA sequencing. The AccI fragment was isolated and replaced in C betaARK1 (26) cloned in pBC. An MscI-RsrII cassette in pcDNA1/Amp C betaARK1 was replaced with the same cassette isolated from the pBC construct to create C betaARK1-K220M in pcDNA1/Amp.

Cell Culture and Transfection

293 cells were grown in minimal essential medium with Earle's salts, supplemented with heat-inactivated fetal bovine serum (10% v/v) and gentamicin (100 µg/ml). The cells were seeded at a density of 2.5 times 10^6 cells/100-mm dish and transiently transfected using a modified calcium-phosphate method(27) . Cells were transfected with either 0.05 µg/dish, for sequestration studies, or with 0.3-0.4 µg/dish, for phosphorylation studies, of human beta(2)AR receptor cDNA expressed in the pcDNA1/Amp expression vector. In studies where beta(2)AR receptor cDNA was coexpressed with bovine betaARK1 cDNA, the total amount of DNA transfected per dish was kept constant by using appropriate amounts of empty vector. Following transfection (18 h), the 293 cells were incubated with fresh culture medium and allowed to recover 7-9 h, before being reseeded in either 6- or 12-well dishes (Falcon) and allowed to grow an additional 15-18 h. Transient transfection of COS7 cells was achieved in the same manner as 293 cells except that COS7 cells were glycerol shocked prior to incubation with fresh media. Permanent transfection of Chinese hamster ovary cells (CHO) and Chinese Hamster Fibroblasts (CHW) was performed as described previously(3, 15) .

Ligand Binding

Whole cell radioligand binding studies were performed as follows. Cells seeded in 12-well dishes were washed twice with ice-cold PBS and then detached from the wells using ice-cold PBS with 5 mM EDTA. Total receptor binding was measured using saturating concentrations of I-pindolol (1 nM), which, because of its hydrophobicity, can measure both surface and intracellular receptors. Nonspecific binding was measured in the presence of 10 µM propranolol. Binding studies were done at 30 °C for 30 min for the measurement of receptor expression, and bound ligand was separated on glass fiber filters (Whatman, GF/C) by vacuum filtration. The filters were washed four times with 4 ml of cold wash buffer (50 mM Tris, 120 mM NaCl, pH 7.2) and counted in a -counter. Protein concentrations were determined using a Bio-Rad assay kit with bovine serum albumin as the standard.

Sequestration

293 cells grown in six-well dishes (Falcon) were washed twice with serum-free culture medium (37 °C). Matching wells were then treated with serum-free culture medium containing 100 µM ascorbate with or without 10 µM isoproterenol (ISO) and incubated 30 min at 37 °C. Receptor sequestration was measured as described previously (3, 15) ; in brief, the cells were washed and prepared for whole cell binding studies as described above, except that binding was performed at 14 °C for 3 h with I-pindolol in the presence or absence of either 100 nM CGP-12177 or 10 µM propranolol. Receptor sequestration was defined as the fraction of specific radioligand binding not competed for by CGP-12177 (a hydrophilic ligand) minus the basal level of sequestration as measured without exposure to agonist.

Whole Cell Phosphorylation

293 cells seeded in six-well dishes were washed twice with phosphate-free DMEM without serum (37 °C) and then labeled for 45 min at 37 °C with 0.5 ml/well of [P]orthophosphate (100 µCi/ml) in the same medium. Duplicate pairs of matching wells containing labeled cells were then treated with an additional 0.5 ml of serum- and phosphate-free DMEM containing 100 µM ascorbate with or without ISO (10 µM final concentration) and incubated at 37 °C for 15 min or for the indicated times. The cells were washed three times with ice-cold PBS and then scraped in 0.4 ml/well of radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.1 mM phenylmethysulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, pH 7.4), following which duplicate wells were combined. The cells were solubilized for 1 h at 4 °C on an inversion wheel. Supernatants were obtained by centrifugation at 436,000 times g for 15 min in a TLA 100.2 rotor in a Beckman Optima TL ultracentrifuge, following which aliquots of each sample were taken to determine total protein content in the supernatant of each sample to be immunoprecipitated. Epitope-tagged receptor was immunoprecipitated for 1 h at 4 °C on protein A-Sepharose beads using 12CA5 monoclonal antibody. The Sepharose beads were washed three times with radioimmune precipitation buffer and receptor was eluted from the beads in 50 µl of SDS sample buffer at 65 °C for 10 min. In each experiment, each lane of the SDS-polyacrylamide gel was loaded with equivalent amounts of receptor protein in a 45-µl volume. The amount of receptor in each sample was calculated as the function of receptor expression (pmol/mg of whole cell protein) times the total protein content of the solubilized fraction of each sample subjected to immunoprecipitation (i.e. (pmol receptor/mg of whole cell protein) times (mg solubilized protein/sample)= pmol receptor/sample). Receptor expression was determined as described above on cell samples for each transfection. The receptor content of each sample was normalized to the sample with the least receptor content by dilution with sample buffer. Samples were then subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. The extent of receptor phosphorylation was quantitated using a Molecular Dynamics phosphorimaging system and ImageQuant software.

