©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Four Consecutive Serines in the Third Intracellular Loop Are the Sites for -Adrenergic Receptor Kinase-mediated Phosphorylation and Desensitization of the -Adrenergic Receptor (*)

(Received for publication, October 25, 1994; and in revised form, December 13, 1994)

Margaret G. Eason (1) Sandra P. Moreira (1) Stephen B. Liggett (1) (2) (3)(§)

From the  (1)Departments of Pulmonary Medicine, (2)Molecular Genetics, and (3)Pharmacology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0564

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

During short term agonist exposure, the alpha-adrenergic receptor (alphaAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the beta-adrenergic receptor kinase (betaARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to betaARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for betaARK phosphorylation are not known.

To delineate such a motif in the human alphaAR (alpha(2)C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 ± 7% and 57 ± 8% of the phosphorylation found for wild type alpha(2)C10. Additional substitution mutants (SSSA and SAAA) underwent 77 ± 1% and 27 ± 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type alpha(2)C10 by 25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonistpromoted desensitization.

Thus, betaARK-mediated phosphorylation of alpha(2)C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonistpromoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for betaARK-mediated phosphorylation of alpha(2)C10, suggesting that these receptors may also be substrates for betaARK.


INTRODUCTION

alpha(2)-Adrenergic receptors (alpha(2)AR) (^1)are present in a large variety of central and peripheral tissues and are known to mediate such diverse physiological functions as platelet aggregation, inhibition of lipolysis and insulin secretion, autoregulation of neurotransmitter release, and modulation of vascular tone(1) . As members of the superfamily of G-protein coupled receptor family, alpha(2)AR share a number of conserved structural features including an extracellular amino terminus, an intracellular carboxyl terminus, and seven transmembrane-spanning domains linked by three extracellular loops and three intracellular loops(2) . Studies that have used chimeric or mutated forms of G-protein coupled receptors have demonstrated the involvement of the transmembrane domains in ligand binding (3) and the cytosolic domains in G-protein coupling (3) and regulatory processes such as receptor sequestration, down-regulation, and phosphorylation(4) .

Receptor phosphorylation by a variety of kinases is one mechanism by which the phenomenon of desensitization, or refractoriness, of the stimulated receptor-mediated response develops during conditions of prolonged agonist exposure. There are essentially two classes of kinases which are involved in the phosphorylation of G-protein coupled receptors: those that are activated by second messenger production (e.g. cAMP-dependent protein kinases and protein kinase C) and those that are second messenger-independent (e.g. G-protein coupled receptor kinases). G-protein coupled receptor kinases phosphorylate the agonist-occupied form of G-protein coupled receptors at serine and threonine residues in the intracellular domains, typically the third intracellular loop or carboxyl terminus(5) . One such kinase, the beta-adrenergic receptor kinase (^2)or betaARK, although originally named for its first defined role in the phosphorylation and desensitization of the beta(2)AR(8) , has been shown to phosphorylate a number of G-protein coupled receptors including the muscarinic m(2) receptor(9) , the substance P receptor(10) , the adenosine A(1) receptor(11) , the thrombin receptor(12) , and germane to this report, the cloned human alphaAR, alpha(2)C10(14, 15, 16) .

Studies from our laboratory (15, 17, 18) and others(14, 16, 19) strongly support a direct role of betaARK-mediated phosphorylation in agonist-promoted desensitization of alpha(2)C10. However, as with other G-protein coupled receptors that are phosphorylated by betaARK, the precise serine or threonine residues in alpha(2)C10 which are phosphorylated are not known. This is largely because of the lack of a defined consensus sequence for betaARK-mediated phosphorylation. For the most part, attempts to delineate discrete residues within a receptor which are phosphorylated by betaARK have been limited to the prototypical substrate, the beta(2)AR. However, despite extensive deletion (20) and substitution (20, 21, 22, 23) mutagenesis studies, the sites for betaARK-mediated phosphorylation for the beta(2)AR remain unidentified. Moreover, in an examination of the intracellular portions of the receptors that have been definitively shown to be phosphorylated by betaARK, there are typically numerous serine and threonine residues and a low amino acid sequence homology between receptors.

