Activation of the β2-Adrenergic Receptor-Gαs Complex Leads to Rapid Depalmitoylation and Inhibition of Repalmitoylation of Both the Receptor and Gαs *

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein α subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory α subunit of trimeric G protein (Gαs) covalently attached to the β2-adrenergic receptor (β2AR) as a carboxyl-terminal extension (β2AR-Gαs) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Gαs moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the β-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the β2AR and Gαs and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of β2AR-Gαs, indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.

Palmitoylation is a post-translational modification that is limited to a small subset of cellular proteins among which proteins involved in signal transduction are prevalent (1). This thioesterification of cysteine residues by palmitate distinguishes itself from other lipid modifications such as prenylation and myristoylation by its reversibility. Indeed, in contrast to myristoyl and prenyl moieties that are added co-translationally and generally remain attached to the proteins until the protein gets degraded, the protein-bound palmitate is added post-translationally and turns over more rapidly than the protein itself (2)(3)(4). Moreover, the palmitoylation state of several proteins has been shown to be dynamically regulated. In particular, biological regulation of the palmitoylation state of heterotrimeric G proteins and of their cognate receptors has been demonstrated (5)(6)(7)(8)(9)(10)(11)(12).
Activation of G␣ s 1 through receptor stimulation, following direct activation with aluminum fluoride and cholera toxin or as a result of site-directed mutagenesis, has been shown to lead to an increased incorporation of [ 3 H]palmitate into G␣ s during pulse labeling experiments. Because pulse-chase labeling experiments clearly indicated that stimulation increased the depalmitoylation rate, the enhanced incorporation was attributed to an accelerated turnover rate of the G␣ s -bound palmitate (10 -12). Interestingly, Jones et al. (8) found that, despite the increased turnover rate, activation of G␣ s did not significantly affect its stoichiometry of palmitoylation, thus challenging the notion that stimulation ultimately favors the depalmitoylation reaction (12). Agonist stimulation of the ␤ 2 -adrenergic receptor (␤ 2 AR) has also been shown to increase the amount of covalently attached [ 3 H]palmitate (5) as a result of an increased turnover rate of the receptor-bound palmitate (7). A similar agonist-promoted increase in the turnover rate of receptorbound palmitate was observed for the ␣ 2A AR (13), the D 2dopamine receptor (14), and the m 2 -muscarinic receptor (9).
Biologically regulated changes in the palmitoylation state of either receptors or G proteins may have important functional consequences. For example, mutations that prevent palmitoylation of various G␣ subunits have been found to inhibit their association with the plasma membrane and thus their signal-ing function (15)(16)(17)(18), suggesting that biological modulation of the G protein palmitoylation state could regulate their signaling properties. Palmitoylation of G␣ s has also been reported to increase its affinity for G␤␥ (19). For receptors, abolition of palmitoylation by site-directed mutagenesis has been shown to either decrease coupling to G proteins (9, 20 -23), affect receptor internalization (24 -26), or modulate receptor phosphorylation by regulatory kinases (5,27,28).
Despite these potentially important roles, very little is known concerning the mechanism that regulates palmitoylation of these proteins. Both enzymatic (29 -32) and nonenzymatic (33,34) acylation reactions have been proposed for G␣, whereas an enzyme that can catalyze the depalmitoylation of G␣ proteins has recently been identified (35). However, the mechanisms by which activation of the signaling pathway could control the acylation/deacylation cycle remain unknown. Analysis of the effects of stimulation on the palmitoylation status of receptors and G proteins is complicated by several factors (for a review, see Ref. 36). These include the fact that, following the initial conformational changes and protein-protein interactions that are promoted by receptor stimulation, multiple processes that limit the extent of the activation and contribute to signal termination come rapidly into play. It follows that it is difficult to temporally distinguish between the early events that lead to activation from the ones involved in rapid desensitization of the signaling system. This is an important problem because these two sets of events could theoretically have opposite effects on the palmitoylation reaction. Indeed, on a time scale that is virtually indistinguishable from that of the activation of the G proteins, stimulation of the receptors leads to their progressive functional inactivation. This desensitization results largely from agonist-promoted phosphorylation, uncoupling, and internalization of the receptors (37,38). Internalization of the G proteins has also been suggested to contribute to desensitization of the signaling unit (39 -43).