Western Blotting

Samples of 293 cells resuspended in PBS with 5 mM EDTA were centrifuged at 4000 rpm for 20 min at 4 °C, pellets resuspended in SDS-sample buffer (50 µg of whole cell protein/25 µl), and sonicated 30 s. 25-µl samples were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting with a Millipore Milliblot semi-dry electroblotting system onto nitrocellulose membranes. The membranes were blocked using PBS with 3% bovine serum albumin for 1 h at room temperature and then incubated with diluted 1:4000 betaARK1/2 specific antiserum (28) in PBS with 3% bovine serum albumin and 0.15% Tween 20 (v/v). After incubation with the antiserum, the membranes were washed three times for 10 min in PBS with 0.2% Tween 20 and then incubated 1 h at room temperature with diluted 1:2000 horseradish peroxidase-conjugated donkey anti-rabbit IgG supplied with the ECL Western blotting analysis system in PBS with 3% bovine serum albumin and 0.15% Tween 20. After removal of the secondary antiserum, the membranes were washed three times in PBS with 0.2% Tween 20 and then were exposed using the ECL Western blotting analysis system.

Data Analysis

Mean and either the standard deviation or standard error of the mean are expressed for values obtained on the number of separate experiments indicated. Statistical significance was determine by both ANOVA and multiple comparisons between groups using a two-tailed t test for independent samples and found to be equivalent. The statistical significance values for the multiple comparisons made are shown in the figure legends. p values were not corrected for multiple comparisons. Phosphorylation time course data was fit to the curve Y = (R(0) times k(1)/(k(1) + k(2))) times (1 - exp(-(k(1) + k(2))) times X), where R(0) = maximal response, k(1) = forward rate constant, and k(2) = reverse rate constant, and analyzed using GraphPad Prism. Dose-response data were analyzed using GraphPad Prism.


RESULTS

Comparison of Wild-type beta(2)AR and beta(2)AR-Y326A Mutant Phosphorylation

Initial experiments tested the time course for the whole cell phosphorylation of wild-type beta(2)AR and beta(2)AR-Y326A expressed at similar levels in 293 cells, in response to 10 µM ISO stimulation (Fig. 1A). This resulted in a maximum increase in phosphorylation of 5.2 ± 1.0-fold over basal for the beta(2)AR, but only 2.6 ± 0.7-fold over basal for the beta(2)AR-Y326A mutant (a similar result was obtained for permanently transfected CHO cells, data not shown). Maximal phosphorylation of each receptor was observed within 3-5 min of agonist stimulation (t = 41 ± 14 s for the beta(2)AR versus 76 ± 13 for the beta(2)AR-Y326A, p < 0.05).


Figure 1: Comparison of the whole cell phosphorylation of wild-type beta(2)ARs and beta(2)AR-Y326A mutants. 293 cells were transfected with pcDNA1/Amp cDNA encoding beta(2)AR and beta(2)AR-Y326A and assayed for whole cell phosphorylation as described under ``Experimental Procedures.'' A, time course for the phosphorylation of wild-type beta(2)AR (725 ± 261 fmol/mg of whole cell protein) and beta(2)AR-Y326A mutant (646 ± 175 fmol/mg of whole cell protein) in response to stimulation with 10 µM ISO (15 s to 20 min). The R values for the curve fits for beta(2)AR and beta(2)AR-Y326A mutant phosphorylation time courses were 0.97 and 0.98, respectively. B, the comparison of agonist-induced (10 µM ISO) phosphorylation with forskolin (FSK)-mediated (25 µM in the presence of 1 mM IBMX) phosphorylation of the wild-type beta(2)AR (772 ± 457 fmol/mg of whole cell protein) and beta(2)AR-Y326A mutants (763 ± 385 fmol/mg of whole cell protein). In each experiment, the basal phosphorylation for each receptor type was subtracted from that seen in the presence of agonist to give the agonist-stimulated increase in the level of phosphorylation. This value, in turn, was compared with that seen with wild-type beta(2)AR in the same experiment and expressed as a percentage of the agonist-induced wild-type beta(2)AR phosphorylation, which was increased 5.7 ± 1.0-fold above basal, following 15-min stimulation with 10 µM ISO. The data from the autoradiographs were analyzed using a Molecular Dynamics phosphorimaging system. Basal phosphorylation (agonist-independent phosphorylation) of the beta(2)AR and beta(2)AR-Y326A mutant in A and B was identical as quantitated using the Molecular Dynamics phosphorimaging system. The data in each panel represent the mean ± S.D. (bars) values from four different experiments; where no error bar is shown, the S.D. was smaller than the symbol. In B, the asterisk indicates p < 0.001 versus wild-type beta(2)AR phosphorylation in response to agonist.