Previously, we have shown that removal of 75 amino acids(257-332), including nine serine and threonine residues, in the middle of the third intracellular loop of alpha(2)C10 ablates the rapid component of agonist-promoted desensitization and receptor phosphorylation(15) , indicating that the sites for betaARKmediated phosphorylation are found within this region. Of the nine serine and threonine residues, four serines occur consecutively and are preceded by two glutamic acid residues (EESSSS). Using a series of synthetic peptides in a reconstituted phospholipid vesicle system with purified betaARK, Onorato et al.(19) showed that betaARK preferentially phosphorylates serines preceded by amino-terminal acidic residues, with the most preferred sequence being EXS. Consequently, we considered that these aforementioned four consecutive serines (Ser-296-299) might serve as sites for betaARK-mediated phosphorylation of alpha(2)C10. To explore this possibility, six mutant alpha(2)C10 receptors lacking all or some combination of the four serines were constructed using site-directed mutagenesis, recombinantly expressed in clonal cell lines, and assessed for their ability to undergo the processes of agonist-promoted desensitization and betaARK-mediated phosphorylation.


EXPERIMENTAL PROCEDURES

cDNA Constructs and Mutagenesis

The human alpha(2)C10 cDNA construct in the mammalian expression vector pBC12BI has been described previously(13) . This construct was used as a template for mutagenesis and for subsequent transient and permanent transfection of the mutant and wild type alpha(2)C10 cDNA into CHO and COS-7 cells as described below. A mutated alpha(2)C10 in which amino acid residues 293-304 (denoted as DEL 293-304) were deleted was constructed using a polymerase chain reaction-based technique as described(15) . Briefly, oligonucleotide primers corresponding to unique BspEI and SacII restriction sites (located at amino acid residues 235-236 and 333-334, respectively) were utilized in combination with two mutagenic primers encoding two new AgeI restriction sites (located at amino acids 292-293 and 303-304, respectively) to allow for polymerase extension of a BspEI/AgeI fragment and an AgeI/SacII fragment. These fragments, when digested and ligated into the alpha(2)AR-pBC12BI construct, eliminate the nucleotides encoding amino acids 293-304. For construction of the substitution mutants denoted as AAAA, AASS, SSAA, SAAA, and SSSA, a BglII/KpnI fragment of alpha(2)C10 was cloned into the phage vector M13, and oligonucleotide-directed mutagenesis was carried out by the method of Kunkel et al.(24) . Mutated fragments were digested with BspEI and KpnI and ligated back into digested alpha(2)C10-pBC12BI. All mutations were verified by dideoxy sequencing. The construct consisting of bovine betaARK in pBC12BI was as described by Benovic et al.(6) .

Transfection and Cell Culture

For transient expression of wild type and mutant alpha(2)AR, COS-7 cells in monolayers at 30-50% confluence were transfected with 10 µg each of the wild type or mutant alpha(2)AR constructs and the betaARK construct via the DEAE-dextran method as described(25) . COS-7 cells transfected with this method routinely expressed mutant and wild type alpha(2)C10 at levels of 10-12 pmol/mg. Transiently transfected COS-7 cells were then used for experiments 48 h following transfection. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a 5% CO(2) atmosphere. For permanent expression of wild type and mutant alpha(2)AR, CHO cells in monolayers at 30% confluence were cotransfected using a calcium precipitation method (25) . Cells were transfected with 3 µg of pSV(2)neo, which provides for G-418 resistance, and 20-40 µg of wild type or mutant alpha(2)AR constructs. Clones were screened for expression of wild type or mutant alpha(2)AR using a [^3H]yohimbine binding assay as described below. CHO cells were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 80 µg/ml G-418 (to maintain selection pressure) at 37 °C in a 5% CO(2) atmosphere. For experiments using permanently expressed mutant and wild type alpha(2)AR in CHO cells, cells in monolayers at 90% confluence were utilized. Multiple clones expressing each receptor were studied.

Desensitization Conditions and Membrane Preparation

CHO cells expressing wild type or mutant alpha(2)AR at 90% confluence in monolayers were washed twice with phosphate-buffered saline (PBS) and fresh Ham's F-12 medium without fetal bovine serum was added. Epinephrine at a final concentration of 100 µM was added, and cells were incubated for the indicated times at 37 °C in a 5% CO(2) atmosphere. To prevent oxidation of epinephrine, 100 µM ascorbic acid was added to all flasks, including controls. Incubations were terminated by placing flasks on ice and washing five times with ice-cold PBS. Membranes were prepared by hypotonic lysis in ice-cold buffer (5 mM Tris, 2 mM EDTA, pH 7.4) and scraping with a rubber policeman, followed by centrifugation at 42,000 times g for 10 min at 4 °C. The crude membrane pellets were then resuspended in the appropriate buffer for use in the assays.