In an effort to distinguish between the effects of activation and desensitization on receptor and G protein palmitoylation, we took advantage of a ␤ 2 AR-G␣ s fusion protein that can be activated but not desensitized, internalized, or down-regulated (44,45). The pharmacological properties of such receptor-G protein fusion constructs has recently attracted considerable attention and many of their properties have been recently reviewed (46,47). The agonist-bound ␤ 2 AR-G␣ s fusion protein presumably mimics an early intermediate in the normal activation cycle. Also of interest to the present study is the fact that complete physical dissociation between the receptor and G␣ s , which normally follows the initial stimulatory interaction, is not permitted in the fusion protein. These features of the fusion protein allow study of the effects of early activation events on the palmitoylation state of the receptor and G protein independently of those resulting from the inactivation processes. Furthermore, the use of fusion protein restricts the analysis to those receptors and G proteins that did physically interact in the course of the experiment. We report that stimulation of ␤ 2 AR-G␣ s with ␤-adrenergic agonists promotes rapid depalmitoylation and inhibits repalmitoylation of both the receptor and the G␣ subunit. This contrasts with the facilitated repalmitoylation that is observed when the two proteins are expressed individually and suggests that early events in the activation process lead to the depalmitoylation of the two proteins, whereas later deactivation mechanisms, that do not occur for the fusion protein, are required for the repalmitoylation reaction.
Recombinant Baculoviruses Construction-The recombinant c-Myc-␤ 2 AR baculovirus was generated by subcloning the cDNA of a c-Myctagged human ␤ 2 AR (5) into the pJVELTZ recombination plasmid (In-Vitrogen). The pBacPAK-pHIS-␤ 2 AR-G␣ s was constructed by inserting in phase the Klenow-filled NcoI-SalI cDNA fragment of the fusion protein ␤ 2 AR-G␣ s (45,51) into the Klenow-filled BamHI-EcoRI pBacPAK1-polyHIS vector (CLONTECH). The constructs were confirmed by DNA sequencing. The viruses were then produced by homologous recombination in Sf9 cells according to standard procedures (52). The recombinant baculovirus encoding the ␤ 2 AR-Thr-G␣ s construct was generated as described previously (53). Following infection of Sf9 cells with the appropriate viruses, expression of ␤ 2 AR and ␤ 2 AR-G␣ s was assessed by radioligand binding assays and Western blot analysis.
Cell Culture, Metabolic Labeling, and Membrane Preparations-Sf9 cells were cultured in Grace's supplemented media containing 10% fetal bovine serum, 0.001% pluronic acid in spinner flasks (Bellco Glass) at 27°C. Cells (2 ϫ 10 6 /ml) were infected with the recombinant baculoviruses at a multiplicity of infection varying between 2 and 5 for 48 to 72 h. [ 3 H]Palmitate labeling was then carried out in cells expressing the ␤ 2 AR or ␤ 2 AR-G␣ s fusion proteins. Cells were harvested and placed in serum-free medium for 1 h prior to the start of metabolic labeling. [ 3 H]palmitate dissolved in a minimal volume of dimethyl sulfoxide was then added (100 Ci/millions of cells), and the cells were incubated at 27°C in the presence or absence of ␤-adrenergic ligands for various periods of time as described previously (7). In some experiments, labeling was allowed to proceed for 45 min before ␤-adrenergic ligands were added. Labeling was stopped by chilling the reaction on ice. Cells were centrifuged at 500 ϫ g for 5 min at 4°C, rinsed twice with ice-cold PBS and resuspended in 20 ml of an ice-cold lysis buffer containing 20 mM Tris-HCl, 5 mM EDTA, pH 7.4, and the following protease inhibitors: 5 g/ml leupeptin, 5 g/ml soybean trypsin inhibitor, and 10 g/ml benzamidine. Cells were disrupted by sonication and the lysate was centrifuged 5 min at 500 ϫ g at 4°C. The supernatant was then centrifuged at 45,000 ϫ g for 20 min at 4°C. The pelleted membranes were then resuspended in 10 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, pH 7.4, containing 0.3% n-dodecyl-␤-D-maltoside and protease inhibitors (solubilization buffer). Solubilization was carried out for 90 min at 4°C, and solubilized receptors were purified as described below.