Although the wild-type beta(2)AR and beta(2)AR-Y326A mutant phosphorylate at somewhat different rates, this could not account for the large differences in the extent of phosphorylation observed for these two receptors. The beta(2)AR serves as substrate for phosphorylation by both PKA and betaARK(3, 4) . Therefore, the beta(2)AR-Y326A mutant was tested for its ability to be phosphorylated by these two types of kinases. This was tested in two ways. First, wild-type beta(2)ARs and beta(2)AR-Y326A mutants, expressed in 293 cells, were challenged for 15 min with either 10 µM ISO or 25 µM forskolin (in the presence of 1 mM IBMX) to stimulate adenylyl cyclase and raise intracellular cAMP levels in the presence or absence of receptor activation, respectively. These results are illustrated in Fig. 1B and are normalized to agonist-induced stimulation of wild-type beta(2)AR phosphorylation. Under these conditions, the magnitude of agonist- and forskolin-induced phosphorylation of the beta(2)Y326A mutant was identical (26 ± 5.7% and 23 ± 7.6% of wild-type beta(2)AR phosphorylation, respectively). This was equivalent to forskolin-induced phosphorylation of the wild-type beta(2)AR (21 ± 7.6%). Second, wild-type beta(2)AR and beta(2)AR-Y326A mutants were prepared in which their putative sites of PKA and betaARK phosphorylation were removed. The removal of the putative PKA and betaARK phosphorylation sites in either the wild-type beta(2)AR or beta(2)AR-Y326A did not prevent the mobilization of an adenylyl cyclase response by any of the phosphorylation site-deficient mutants ((3) ; data not shown). The agonist induced-phosphorylation of these phosphorylation site-deficient mutants is illustrated in Fig. 2, A and B. In 293 cells, the wild-type beta(2)AR was phosphorylated predominantly at betaARK phosphorylation sites (69.3 ± 15.3%) rather than PKA phosphorylation sites (22 ± 7.9%). However, the beta(2)AR-Y326A served solely as a PKA substrate, as the level of its phosphorylation was unchanged by the removal of betaARK phosphorylation sites (20 ± 7.5% versus 17 ± 2.3%). beta(2)AR-Y326A mutant phosphorylation was essentially inhibited upon the removal of its PKA phosphorylation sites (Fig. 2).


Figure 2: Phosphorylation of wild-type beta(2)AR and beta(2)AR-Y326A phosphorylation site-deficient mutants. 293 cells were transfected with pcDNA1/Amp cDNA encoding wild-type (WT), betaARK phosphorylation site-deficient (betaARK-) or PKA phosphorylation site-deficient (PKA-) beta(2)AR and beta(2)AR-Y326A and assayed for whole cell phosphorylation as described under ``Experimental Procedures.'' A, autoradiograph from a representative experiment showing the whole cell phosphorylation of each receptor following a 15-min incubation in the absence(-) or presence (+) of 10 µM ISO. Expression of each of the mutant receptors was equivalent in these experiments, and each lane was loaded with equivalent amounts of receptor protein as described under ``Experimental procedures.'' B, The mean ± S.D. (bars) for the quantitative analysis of four different experiments. In these experiments, the data were normalized to the agonist-induced wild-type beta(2)AR phosphorylation which was increased 6.6 ± 1.2-fold above basal (see legend to Fig. 1). The expression levels for each receptor (fmol/mg of whole cell protein) were as follows beta(2)AR-WT = 1725 ± 455, beta(2)AR-betaARK = 1729 ± 122, beta(2)AR-PKA = 1995 ± 183, beta(2)AR-Y326A-WT = 1665 ± 268, beta(2)AR-Y326A-betaARK = 1786 ± 128 and beta(2)AR-Y326A-PKA = 1879 ± 283. As seen in the autoradiograph there was no difference in the basal phosphorylation for any of the receptors. Asterisk, p < 0.05; double asterisks, p < 0.0005 versus agonist-stimulated wild-type beta(2)AR phosphorylation. , p < 0.01 versus agonist-stimulated wild-type beta(2)AR-Y326A phosphorylation.



Rescue of beta(2)AR-Y326A Mutant Phosphorylation and Sequestration

It was clear that the beta(2)AR-Y326A mutant, under the experimental conditions tested, did not serve as a substrate for phosphorylation at betaARK phosphorylation sites and served only as a substrate for PKA in 293 cells. However, the possibility existed that the Y326A mutation reduced the effectiveness of the activated mutant receptor as a substrate for betaARK phosphorylation. Therefore, we tested whether betaARK1 overexpression could overcome the presumed impairment as a substrate for betaARK phosphorylation of the beta(2)AR-Y326A mutant. Transfection of 293 cells with increasing amounts of betaARK1 cDNA in a pcDNA1 expression vector resulted in increased betaARK1 expression levels (Fig. 3A). As shown in Fig. 3B, betaARK1, when overexpressed, rescued the phosphorylation of the beta(2)AR-Y326A mutant, such that the extent of phosphorylation of the mutant receptor was equivalent to wild-type beta(2)AR phosphorylation if both receptors where coexpressed with 10 µg of betaARK1 cDNA. The level of agonist-induced beta(2)AR-Y326A mutant phosphorylation was increased >5-fold from 26.9 ± 2.6% to a maximum of 146 ± 36% of wild-type beta(2)AR phosphorylation (Fig. 3C). Therefore, the Y326A mutation appears to decrease the effectiveness of beta(2)AR as a substrate for betaARK phosphorylation. Overexpressed betaARK1 also increased wild-type beta(2)AR phosphorylation by 50-70% above control phosphorylation levels, indicating that the availability of betaARK in these cells may be limiting in some fashion.