Adenylyl Cyclase Assay

Adenylyl cyclase activities were determined in the presence of buffer alone (basal activity), 1.0 µM forskolin, or 1.0 µM forskolin with the indicated concentrations of agonist via the method of Salomon et al.(26) as modified(17) . Briefly, membranes from CHO cells expressing the wild type and mutant alpha(2)AR, which had been exposed to either media alone or 100 µM epinephrine for 30 min, were prepared as described above and resuspended in a buffer that provided for a final assay concentration of 25 mM NaCl, 1.6 mM MgCl(2), 0.8 mM EDTA, and 40 mM HEPES, pH 7.4. Since alpha(2)AR expressed in CHO cells can also couple to G(s) and result in a stimulation of adenylyl cyclase at high agonist concentrations in the assay(27, 28) , cells were pretreated with 20 µg/ml cholera toxin as described (27) for 24 h prior to agonist exposure to ablate G(s) coupling. Membranes (20-30 µg) were incubated in the above buffer with 2.7 mM phosphoenolpyruvate, 50 µM GTP, 0.1 mM cAMP, 0.12 mM ATP, 50 µg/ml myokinase, 0.05 mM ascorbic acid, and 1.0 µCi of [alpha-P]ATP for 45 min at 37 °C. Reactions were stopped by the addition of 1.0 ml of an ice-cold solution containing [^3H]cAMP (100,000 dpm) and excess ATP and cAMP. [P]cAMP and [^3H]cAMP were isolated by sequential chromatography over Dowex and alumina columns, with [^3H]cAMP used to quantitate individual column recovery. Dose-response data were analyzed using iterative least squares techniques(29) . Agonist-promoted desensitization of wild type alpha(2)C10 expressed in CHO cells under the above conditions typically is manifested by a 5-fold increase in the EC for epinephrine-mediated inhibition of adenylyl cyclase activity, without a change in the maximal extent of inhibition(17) . The relative overexpression of mutant and wild type alpha(2)C10 in the current studies probably accounts for this degree of desensitization as compared to the more robust desensitization reported in lower expressing cells(30, 31) .

Radioligand Binding Assays

Expression levels of wild type and mutant alpha(2)AR were determined using a [^3H]yohimbine binding assay. Membranes prepared as described above were incubated with 25 nM [^3H]yohimbine in the absence or presence of 10 µM phentolamine, which was used to define nonspecific binding, in a buffer containing 75 mM Tris, pH 7.4, 12.5 mM MgCl(2), and 2 mM EDTA for 30 min at 37 °C. Specific binding was normalized for protein. Competition studies were performed by incubation of membranes with 6 nM [^3H]yohimbine in the presence of 12 concentrations of agonist, ranging from 10 nM to 1 mM, in a 50 mM Tris, 10 mM MgSO(4), pH 7.6, 0.5 mM EDTA buffer for 30 min at room temperature in both the absence and presence of 100 µM GTP. Saturation binding studies were carried out by incubation of membranes with various concentrations of [^3H]yohimbine ranging from 0.5 to 30 nM, in the absence and presence of 10 µM phentolamine in a 25 mM glycylglycine buffer, pH 7.4, for 30 min at room temperature. Determination of agonist-promoted receptor sequestration was carried out using a whole cell [^3H]yohimbine binding assay at 4 °C exactly as described(17) . For all radioligand binding assays, reactions were terminated by dilution with ice-cold 10 mM Tris, pH 7.4, followed by vacuum filtration through Whatman GF/C glass fiber filters.