Receptor Affinity Purification-Alprenolol-Sepharose affinity purification matrix was synthesized according to the method of Benovic et al. (54). This matrix was used to purify the Sf9-derived ␤ 2 AR, ␤ 2 AR-G␣ s , and ␤ 2 AR-Thr-G␣ s as described previously (5). The affinity purified preparations were concentrated using Centriprep and Centricon cartridges (Amicon), and the amount of ␤ 2 AR, ␤ 2 AR-G␣ s , or ␤ 2 AR-Thr-G␣ s in each sample was determined by [ 125 I]CYP soluble radioligand binding assay as described elsewhere (55).
Hydroxylamine Treatment, Chemical and Enzymatic Cleavages-For hydroxylamine treatment, purified ␤ 2 AR or ␤ 2 AR-G␣ s was mixed to an equal volume of 1 M Tris, pH 7.0, containing or not 1 M NH 2 OH and incubated overnight at 4°C. Cyanogen bromide (CNBr) cleavage was carried out following a protocol described by Luo et al. (56). Briefly, affinity purified ␤ 2 AR-G␣ s were separated by SDS-PAGE in nonreducing condition. The proteins were then transferred electrophoretically to nitrocellulose membrane. The band corresponding to ␤ 2 AR-G␣ s was cut out and the strip submerged in 500 l of 70% (v/v) formic acid. The cleavage reaction was started by adding 10 l of 5 M CNBr in acetonitrile. The reaction was allowed to proceed for 180 min at room temperature in the dark. The thrombin cleavage of the ␤ 2 AR-Thr-G␣ s construct was carried out on affinity purified fusion protein using 10 NIH units/ml of thrombin from human placenta in the solubilization buffer containing only 0.03% n-dodecyl-␤-D-maltoside for 30 min at room temperature.
SDS-PAGE and Western Blot Analysis-Affinity purified ␤ 2 AR, ␤ 2 AR-G␣ s , ␤ 2 AR-Thr-G␣ s , ␤ 2 AR-G␣ s cleaved with CNBr, or ␤ 2 AR-Thr-G␣ s cleaved with thrombin were resolved on nonreducing (or mildly reducing, 10 mM dithiothreitol, when specified) 10 -15% slab gels containing 6 M urea. The gels were then fixed, incubated in Enlightning (DuPont), or in 1 M salicylic acid, dried, and exposed to DuPont RE-FLECTION films from 10 to 60 days at Ϫ80°C. Fluorograms were scanned and digitized (Hewlett Packard laser scanner), and densitometric analysis was carried out using the NIH Image program. When Western blot analyses were performed, aliquots of the samples were loaded in parallel gels and transferred onto nitrocellulose membranes. The ␤ 2 AR-Thr-G␣ s and ␤ 2 AR moiety of the fusion protein were first visualized using the Flag M2 antibody (dilution 1:10,000) and the Renaissance chemiluminescence reagent plus (Mandel). The same membrane was then stripped using 100 mM glycine, pH 2.2 (57), and reprobed, using a rabbit polyclonal antibody against the carboxyl-terminal portion of G␣ s subunit (dilution 1:8, 500) (a generous gift of Dr. A. D. Strosberg, Institut Cochin de Génétique Moléculaire, Paris), to reveal the ␤ 2 AR-Thr-G␣ s and G␣ s moiety of the fusion protein.
Radioligand Binding Assay-Sf9 cells infected with the recombinant ␤ 2 AR or ␤ 2 AR-G␣ s baculoviruses were harvested and rinsed twice with ice-cold phosphate-buffered saline, and the membranes were prepared according to Mouillac et al. (5). Membrane suspensions were added to obtain a concentration of 2-10 g/ml in a final volume of 500 l of 75 mM Tris, 12.5 mM MgCl 2 , 2 mM EDTA containing a saturating concentration (250 pM) of the radiolabeled ␤-adrenergic antagonist [ 125 I]CYP. Nonspecific binding was determined as the residual binding observed in the presence of 10 M alprenolol. Binding reactions carried out at room temperature for 90 min were stopped by rapid filtration over glass-fiber filters.
Adenylyl Cyclase Assay-Adenylyl cyclase activity was determined in membrane preparations according to the method of Salomon et al. (58). Activities were determined in the presence or absence of the following activators: 1 M isoproterenol, 10 M alprenolol, 10 M dichloroisoproterenol, or 100 M forskolin. Data were expressed as picomoles of cAMP produced per min per mg of protein. Protein concentrations were measured by the method of Bradford (Bio-Rad) using bovine serum albumin as standard (59).