Figure 3: Effect of the overexpression of betaARK1 on beta(2)AR and beta(2)AR-Y326A mutant phosphorylation. 293 cells were transfected with 0, 1, 2.5, or 10 µg of betaARK1 cDNA in a pcDNA1 expression vector along with pcDNA1/Amp cDNA either coding for wild-type beta(2)AR (1237 ± 142 fmol/mg of whole cell protein) or beta(2)AR-Y326A (2071 ± 250 fmol/mg of whole cell protein) and assayed for whole cell phosphorylation as described under ``Experimental Procedures.'' A, immunoblot of the overexpression of betaARK1 protein with increasing amounts of transfected betaARK1/2 cDNA. The far right lane shows the appropriate migration of 10 ng of purified betaARK1. Two major molecular weight species were detected with betaARK1/2 antibody, when betaARK1 was overexpressed using the pcDNA1/Amp expression vector. The lower band migrates at the same molecular weight as the purified betaARK1. The upper band is presumed to represent improperly processed betaARK1 resulting from its overexpression in 293 cells. Nontransfected 293 cells express both betaARK1 and betaARK2 but cannot be easily visualized in the immunoblot without saturating the lanes containing samples of cells overexpressing betaARK1. B, autoradiograph showing a representative whole cell phosphorylation of wild-type beta(2)AR and beta(2)AR-Y326A following coexpression with increasing amounts of betaARK1 following incubation for 15 min in the absence(-) or presence (+) of 10 µM ISO. Each lane was loaded with equivalent amounts of wild-type and Y326A mutant receptor protein as described under ``Experimental procedures.'' The major species of the beta(2)AR expressed in 293 cells is a glycoprotein of a molecular mass ranging from 56 to 85 kDa. Immunoblots with biotinylated 12CA5 identified each of the major phosphorylated bands (data not shown). C, the mean ± S.D. (bars) of the quantitative analysis of three different experiments. In these experiments, the data were normalized to the agonist-induced wild-type beta(2)AR phosphorylation, which was increased 4.2 ± 1.4-fold above basal (see legend to Fig. 1). The basal phosphorylation of either receptor was unaffected by the coexpression of increasing amounts of betaARK1. p < 0.05 (asterisk) or p < 0.001 (double asterisks) versus control agonist-stimulated beta(2)AR phosphorylation.



Since the beta(2)AR-Y326A mutant phosphorylation deficit could be reversed by overexpressing betaARK1, we reasoned that agonist-promoted sequestration might also be influenced by the overexpression of betaARK1. Under control conditions, 42 ± 3.1% of wild-type beta(2)ARs and 5 ± 1.9% of beta(2)AR-Y326A mutants sequestered in response to 10 µM ISO stimulation (Fig. 4). However, as shown in Fig. 4, the increase in betaARK1 expression (Fig. 3A) produced a progressive rescue of beta(2)AR-Y326A mutant sequestration essentially back to wild-type beta(2)AR levels. Overexpression of betaARK1 had no effect on the sequestration of the wild-type beta(2)AR.


Figure 4: Effect of the overexpression of betaARK1 on beta(2)AR and beta(2)AR-Y326A mutant sequestration. 293 cells were transfected with 0, 1, 2.5, or 10 µg of betaARK1 cDNA in a pcDNA1 expression vector along with pcDNA1/Amp cDNA either coding for wild-type beta(2)AR (967 ± 85 fmol/mg of whole cell protein) or beta(2)AR-Y326A mutant (812 ± 94 fmol/mg of whole cell protein) and assayed for agonist-promoted sequestration as described under ``Experimental Procedures.'' Basal sequestration of both the beta(2)AR and beta(2)AR-Y326A mutant were unaffected by coexpression with increasing amounts of betaARK1. Basally sequestered receptors represented 27 ± 4% and 31 ± 2.4% of total cellular beta(2)AR and beta(2)AR-Y326A, respectively, in these experiments. The data represents the mean ± S.E. (bars) for three different experiments. p < 0.01 (asterisk) or p < 0.0001 (double asterisks) versus control agonist-promoted beta(2)AR sequestration. p < 0.005 () or p < 0.0005 () versus control agonist-promoted beta(2)AR-Y326A mutant sequestration.