Receptor Phosphorylation Studies

Whole cell phosphorylation assays were carried out using COS-7 cells transiently coexpressing betaARK with the wild type or one of the mutated alpha(2)C10 receptors. For these studies, cells in monolayers were washed twice with PBS, placed in fresh serum-free medium containing 0.3 Ci/ml P(i), and incubated for 2 h at 37 °C in a 5% CO(2) atmosphere. Cells were then treated with vehicle or a 10 µM concentration of the alpha(2)AR agonist UK-14304 for 15 min. Incubations were terminated by placing flasks on ice and washing five times with ice-cold PBS. Membranes were prepared by scraping with a rubber policeman in an ice-cold hypotonic lysis buffer (10 mM Tris, pH 7.4 at 4 °C, 5 mM EGTA, 5 mM EDTA) supplemented with phosphatase inhibitors (10 mM sodium pyrophosphate and 10 mM NaF) and protease inhibitors (10 µg/ml soybean trypsin inhibitor, 10 µg/ml benzamidine, and 5 µg/ml leupeptin), followed by centrifugation at 40,000 times g. Crude membrane pellets were then resuspended in the above lysis buffer, sonicated for 15 s, and then repelleted by centrifugation at 40,000 times g. For solubilization, membrane fractions were resuspended in PBS with 1% Triton X-100, 0.05% SDS, 1 mM EDTA, 1 mM EGTA, and the aforementioned phosphatase and protease inhibitors and stirred for 2 h at 4 °C. Samples were cleared of unsolubilized material by centrifugation at 40,000 times g. Receptors were purified via immunoprecipitation with antisera directed against alpha(2)C10 as described previously(15) . Solubilized material was first rotated at room temperature with preimmune serum (1:200) and protein A-Sepharose 6MB beads for 1 h, and the beads were removed by centrifugation. The supernatant was then rotated at 4 °C with alpha(2)C10 antiserum (1:200) and 50 µl of protein A-Sepharose 6MB beads overnight. Beads complexed to immunoprecipitated material were washed five times with 1 ml of ice-cold solubilization buffer, resuspended in SDS sample buffer, sonicated for five minutes, centrifuged, and supernatants containing equivalent amounts of protein were subjected to 10% discontinuous SDS-polyacrylamide gel electrophoresis. Gels were dried and exposed to Amersham Hyperfilm-MP for 6 h with an intensifying screen at -70 °C. Dried gels were also exposed to storage Phosphor screens for 2 h, and incorporated P(i) was quantitated using a Molecular Dynamics PhosphorImager (Sunnyvale, CA). To confirm the total lack of agonist-promoted phosphorylation observed for the AAAA mutant expressed in COS-7 cells (see ``Results''), a limited number of phosphorylation studies were also carried out in CHO cells using the same methods as described above.

Data Analysis

Adenylyl cyclase dose-response data and radioligand binding data from competition and saturation experiments were analyzed by iterative least squares techniques, as described by De Lean et al.(29) . For determination of whether agonist competition data was best fit by a simple model (one-site curve) or more complex model (two-site curve), F-test analysis was used with the assignment of the more complex model when p < 0.05. For adenylyl cyclase studies, data are reported as mean ± standard error of the R(max) (maximal response) and the EC for epinephrine-mediated inhibition from the indicated number of individual experiments. Comparisons for all experiments were made by two-tailed t tests, with significance imparted at p < 0.05.

Materials

[^3H]Yohimbine (80 Ci/mmol), [alpha-P]ATP (30 Ci/mmol), [^3H]cAMP (31 Ci/mmol), and P(i) (9,000 Ci/mmol) were from DuPont NEN. Forskolin,(-)-epinephrine, cholera toxin, and protein A-Sepharose 6MB beads were from Sigma. UK-14304 and phentolamine were from Research Biochemicals. The cDNA encoding bovine betaARK was a generous gift from Jeffrey L. Benovic (Thomas Jefferson University). Geneticin (G-418) was from Life Technologies, Inc. Ham's F-12 medium, Dulbecco's modified Eagle's medium, and fetal calf serum were from JRH Biosciences. All other reagents were obtained from standard commercial sources.


RESULTS

To ascertain the role of Ser-296-299 in the agonist-promoted desensitization and betaARK-mediated phosphorylation of alpha(2)C10, we initially constructed a deletion mutant (denoted as DEL 293-304) lacking 12 amino acids, including the four serines and preceding glutamic acids (Fig. 1). This mutant failed to undergo agonist-promoted desensitization (see below), and so we focused our efforts on the four serines within this region with five additional mutants having direct substitution of the potentially phosphorylated serines (Ser-296, 297, 298, and 299). For one of these substitution mutants, all four serines in this region were replaced by alanine residues (denoted as receptor AAAA). Two other mutants had two consecutive serines of the four serines replaced by alanine residues (denoted as receptors AASS and SSAA) (Fig. 1). Mutant and wild type alpha(2)C10 were transfected into CHO cells, and clones stably expressing receptors at 3100 ± 200 fmol/mg were utilized for pharmacological characterization and agonist-promoted desensitization studies.


Figure 1: Schematic for deletion and substitution of potential betaARK phosphorylation sites in alpha(2)C10. Shown is the proposed membrane topology of alpha(2)C10 with potential betaARK phosphorylation sites in the third intracellular loop. Roman numerals indicate transmembrane domains I-VII. Circles with single letters represent the primary amino acid sequence of the third intracellular loop with serines and threonines indicated by darkened circles. Asterisks indicate Ser-296, 297, 298, and 299. The six mutants constructed include a deletion mutant lacking amino acids 293-304 (DEL 293-304) and five substitution mutants with various substitutions of Ser-296-299 with alanines (AAAA, AASS, SSAA, SSSA, and SAAA).