RESULTS AND DISCUSSION
Functional Characteristics of the ␤ 2 AR-G␣ s Fusion- Fig. 1 illustrates the two fusion protein constructs between ␤ 2 AR and G␣ s (␤ 2 AR-G␣ s and ␤ 2 AR-Thr-G␣ s ) that were used in the present study. Infection of Sf9 cells with recombinant baculoviruses encoding either of the two ␤ 2 AR-G␣ s fusion proteins conferred both ␤ 2 AR binding (data not shown) and ␤-adrenergic-stimulated adenylyl cyclase activities (Fig. 2) confirming that, as observed in mammalian systems (44,45,48), the fusion proteins are synthesized, translocated to the plasma membranes, and functional. Also in agreement with what was observed in mammalian systems, sustained stimulation of ␤ 2 AR-G␣ s with an agonist does not promote any desensitization of the ␤-adrenergic-stimulated adenylyl cyclase activity. This is in sharp contrast with the rapid desensitization observed in Sf9 cells expressing the wild type ␤ 2 AR. Indeed, as seen in Fig. 2, pretreatment of ␤ 2 AR expressing cells with 1 M isoproterenol for 30 min reduced the isoproterenol-stimulated adenylyl cyclase activity by 23% without significantly affecting the basal activity, thus leading to a desensitization of 40% of the net agonist-stimulated adenylyl cyclase activity. The same treatment was without effect on the isoproterenol-stimulated adenylyl cyclase activity in cells expressing the ␤ 2 AR-G␣ s fusion protein. Agonist pre-treatment for as long as 24 h was also unable to promote any desensitization of the fusion protein. This characteristic of ␤ 2 AR-G␣ s was interpreted by Bertin et al. (44) as an indication that the covalent complex can become activated but does not enter the deactivation path upon agonist stimulation. This contention is also supported by the observation that the formation of the nucleotide-sensitive high affinity state for agonist can be readily observed for ␤ 2 AR-G␣ s in both mammalian (44) and Sf9 (49,50) cells but that no agonist-promoted internalization or down-regulation was observed upon sustained stimulation (Ref. 44, and data not shown).
It follows that ␤ 2 AR-G␣ s provides a convenient model to study the early events involved in the activation of the ␤ 2 AR-Gs complex without the confounding effects of the regulatory events leading to deactivation. This may be particularly important when considering that, following their initial activating interaction, the receptor and G␣ s may be targeted to distinct cellular compartments upon dissociation. Therefore, ␤ 2 AR-G␣ s allows study of the early events linked to the activation of a single G protein by a unique receptor molecule at equimolar ratio in a common cellular compartment.
Palmitoylation of ␤ 2 AR-G␣ s -Based on the premises described above, we undertook study of the dynamics of ␤ 2 AR-G␣ s palmitoylation and the effect of agonist activation on the palmitoylation state of this complex. As shown in Fig. 3, metabolic labeling of Sf9 cells expressing either ␤ 2 AR or ␤ 2 AR-G␣ s led to the incorporation of [ 3 H]palmitate in the two proteins. The major radiolabeled bands of ϳ45 and ϳ96 kDa obtained following alprenolol-Sepharose affinity purification corresponded to the expected molecular masses for the ␤ 2 AR and ␤ 2 AR-G␣ s , respectively, when expressed in Sf9 cells (5). Molecular species with identical electrophoretical mobilities were detected in Western blot analysis using anti-␤ 2 AR and anti-␣ s antibodies (data not shown), thus confirming the identity of the fusion protein. In Fig. 3A, identical numbers of ␤ 2 AR and of ␤ 2 AR-G␣ s , as assessed by radioligand binding, were loaded but densitometric analysis revealed around 1.7 times higher [ 3 H]palmitate incorporation into the ␤ 2 AR-G␣ s than ␤ 2 AR, consistent with the fact that two palmitoylation sites are present in the fusion protein as compared with only one in the receptor. Fig. 3B illustrates   FIG. 1. The ␤ 2 AR-G␣ s fusion constructs. Schematic representation of the two ␤ 2 AR-G␣ s fusion proteins used in this study. Upper panel, the ␤ 2 AR-G␣ s fusion protein constructed as in Bertin et al. (45). This construct links the bovine G␣ s through its amino-terminal methionine to the carboxyl terminus of the poly-His-tagged human ␤ 2 AR. Lower panel, the ␤ 2 AR-Thr-G␣ s fusion protein constructed as in Seifert et al. (53). This construct links the human ␤ 2 AR, tagged at its amino and carboxyl termini by the Flag epitope and a histidine hexamere, respectively, and bovine G␣ s through an engineered thrombin cleavage site. The wavy lines represent the sites of palmitoylation. the sensitivity of the labeling to hydroxylamine treatment, indicating that the [ 3 H]palmitate was covalently attached to both ␤ 2 AR and ␤ 2 AR-G␣ s via thioester bonds.