In addition to 293 cells, the ability of the beta(2)AR-Y326A mutant to sequester was also tested in CHO, CHW, and COS7 cells. In each case, the sequestration of the beta(2)AR-Y326A mutant was impaired to an equivalent or greater extent than reported here for its sequestration in 293 cells, indicating that its inability to sequester in response to agonist was not the consequence of the cellular environment in which it was tested (data not shown).

Role of Phosphorylation in Sequestration

betaARK1 rescued both the phosphorylation and sequestration of the beta(2)AR-Y326A mutant. The idea that betaARK-mediated receptor phosphorylation might facilitate agonist-promoted sequestration was confirmed by two approaches. First, we tested the ability of betaARK1 overexpression to rescue the sequestration of phosphorylation site-deficient beta(2)AR-Y326A mutants. Second, a dominant-negative betaARK1 was assessed for its ability to inhibit both phosphorylation and sequestration of wild-type beta(2)AR.

For these experiments, geranylgeranylated (C isoprenylated) versions of betaARK1 or betaARK1-K220M (C betaARK1) in pcDNA1/Amp were used. Previously, Kong et al.(29) demonstrated that betaARK1-K220R did not inhibit beta(2)AR sequestration. However, this dominant-negative betaARK1 mutant appeared to be impaired in its interaction with G protein beta subunits(29) . Isoprenylation of betaARK1 directly targets the cytosolic kinase to the membrane, without the need for coupling of the receptor to G(s), resulting in the subsequent dissociation of G which then mediates translocation of betaARK to the plasma membrane(5, 26, 30) . This suggested that a C isoprenylated dominant-negative betaARK1 might be more effective at inhibiting beta(2)AR phosphorylation and sequestration. When tested, C BARK1, like wild-type betaARK1, could rescue both wild-type beta(2)AR-Y326A mutant sequestration (compare Fig. 4and Fig. 5) and phosphorylation (data not shown). C betaARK1 was used to test the rescue of phosphorylation site-deficient beta(2)AR and beta(2)AR-Y326A mutants. Removal of PKA phosphorylation sites had no effect on the sequestration of the wild-type beta(2)AR, but removal of betaARK phosphorylation sites reduced agonist-induced sequestration by 50%, again hinting toward a potential role for phosphorylation in the process. Overexpression of C betaARK1 was unable to rescue the sequestration of the betaARK phosphorylation-deficient beta(2)AR. In the absence of C betaARK1 overexpression, all of the beta(2)AR-Y326A mutants were impaired in their ability to sequester. However, overexpression of C betaARK1 rescued the sequestration of the wild-type beta(2)AR-Y326A and PKA phosphorylation site-deficient beta(2)AR-Y326A mutants, but was unable to rescue the sequestration of the betaARK phosphorylation site-deficient beta(2)AR-Y326A mutant.


Figure 5: Effect of C betaARK1 on the sequestration of wild-type beta(2)AR and beta(2)AR-Y326A phosphorylation site-deficient mutants. 293 cells were transfected with 1 µg of pcDNA1/Amp C betaARK1 cDNA along with pcDNA1/Amp cDNA encoding one of the following: wild-type (WT), betaARK phosphorylation site-deficient (betaARK-), or PKA phosphorylation site-deficient (PKA-) beta(2)AR and beta(2)AR-Y326A and assayed for agonist-promoted sequestration as described under ``Experimental Procedures.'' The basal sequestration of each of the receptors was equivalent and was not affected by coexpression of 1 µg of C betaARK1 cDNA. Basally sequestered receptors represented 30 ± 1.8% of total cellular receptors in these experiments. The expression levels for each receptor (fmol/mg of whole cell protein) were as follows beta(2)AR-WT = 1102 ± 173, beta(2)AR-betaARK = 1108 ± 243, beta(2)AR-PKA = 1440 ± 252, beta(2)AR-Y326A-WT = 757 ± 112, beta(2)AR-Y326A-betaARK = 795 ± 161 and beta(2)AR-Y326A-PKA = 951 ± 161. The data represent the mean ± S.E. (bars) for three to five different experiments. p < 0.05 (asterisk) or p < 0.005 (double asterisks) versus agonist-promoted wild-type beta(2)AR sequestration. p < 0.05 () or p < 0.005 () versus agonist-promoted wild-type beta(2)AR-Y326A mutant sequestration.