Prior to functional studies, the pharmacological characteristics of the mutant and wild type alpha(2)C10 were assessed. As summarized in Table 1, all receptors bound [^3H]yohimbine with high affinity and displayed the ability to form the agonist-receptor-G-protein high affinity complex in [^3H]yohimbine competition studies with epinephrine, with 60-70% of the receptors occupying the high affinity state. Both high and low affinity binding constants of the mutant receptors for epinephrine were similar to those of the wild type alpha(2)C10, such that similar occupancy would be observed during epinephrine exposure of 100 µM (Table 1). This high affinity binding was sensitive to guanine nucleotide, as curves for both mutant and wild type receptors were monophasic and were best fit to one-site models when 100 µM GTP was included in the assay (Table 1). In addition (see below), the mutated alpha(2)ARs functionally coupled to G(i) as determined in adenylyl cyclase assays.



For studies of agonist-promoted desensitization, wild type alpha(2)C10 (alpha(2)C10), the deletion mutant (DEL 293-304), and the substitution mutants (AAAA, AASS, and SSAA) were exposed to 100 µM epinephrine for 30 min, and the effects of agonist pretreatment on alpha(2)AR-mediated inhibition of adenylyl cyclase activity were assessed in the membranes. As shown in Fig. 2, wild type alpha(2)C10-mediated inhibition of adenylyl cyclase activity underwent desensitization following a 30-min exposure to agonist. This desensitization was manifested as a 5-fold increase in the EC for epinephrine-mediated inhibition (0.26 ± 0.03 µMversus 1.25 ± 0.12 µM, p < 0.002, Table 2) with no change in the maximal inhibition (44 ± 2% versus 48 ± 2% decrease in forskolin-stimulated cyclase activity, p = NS, Table 2). In contrast, all of the mutant receptors failed to undergo agonist-promoted desensitization, with no significant change in the EC or maximal inhibition of adenylyl cyclase activity ( Fig. 2and Table 2).


Figure 2: Effects of substitution or deletion of Ser 296-299 on agonist-promoted desensitization of alpha(2)C10. CHO cells expressing wild type alpha(2)C10 (WT alpha(2)C10) and the mutants DEL 293-304, AAAA, AASS, and SSAA were exposed to 100 µM epinephrine for 30 min, membranes prepared, and adenylyl cyclase activities determined in the presence of 1.0 µM forskolin and the indicated concentrations of epinephrine (EPI). Data are presented as the percent of forskolin-stimulated activity. Desensitization was manifested as an increase in the EC for epinephrine-mediated inhibition for wild type alpha(2)C10 and was not detected in any of the mutated receptors. Results shown represent the mean ± S.E. of four individual experiments performed.





To establish whether the loss of desensitization was, in fact, due to a loss of phosphorylation, whole cell phosphorylation studies were carried out using wild type alpha(2)C10 and the three substitution mutants AAAA, AASS, and SSAA. These were carried out using COS-7 cells transiently expressing betaARK and mutant or wild type alpha(2)C10. Cotransfection with betaARK was utilized for several reasons. First, it has been reported recently by Kurose and Lefkowitz (16) that in Western blotting experiments with antibodies specific for betaARK and betaARK2, betaARK is by far the most predominant of the two betaARK isoforms expressed in CHO cells. Thus, phosphorylation studies carried out with betaARK would more likely correlate with the functional studies of agonist-promoted desensitization which were performed in CHO cells. Second, thus far definitive phosphorylation of purified, reconstituted alpha(2)C10 has only been demonstrated using betaARK(14) . Finally, since these receptors were each overexpressed, it seemed prudent also to overexpress the kinase to ensure its availability for receptor phosphorylation.

For these studies, transfected COS-7 cells were preincubated with P(i) and then exposed to either vehicle alone or vehicle plus 10 µM UK-14304. Mutant and wild type alpha(2)C10 were then purified and electrophoresed through 10% discontinuous SDS-polyacrylamide gels. Incorporation of P(i) under basal and agonist-treated conditions for mutant and wild type alpha(2)C10 was quantitated using a PhosphorImager. Comparisons of the extent of agonist-promoted phosphorylation between mutant and wild type alpha(2)C10 were made using the net phosphorylation, i.e. after subtraction of basal phosphorylation. As shown in Fig. 3, all receptors migrated at a molecular mass of 78 kDa. Wild type alpha(2)C10 clearly underwent agonist-promoted phosphorylation with a 3-4-fold increase in receptor phosphorylation during agonist exposure as compared with basal. In contrast, the complete substitution mutant AAAA displayed a virtual complete loss in the extent of agonist-promoted phosphorylation, which amounted to only 9 ± 2% of the phosphorylation found with wild type alpha(2)C10 (n = 4, p < 0.001, Fig. 3). The partial substitution mutants AASS and SSAA, which also did not undergo agonist-promoted desensitization, were phosphorylated 55 ± 7% and 57 ± 8% as compared with the wild type alpha(2)C10, respectively (n = 4, p < 0.002, Fig. 3). Studies performed in the same manner using CHO cells stably expressing the complete substitution mutant AAAA and wild type alpha(2)C10 yielded similar results wherein the wild type alpha(2)C10 readily underwent agonist-promoted phosphorylation (4-fold), while the mutant AAAA did not (-0.5 ± 1.6% net phosphorylation as compared with the wild type alpha(2)C10, n = 3, p < 0.001).