Agonist-promoted Depalmitoylation of ␤ 2 AR-G␣ s -To assess the effect of receptor stimulation on the dynamics of palmitoylation, pulse labeling experiments were carried out in the presence or absence of agonists for periods varying between 5 and 60 min. In the absence of agonist, incorporation of palmitate into ␤ 2 AR-G␣ s increased almost linearly for the first 30 min of labeling and remains stable thereafter (Fig. 4). The presence of isoproterenol during the labeling period greatly inhibited the incorporation of [ 3 H]palmitate in the fusion protein. This unexpected result contrasts sharply with the agonist-promoted increase in palmitate incorporation observed on ␤ 2 AR and G␣ s when these proteins are expressed individually (5, 7, 10 -12).
For the ␤ 2 AR and G␣ s expressed separately, the increase in palmitate turnover was linked to a faster rate of depalmitoylation upon agonist stimulation (7,12). The apparent increase in [ 3 H]palmitate incorporation was thus attributed to a concomitant acceleration of the repalmitoylation reaction. It follows that the agonist-promoted reduction of [ 3 H]palmitate incorporation into the ␤ 2 AR-G␣ s fusion protein could result from a slower depalmitoylation or reflect an inhibition of the repalmitoylation reaction. To distinguish between these two hypotheses, cells were metabolically labeled with [ 3 H]palmitate in the absence of agonist. Following a 45-min pulse period, corresponding to the period required to attain steady state labeling, isoproterenol was added or not in the continued presence of [ 3 H]palmitate and incubated for an additional 5 or 15 min. As seen in Fig. 5, incubation with isoproterenol rapidly reduced the extent of ␤ 2 AR-G␣ s palmitoylation, thus suggesting that agonist stimulation promotes its rapid depalmitoylation and that repalmitoylation of the active complex cannot occur. This is in sharp contrast with the increased repalmitoylation that is observed when identical treatment is carried out in cells expressing the wild type ␤ 2 AR as an individual protein (Fig. 5B).
Because the ␤ 2 AR-G␣ 2 fusion protein can be activated but that later processes of inactivation such as G protein dissociation, desensitization, or internalization do not occur, it could be hypothesized that early events leading to activation of the receptor-G protein complex promote depalmitoylation but that later processes are required for repalmitoylation. The fact that the depalmitoylation and repalmitoylation reactions may occur with very similar kinetics when the ␤ 2 AR and G␣ s are expressed as individual proteins may explain why Jones et al. (8) did not observe any change in the stoichiometry of palmitoylation of G␣ s upon activation despite the universally observed increase in the turnover rate of the G␣ s -bound palmitate. The stabilization of the activated receptor-G protein complex using ␤ 2 AR-G␣ s allowed isolation of the effects that resulted solely from the activation process.
The effect of ␤-adrenergic ligands of various levels of intrinsic activity was then assessed on the palmitoylation of ␤ 2 AR-G␣ s . As shown in Fig. 6, the addition of all ligands caused a significant reduction in the incorporation of the labeled fatty acid into ␤ 2 AR-G␣ s . Interestingly, the extent of the decrease in labeling was directly correlated (r 2 ϭ 0.992) to the intrinsic activity of the compounds toward ␤ 2 AR-G␣ s as assessed in a membrane adenylyl cyclase assay (Fig. 6B). However, direct stimulation of cAMP production by forskolin did not affect the palmitoylation of ␤ 2 AR-G␣ s (Fig. 6C), thus suggesting that the agonist-promoted depalmitoylation is linked to conformational changes imposed by the agonists and not to second messenger generation.