A kinase mutant with a methionine residue substituted for lysine 220 in the catalytic domain of C betaARK1 (C betaARK1-K220M) was overexpressed using a pcDNA1/Amp expression vector (Fig. 6A) and tested for its ability to inhibit agonist-induced wild-type beta(2)AR phosphorylation (Fig. 6B) and sequestration (Fig. 7). Agonist-induced phosphorylation of the beta(2)AR was inhibited significantly following cotransfection with either 1 µg (31 ± 11%, p < 0.01) or 2.5 µg (40 ± 6.8%, p < 0.001) of dominant-negative kinase DNA, but beta(2)AR-Y326A mutant phosphorylation was unaffected (Fig. 6C). Cotransfection with larger amounts of DNA did not further enhance the inhibition of wild-type beta(2)AR phosphorylation by the dominant-negative kinase. Shown in Fig. 7is a dose-response curve for agonist-promoted wild-type beta(2)AR sequestration in either the presence or absence of 1 µg of cotransfected C betaARK1-K220M cDNA. Overexpression of C betaARK1-K220M significantly reduced the maximal response for sequestration, 25 ± 5.8% (p < 0.05). Expression of the dominant-negative kinase had no effect on the EC for sequestration. The EC values in the presence and absence of C betaARK1-K220M were 11 ± 2.9 nM and 12 ± 1.7 nM, respectively. Under similar conditions, beta(2)AR-Y326A mutant sequestration was unaffected by C betaARK1-K220M, which is presumably consistent with the inability of C betaARK1-K220M to rescue phosphorylation of the mutant receptor (data not shown).


Figure 6: The effect of C betaARK1-K220M overexpression on the phosphorylation of wild-type beta(2)AR and beta(2)AR-Y326A mutant. 293 cells were transfected with 0, 1, or 2.5 µg of pcDNA1/Amp C betaARK1-K220M cDNA along with pcDNA1/Amp cDNA coding for wild-type beta(2)AR (1409 ± 104 fmol/mg of whole cell protein) or beta(2)AR-Y326A (994 ± 164 fmol/mg of whole cell protein) and assayed for whole cell phosphorylation as described under ``Experimental Procedures.'' A, immunoblot of the overexpression of C betaARK1-K220M protein with increasing amounts of transfected cDNA. Two major molecular weight species were also detected with betaARK1/2 antibody, when C betaARK1-K220M was overexpressed using the pcDNA1/Amp expression vector (see Fig. 3legend). B, autoradiograph showing a representative whole cell phosphorylation of wild-type beta(2)AR following coexpression with increasing amounts of C betaARK1-K220M incubated for 15 min in the absence(-) or presence (+) of 10 µM ISO. Each lane was loaded with equivalent amounts of receptor protein as described under ``Experimental procedures.'' C, the mean ± S.D. (bars) of the quantitative analysis of four different experiments. In these experiments, the data were normalized to the agonist-induced wild-type beta(2)AR phosphorylation which was increased 5.1 ± 1.9-fold over basal (see legend to Fig. 1). The basal phosphorylation of either receptor was unaffected by the coexpression of increasing amounts of C betaARK1-K220M. p < 0.01 (asterisk) or p < 0.001 (double asterisks) versus control agonist-stimulated beta(2)AR phosphorylation.




Figure 7: Dose response for the inhibition of wild-type beta(2)AR sequestration by C betaARK1-K220M. 293 cells were transfected with pcDNA1/Amp cDNA either coding wild-type beta(2)AR with (1435 ± 319 fmol/mg of whole cell protein) or without (1345 ± 150 fmol/mg of whole cell protein) 1 µg of pcDNA1/Amp C betaARK1-K220M cDNA and assayed for agonist-promoted sequestration as described under ``Experimental Procedures'' except that sequestration was measured following 30-min stimulation with 10 to 10M ISO. The basal sequestration of the beta(2)AR in these experiments in the presence or absence C betaARK1-K220M cDNA was 33 ± 1.2% and 30 ± 0.8%, respectively. The data represent the mean ± S.E. (bars) for three different experiments. The R values for the curve fits in the presence and absence of C betaARK1-K220M were 0.96 and 0.98, respectively.




DISCUSSION

The present experiments clearly demonstrate a role for betaARK-mediated phosphorylation in facilitating beta(2)AR sequestration. This idea is supported by three observations. First, betaARK1, when overexpressed, rescues both the phosphorylation and the sequestration of the beta(2)AR-Y326A mutant which was defective in its ability to be phosphorylated by betaARK and to sequester in response to agonist stimulation. Second, overexpressed betaARK1 can rescue the sequestration of PKA phosphorylation site-deficient, but not betaARK phosphorylation site-deficient, beta(2)AR-Y326A mutants. Third, sequestration of the wild-type beta(2)AR can be attenuated by overexpressing a dominant-negative betaARK1, which also diminishes agonist-induced phosphorylation of the receptor to a similar extent. These results are in agreement with the work of Tsuga et al.(23) where they describe the ability of betaARK1 to augment m2 muscarinic acetylcholine receptor sequestration.

The conclusion that betaARK1 phosphorylation is involved in beta(2)AR sequestration evolved from experiments testing the role of sequestration in receptor dephosphorylation using the beta(2)AR-Y326A mutant. Upon investigation, we found that the phosphorylation of the receptor mutant was reduced and that it served predominantly as a substrate for PKA-mediated phosphorylation, when expressed in 293 cells. This result indicated that the beta(2)AR-Y326A mutant could not be used to study receptor dephosphorylation. Nonetheless, these results suggested that mutation of tyrosine residue 326 to an alanine not only inhibits the ability of the beta(2)AR to sequester but also abolished its ability to act as a substrate for betaARK phosphorylation. Therefore, the desensitization of the beta(2)AR-Y326A mutant previously reported (15) was likely the consequence of PKA- rather than betaARK-mediated phosphorylation of the receptor, since both mechanisms have been demonstrated to effectively desensitize the wild-type beta(2)AR(3, 4) .