Figure 3: Agonist-promoted phosphorylation of wild type alpha(2)C10 and mutant receptors AAAA, AASS, and SSAA. COS-7 cells transiently coexpressing betaARK with wild type alpha(2)C10 (WT alpha(2)C10) and the mutant receptors AAAA, AASS, and SSAA were incubated for 2 h with 0.3 Ci/ml P(i) and then exposed to either vehicle or 10 µM UK-14304 for 15 min at 37 °C. Wild type and mutant alpha(2)C10 were purified as described under ``Experimental Procedures'' and subjected to 10% discontinuous SDS-polyacrylamide gel electrophoresis. Dried gels were quantitated using a PhosphorImager and exposed to x-ray film. Shown is a representative experiment of four performed.



Based on the complete loss of agonist-promoted desensitization and phosphorylation found with the mutant AAAA, we concluded that agonist-promoted phosphorylation occurs solely within this cluster of four serine residues and not at other serines and threonines within the receptor. However, since the mutants AASS and SSAA were partially phosphorylated to an equivalent extent (50%), the precise residues of Ser-296-299 which are phosphorylated remained unclear. Since AASS and SSAA, with two of the four serines remaining, underwent 50% of the agonist-promoted phosphorylation as seen with the wild type alpha(2)C10, we presumed that the total number of serines that are phosphorylated in wild type alpha(2)C10 would be either four or two. To address these possibilities, we constructed two additional mutants, denoted as receptors SAAA and SSSA, and performed whole cell phosphorylation experiments in parallel with the wild type alpha(2)C10 and the SSAA mutant.

The mutants SSSA, SSAA, and SAAA and wild type alpha(2)C10 were transiently coexpressed with betaARK in COS-7 cells, and whole cell phosphorylation studies carried out as before. Comparison of P incorporation revealed an agonist-promoted phosphorylation of SSSA, SSAA, and SAAA of 77 ± 1%, 49 ± 6%, and 27 ± 4% of that found with the wild type alpha(2)C10, respectively (p < 0.001 compared with wild type alpha(2)C10, n = 3, Fig. 4). Thus, as summarized in Fig. 5, substitution of alanine for each additional serine decreased overall phosphorylation compared with wild type alpha(2)C10 by 25%. These data, together with the absence of agonist-promoted phosphorylation found with the mutant AAAA and 50% agonist-promoted phosphorylation found with the mutant AASS relative to wild type alpha(2)C10, are consistent with all four serines, Ser-296-299, being phosphorylated in the wild type alpha(2)C10.


Figure 4: Agonist-promoted phosphorylation of wild type alpha(2)C10 and the mutant receptors SSSA, SSAA, and SAAA. Whole cell phosphorylation studies using COS-7 cells transiently coexpressing betaARK with wild type alpha(2)C10 (WT alpha(2)C10) and the mutant receptors SSSA, SSAA, and SAAA were carried out as described for the experiments in Fig. 3. Shown is a representative experiment of three performed.




Figure 5: Summary of agonist-promoted phosphorylation for mutant and wild type alpha(2)C10. Data from whole cell phosphorylation experiments described in Fig. 3and Fig. 4using wild type alpha(2)C10 (WT alpha(2)C10) and the mutants SSSA, SSAA, AASS, SAAA, and AAAA are summarized. For each receptor, the extent of agonist-promoted phosphorylation over basal (net phosphorylation) is expressed as a percentage of the net phosphorylation obtained with wild type alpha(2)C10. Results shown are the mean ± S.E. from four to seven experiments performed. All mutant receptors displayed significantly less agonist-promoted desensitization as compared with wild type alpha(2)C10 (p < 0.002). The agonist-promoted phosphorylation obtained with the mutant AAAA was not significantly greater than basal phosphorylation (p = NS).