[ 3 H]Palmitate Incorporation into the ␤ 2 AR and G␣ s Moieties of the Fusion Protein-Cysteine 341 of ␤ 2 AR and cysteine 3 of G␣ s , corresponding to position 358 and 428 in ␤ 2 AR-G␣ s , respectively, represent the confirmed palmitoylation sites of these two proteins (20,29). In the experiments described above, palmitoylation of ␤ 2 AR-G␣ s was studied as a whole with no specific consideration of the individual palmitoylation sites. To determine whether the two sites were indeed palmitoylated and to assess if agonist treatment had similar effects on the palmitoylation state of the two proteins, we took advantage of another fusion protein construct in which a thrombin cleavage site was engineered between the receptor and G␣ s (␤ 2 AR-Thr-G␣ s ; see Fig. 1). As a control, thrombin treatment was performed on wild type ␤ 2 AR without any effect on the palmitoylation state nor the integrity of the receptor (data not shown). Fig. 7A shows that thrombin treatment of the purified fusion protein, following metabolic labeling, generated two labeled proteins corresponding to the expected mobility for ␤ 2 AR and G␣ s , indicating that the two proteins were palmitoylated within the fusion construct. The identity of the cleaved frag- ments was further confirmed by Western blot analysis using the anti-␣ s antibody to detect G␣ s and the anti-Flag M2 antibody to detect the Flag epitope-bearing ␤ 2 AR. The apparently higher [ 3 H]palmitate incorporation observed into the ␤ 2 AR band when compared with G␣ s most likely reflects the presence of some background labeling observed in this region of the gel even in the absence of thrombin.
As previously observed for ␤ 2 AR-G␣ s , the presence of isoproterenol during the metabolic labeling induced a significant reduction of the [ 3 H]palmitate incorporation into ␤ 2 AR-Thr-G␣ s . Thrombin cleavage revealed that the overall decrease in the radiolabeling of the fusion protein was the consequence of a reduction of [ 3 H]palmitate incorporation into both the receptor and G␣ s . The agonist-induced G␣ s depalmitoylation that we observed could be mediated, in part, by the receptor-promoted dissociation of ␤␥ subunits from the activated fusion protein. Indeed, as reported by Iiri et al. (19), ␤␥ did protect GDP-bound ␣ s but not ␣ s -GTP[␥S] from depalmitoylation by a recombinant esterase. Because, nonreducing SDS-PAGE conditions could lead to aggregation of some proteins, including the receptor, reducing conditions were also used. As shown in Fig. 7B, identical results were obtained when receptor and G␣ s were resolved under mildly reducing conditions (10 mM dithiothreitol) that diminished aggregation and promoted only partial chemical depalmitoylation.
Palmitoylation of both receptor and G␣ s and the effect of isoproterenol on the two proteins was further confirmed using CNBr hydrolysis of the 3 H-palmitoylated ␤ 2 AR-G␣ s construct. The primary sequence of ␤ 2 AR-G␣ s containing 18 methionines, complete cleavage should generate 19 fragments (Fig. 8A). Given that the two palmitoylation sites are located on two distinct fragments, two peptides distinguishable by their size are expected to be 3 H-palmitoylated. Calculated masses for the expected palmitoylated fragments are 16.4 and 7.2 kDa corresponding to the ␤ 2 AR-and the G␣ s -derived peptides, respectively. As shown in Fig. 8B, CNBr treatment yielded two peptides of the expected electrophoretic mobility, confirming that both the receptor and G␣ s were palmitoylated within the fusion protein. The difference in the labeling intensity of the two bands most likely results from quantitatively different elution and recovery of the two fragments from the nitrocellulose membrane during the hydrolysis. Weakly labeled bands at 21 and 44 kDa represent partial cleavage products. Addition of isoproterenol following a 45-min pulse labeling with [ 3 H]palmitate promoted the depalmitoylation of both the 16.4 and 7.2 kDa fragments, confirming once more that agonist activation favored the depalmitoylation of both the receptor and G␣ s .
Taken together, the data presented in this study show that the initial events leading to activation of the ␤ 2 AR-G␣ s complex promote the rapid depalmitoylation of both receptor and G␣ s and that sustained activation prevents repalmitoylation occurring. The extent of depalmitoylation is directly proportional to the intrinsic activity of the agonist and most likely depends on conformational changes and protein-protein interactions that are stabilized by the activating ligands. Also, this study demonstrates for the first time that depalmitoylation and repalmitoylation occur during distinct phases of the receptor-G protein activation/inactivation cycle. Further studies are now required to determine how receptor activation leads to depalmitoylation of the receptor-G protein complex and why deactivation is required for repalmitoylation to occur. In particular, the role played by the newly characterized acyl-protein thioesterase (35) in the activation driven depalmitoylation will need to be assessed.