Historically, most studies have found that receptor phosphorylation was not essential for beta(2)AR sequestration. In particular, Hausdorff et al.(3) demonstrated that neither betaARK nor PKA phosphorylation sites were required for sequestration of beta(2)AR stably expressed by CHW cells. In addition, truncation of the carboxyl tail of the hamster beta(2)AR, which removes its putative betaARK phosphorylation sites, resulted in normal sequestration when expressed in mouse L cells(22) , although further truncation of this receptor did result in some impairment of sequestration(31) . Finally, Lohse et al.(4) , using permeabilized A431 cells, demonstrated that sequestration was unaffected by inhibitors of either PKA and betaARK phosphorylation. Indeed, in the present study, beta(2)ARs lacking putative PKA and betaARK phosphorylation sites, when expressed in 293 cells, also sequestered in response to agonist exposure, although the beta(2)ARs lacking putative betaARK phosphorylation sites were somewhat impaired in their sequestration (50% of control). The observed impairment in the sequestration of beta(2)ARs lacking putative betaARK phosphorylation sites might be related to the fact that we have used transient transfections in the present study to examine their sequestration, whereas Hausdorff et al.(3) selected permanently transfected clonal cell lines which might vary in their sequestration properties depending upon the clone selected. Nonetheless, these mutant receptors do sequester in response to agonist stimulation in 293 cells in the absence of betaARK-mediated phosphorylation which is consistent with previously described work. We have tested the ability of the beta(2)AR-Y326A mutant to sequester in four different cell lines (CHO, 293, CHW, and COS7) and in each case the mutant was impaired in its ability to sequester. This suggests that the inability of this receptor to sequester and be phosphorylated by betaARK is likely an intrinsic property of the beta(2)AR-Y326A mutant receptor rather than the consequence of the cell type in which it has been expressed

It is likely that the intrinsic properties of the beta(2)AR-Y326A mutant have allowed us to uncover a previously unappreciated role for phosphorylation in the beta(2)AR sequestration process. The results suggest that phosphorylation of the beta(2)AR by betaARK is facilitory rather than required for sequestration. betaARK1 phosphorylation of the beta(2)AR-Y326A mutant effectively rescues its complete lack of sequestration, whereas removal of the betaARK phosphorylation sites in the wild-type beta(2)AR only reduces sequestration by 50% in 293 cells. This indicates that the basis for the sequestration impairment of the beta(2)AR-Y326A mutant goes beyond a simple lack of phosphorylation. We suggest that mutation of tyrosine residue 326 to an alanine alters the ability of the agonist-occupied receptor to achieve and/or maintain a conformational state required for receptor phosphorylation by betaARK as well as agonist-promoted sequestration. In fact, a smaller proportion of beta(2)AR-Y326A mutant receptors exhibit high-affinity agonist binding(15) . The isomerization of the receptor from its low- to high-affinity state (R R*) might serve to trigger such a change in receptor conformation. Initiation of beta(2)AR sequestration requires the occupancy of the receptor with agonist; antagonist occupancy is not sufficient. Previous results with a cyc variant line of S49 lymphoma cells, which lack functional G(s), provide support for the idea that agonist occupancy is sufficient for both homologous desensitization (betaARK-dependent phosphorylation) and sequestration in the absence of coupling to adenylyl cyclase(32) . Certainly, constitutively active receptors which achieve R* in the absence of agonist occupancy are both constitutively phosphorylated and desensitized(33) . Thus, an impairment in the active conformation of the beta(2)AR-Y326A mutant may explain a lack of phosphorylation by the normal endogenous complement of G protein-coupled receptor kinase in 293 cells, but that overexpression of betaARK1 in these cells can overcome this deficit. In fact, Ungerer et al.(34) have suggested that in the heart betaARK might be the limiting component in beta-adrenergic receptor desensitization. This might explain why overexpression of betaARK leads to increased phosphorylation of the wild-type beta(2)AR in several cell lines.

The observation that beta(2)ARs lacking putative betaARK phosphorylation sites can sequester in response to agonist stimulation clearly indicates that betaARK-mediated phosphorylation neither serves as the signal initiating the sequestration process nor is it an absolute requirement. Instead, we hypothesize that betaARK phosphorylation either stabilizes the conformation of the receptor or promotes the interaction of the receptor with some as yet unidentified cellular element that mediates beta(2)AR internalization, even in the absence of betaARK phosphorylation. Since arrestins appear to be required for desensitization, and phosphorylation leads to increased affinity of rhodopsin, beta(2)AR, and m2 muscarinic receptor for members of the arrestin family(11, 35, 36, 37, 38) , it is tempting to speculate that arrestins might also play a role in sequestration as they have also been shown to interact with agonist-occupied nonphosphorylated receptor, albeit less effectively(35, 36, 37, 38) .