Receptor sequestration (internalization) has been thought to play little, if any, role in short term agonist-promoted desensitization of alpha(2)C10(17) . Nevertheless, since desensitization was so dramatically affected by these mutations, we assessed agonist-promoted sequestration in the mutant AAAA using a [^3H]yohimbine binding assay as described previously(17) . For wild type alpha(2)C10 and the mutant AAAA, receptor distribution in untreated cells was similar with 8.5 ± 2.2% versus 13.3 ± 0.3% sequestered, respectively (n = 3, p = NS). Following exposure to 100 µM epinephrine for 30 min, both wild type alpha(2)C10 and the mutant AAAA underwent agonist-promoted sequestration to a similar extent with 23.0 ± 1.7% versus 26.3 ± 3.3% sequestered, respectively (n = 3, p = NS). From these data it appears that the lack of desensitization found in the mutants results from a loss of receptor phosphorylation and not sequestration.


DISCUSSION

Agonist-promoted desensitization of alphaAR has been described in a transfected Chinese hamster fibroblast cell line (CHW cells)(15) , transfected CHO cells(17, 18) , NG108-15 cells(30) , HT-29 cells(31) , brain(32) , vascular smooth muscle(33) , and vas deferens (34) . Studies from our laboratory have demonstrated that agonist-promoted desensitization of alpha(2)C10-mediated inhibition of adenylyl cyclase activity occurs rapidly and that this short term component is due solely to receptor phosphorylation (15, 16, 17) . There are multiple lines of evidence which strongly implicate betaARK as the phosphorylating kinase.

Prior to the cloning of the alphaAR, Benovic and colleagues (14) demonstrated that purified platelet alphaAR (which is alpha(2)C10) in an in vitro reconstituted phospholipid vesicle system is a specific substrate for betaARK and is phosphorylated in an agonist-dependent manner to a degree comparable to that of the beta(2)AR. Using a similar system, Onorato et al.(19) reported that the synthetic peptide LEESSSSDHAERPPG, based on a stretch of sequence present in the third intracellular loop of the alpha(2)C10, is also a substrate for betaARK. Subsequently, we have found that agonist-promoted desensitization of recombinantly expressed alpha(2)C10 occurs with a rapid onset (minutes), requires exposure to saturating concentrations of agonist(15, 17, 18) , and is selectively blocked by the betaARK inhibitor heparin(15) . Short term agonist-promoted desensitization of alpha(2)C10 is accompanied by receptor phosphorylation (15) and a loss of high affinity agonist binding of the receptor(17) , which is indicative of receptor-G-protein uncoupling. Loss of agonist-promoted phosphorylation in a mutated alpha(2)C10 lacking a serine- and threonine-rich 75-amino acid portion in the third intracellular loop resulted in a loss of short term desensitization(15) . Taken together, the above provide convincing evidence that betaARK-mediated phosphorylation is the principal mechanism of short term agonist-promoted desensitization of alpha(2)C10.

Despite the above, a precise sequence for betaARK-mediated phosphorylation of the alpha(2)C10, or any other G-protein coupled receptor, had not been delineated. We approached this by first deleting 12 amino acids in the third intracellular loop of alpha(2)C10, which removed the EESSSS sequence but left the other 12 serine and threonine residues in the loop (Fig. 1). This receptor failed to undergo agonist-promoted desensitization, establishing that the determinants of this process were confined to this region. We then systematically substituted the serines within this sequence with alanines, and in a series of five mutated receptors, assessed agonist-promoted receptor phosphorylation and desensitization. The AAAA substitution mutant failed to phosphorylate and to desensitize. Substitutions with alanines leaving one, two, or three serines in place resulted in mutant receptors that phosphorylated 25, 50, and 75% of the wild type receptor, respectively, which is consistent with each of the four serines being phosphorylated (Fig. 5). In functional desensitization studies, we found that agonist-promoted desensitization was completely ablated in the AASS and SSAA mutants (Fig. 2), despite the fact that they were partially (50%) phosphorylated as compared with wild type alpha(2)C10. There are two possibilities for this. It may be that the conformational change induced by phosphorylation of alpha(2)C10 resulting in receptor-G-protein uncoupling requires four phosphoserines. Partial phosphorylation, then, would not result in partial desensitization since the conformational change induced by such partial phosphorylation is not sufficient to induce uncoupling. It may also be that we are not able to detect small degrees of desensitization in the current assay system. The latter possibility is less likely, since we have reproducibly detected as little as 2-fold changes in the EC for alpha(2)C2-mediated inhibition of adenylyl cyclase activity in an identical assay(17) .