The dominant-negative betaARK1-K220M, while able to inhibit both the phosphorylation and sequestration of beta(2)AR to equivalent extents in 293 cells, was not overwhelmingly effective at inhibiting either process, 31 ± 11% and 25 ± 6%, respectively. However, a modest effect of betaARK1-K220M on the sequestration of the wild-type beta(2)AR might be expected, since complete blockade of betaARK phosphorylation should lead at most to a 50% decrease in sequestration (see Fig. 5, betaARK phosphorylation site-deficient beta(2)AR mutant). In two recent studies(23, 29) , dominant-negative betaARKs have been tested for their ability to affect the sequestration and phosphorylation of beta(2)AR and m2 muscarinic acetylcholine receptors. Kong et al.(29) reported that betaARK-K220R, while able to inhibit in vitro beta(2)AR phosphorylation, was impaired in its G targeting and had no effect on sequestration. However, Tsuga et al.(23) demonstrated that betaARK1-K220W could inhibit both the phosphorylation and sequestration of the m2 muscarinic acetylcholine receptor subtype, but this was dependent upon the level of endogenously expressed kinase. In the present study, a similar result was obtained for C betaARK1-K220M, which attenuated both whole cell phosphorylation and sequestration of the beta(2)AR. The apparent discordance in the efficacy of a particular dominant-negative betaARK mutant to inhibit sequestration might be dependent on the nature of the residue substituted for lysine 220 or the cellular background in which they are tested. In the work by Tsuga et al.(23) , the authors concluded that betaARK-facilitated sequestration was unique to the G(i)-coupled m2 muscarinic receptor, since putative betaARK phosphorylation sites are likely found in the third intracellular loop of this receptor rather than in the short cytoplasmic tail(39) . Our findings indicate that, in contrast to what is described in the literature for the beta(2)AR(3, 4, 21, 22) , a role for betaARK phosphorylation in facilitating receptor sequestration might be more general.

Other studies also support the idea that phosphorylation might be important for sequestration of the beta(2)AR. For example, the beta(3)AR does not sequester or phosphorylate in response to agonist stimulation, yet, replacing its carboxyl tail with the tail of the beta(2)AR rescues both sequestration and phosphorylation of the chimeric beta(3)/beta(2)AR(40) . In addition, the sequestration of other G protein-coupled receptors, such as the angiotensin II, neurotensin, and alpha-adrenergic receptors, as well as the receptor for parathyroid hormone and parathyroid hormone-related protein, are inhibited by the truncation of their carboxyl tails(41, 42, 43, 44) . However, the possibility exists that specific sequestration motifs might exist in the tails of these receptors. Interestingly though, a regulatory sequence identified in the tail of the receptor for parathyroid hormone and parathyroid hormone-related protein contains several serine and threonine residues that might act as potential betaARK phosphorylation sites(42) .

In summary, the present studies demonstrate a clear role for betaARK1-mediated phosphorylation in the facilitation of beta(2)AR sequestration. It will be of interest to determine whether this property is unique to betaARK1 or if phosphorylation by other members of the G protein-coupled receptor kinase family can promote beta(2)AR sequestration as well. We suggest that betaARK phosphorylation facilitates, rather than initiates, sequestration as a betaARK phosphorylation site-deficient beta(2)AR mutant can sequester, albeit not normally. betaARK phosphorylation has now been shown to facilitate the sequestration of two different G protein-coupled receptors, indicating that phosphorylation plays a broader role in agonist-promoted receptor sequestration than originally envisaged.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS 19576 (to M. G. C.). 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.

§
These authors contributed equally to this work.

Fellow of the Medical Research Council of Canada.

**
Recipient of a Howard Hughes postdoctoral fellowship.

§§
To whom correspondence should be addressed: Duke University Medical Center, Box 3287, Durham, NC 27710. Tel.: 919-684-5433; Fax.: 919-681-8641.

(^1)
The abbreviations used are: beta(2)AR, beta(2)-adrenergic receptor; PKA, cAMP-dependent protein kinase; betaARK1, beta-adrenergic receptor kinase 1; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; 293 cells, human embryonic kidney cells; CHW, Chinese hamster fibroblasts; CHO, Chinese hamster ovary cells; IBMX, 1-methyl-3-isobutylxanthine; ISO, isoproterenol.


ACKNOWLEDGEMENTS

We especially thank Dr. Robert J. Lefkowitz for his continued encouragement and support and his laboratory for making available betaARK1/2 antisera, purified betaARK protein, wild-type and phosphorylation mutant beta(2)-adrenergic receptor, as well as wild-type and C-betaARK1/2. We also thank Dr. Mario Tiberi for his constructive input into the preparation of this manuscript and Lucie Bertrand for expert assistance in providing cells.


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