As discussed earlier, one limitation in the identification of sites for betaARK-mediated phosphorylation of G-protein coupled receptors is the lack of a defined consensus sequence. Based on in vitro betaARK phosphorylation studies using synthetic peptides(19) , and our current work, we propose that EESSSS in alpha(2)C10 represents such a sequence. It may be that similar sequences in other G-protein coupled receptors are recognized by betaARK. In this regard, as shown in Table 3, we have examined the third intracellular loop and carboxyl-terminal portions of members of several representative classes of the G-protein coupled receptor family for amino acid sequences similar to the EESSSS of alpha(2)C10 as potential sites for betaARK-mediated phosphorylation.



Notably, several of the G-protein coupled receptors that contain such sequences including the beta(2)AR(6, 8) , the muscarinic m(2) receptor(9) , the thrombin receptor(12) , the substance P receptor(10) , and the beta(1)AR (35) have been shown to be substrates for betaARK. Moreover, for the beta(2)AR (20) and the thrombin receptor(12) , removal of most of the carboxyl terminus or substitution of serines and threonines in the carboxyl terminus ablates betaARK-mediated desensitization. Several other receptors in Table 3, including the opossum alphaAR(36) , the D(1)(37, 38, 39) and D(2)(40, 41) dopamine receptors, even though betaARK-mediated phosphorylation has not been definitively demonstrated, display a rapid stage of agonist-promoted desensitization, which in some cases has been shown to be accompanied by receptor phosphorylation. Conversely, there are several receptors known to be phosphorylated by betaARK which do not contain an analogous sequence to EESSSS, including the adenosine A(1) receptor(11) , and the alpha(2)AR subtype, alpha(2)C2(16) . In this respect it is important to note that the preference by betaARK for acidic residues immediately adjacent to the phosphorylated moiety was established using peptides of 12 amino acids which lack the complex structure of an intact receptor. So, it may be that within the intracellular portions of a receptor there are residues separate from potential phosphorylation sites in primary sequence, but nevertheless within the context of overall receptor conformation, which provide an acidic milieu for betaARK. For instance, the third intracellular loop of alpha(2)C2 contains a large stretch of 16 consecutive glutamic acid residues as well as numerous serine and threonine residues. Also, the human alphaAR (alpha(2)C4) which has the sequence ESS in the third intracellular loop, fails to undergo both agonist-promoted desensitization (16, 17, 18) and betaARK-mediated phosphorylation(16) . However, this sequence is not in the analogous position to the EESSSS of alpha(2)C10, and there is marked diversity in the amino acid sequences of the third intracellular loops of these two receptors Thus, although we have now defined a precise receptor sequence of amino acids that undergo phosphorylation by betaARK in intact cells, and similar sequences are present in a number of G-protein coupled receptors, the location of such a sequence and the surrounding milieu are also important variables when considering whether a given receptor is a candidate for betaARK-mediated phosphorylation.

In summary, from the present studies two conclusions may be drawn. First, the precise sites for betaARK-mediated phosphorylation of alpha(2)C10 are the four consecutive serines Ser-296-299 in the third intracellular loop. And second, even partial phosphorylation of the receptor, as seen with the mutants AASS and SSAA, does not result in detectable agonist-promoted desensitization. These four amino acids provide the structural basis for the primary mechanism of short term agonist-promoted desensitization of alpha(2)C10, and they represent the first discrete motif for betaARK-mediated phosphorylation to be identified in a G-protein coupled receptor.


FOOTNOTES

*
This work was supported in part by a Department of Defense National Defense Science and Engineering Graduate fellowship (to M. G. E.). 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.

§
To whom correspondence and reprint requests should be addressed: 231 Bethesda Ave., Rm. 7511, Post Office Box 670564, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835.

(^1)
The abbreviations are: alpha(2)AR, alpha(2)-adrenergic receptor; alpha(2)C10, alpha(2)C4, and alpha(2)C2, alpha(2)AR subtypes localized to human chromosomes 10, 4, and 2, respectively; betaARK, beta-adrenergic receptor kinase; G(i) and G(s), inhibitory and stimulatory G-protein, respectively; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.

(^2)
In this report, betaARK refers to the cloned bovine betaARK, EC 2.7.1.126(6) . With the cloning of a similar kinase (betaARK2(7) ), betaARK has also been referred to as betaARK1.


ACKNOWLEDGEMENTS

We thank Dr. Jeffrey L. Benovic for providing the betaARK-pBC12BI construct, Marie T. Jacinto for technical assistance with adenylyl cyclase assays, and Cheryl T. Theiss for tissue culture and transfection.


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