S-Palmitoylation of a Novel Site in the β2-Adrenergic Receptor Associated with a Novel Intracellular Itinerary*

We report here that a population of human β2-adrenergic receptors (β2AR), a canonical G protein-coupled receptor, traffics along a previously undescribed intracellular itinerary via the Golgi complex that is associated with the sequential S-palmitoylation and depalmitoylation of a previously undescribed site of modification, Cys-265 within the third intracellular loop. Basal S-palmitoylation of Cys-265 is negligible, but agonist-induced β2AR activation results in enhanced S-palmitoylation, which requires phosphorylation by the cAMP-dependent protein kinase of Ser-261/Ser-262. Agonist-induced turnover of palmitate occurs predominantly on Cys-265. Cys-265 S-palmitoylation is mediated by the Golgi-resident palmitoyl transferases zDHHC9/14/18 and is followed by depalmitoylation by the plasma membrane-localized acyl-protein thioesterase APT1. Inhibition of depalmitoylation reveals that S-palmitoylation of Cys-265 may stabilize the receptor at the plasma membrane. In addition, β2AR S-palmitoylated at Cys-265 are selectively preserved under a sustained adrenergic stimulation, which results in the down-regulation and degradation of βAR. Cys-265 is not conserved in β1AR, and S-palmitoylation of Cys-265 may thus be associated with functional differences between β2AR and β1AR, including relative resistance of β2AR to down-regulation in multiple pathophysiologies. Trafficking via the Golgi complex may underlie new roles in G protein-coupled receptor biology.

The function of G protein-coupled receptors (GPCR) 3 is regulated by diverse posttranslational modifications, which in the case of the canonical GPCR, the ␤ 2 -adrenergic receptor (␤ 2 AR), include prominent phosphorylation and S-palmitoylation. The dynamic regulation of individual posttranslational modifications and potential cross-talk between them remain incompletely understood. A number of GPCR have been shown to be S-palmitoylated at 1-3 Cys residues located within the C-terminal tail at the cytoplasmic end of the seventh transmembrane segment (1). The first example of S-palmitoylation of a ligandactivated GPCR (following the seminal demonstration of S-palmitoylation of the light-activated photoreceptor GPCR rhodopsin (2)) was provided by the ␤ 2 AR. Metabolic labeling with [ 3 H]palmitic acid in combination with mutational analysis revealed that the ␤ 2 AR incorporates palmitate at Cys-341 (numbering here and below is according to the human sequence) within the C-terminal cytoplasmic tail under basal conditions (3), and it was subsequently suggested that agonist stimulation increases the rate of palmitate turnover (4). It is also reported that mutation of Cys-341 inhibits coupling of the ␤ 2 AR to G s (3) and suppresses agonist-induced recruitment of ␤-arrestin (5), although the extent to which those effects of mutation reflect an absence of palmitoylation per se has not been determined.
In the 27 years following the initial report (3), it has been assumed that Cys-341 represents the sole site of S-palmitoylation within the ␤ 2 AR, consistent with elimination of metabolic labeling with [ 3 H]palmitic acid under basal conditions by mutation of Cys-341 (3). We report here the discovery of a second site of ␤ 2 AR S-palmitoylation, at Cys-265 within the third intracellular loop. S-Palmitoylation of Cys-265 is predominantly dependent upon agonist activation of the ␤ 2 AR and requires both phosphorylation by cAMP-dependent kinase (PKA) of Ser-261/Ser-262 and receptor internalization. S-Palmitoylation of Cys-265 is rapidly reversed upon withdrawal of agonist. Agonist-induced turnover of palmitate on the ␤ 2 AR occurs predominantly on Cys-265. We identified the enzymes responsible for S-palmitoylation of Cys-265 as zDHHC9/14/18, which constitute a distinct subfamily of the 23 mammalian zDHHC defined by phylogenetic tree analysis, and we identified the enzyme responsible at least in large part for depalmitoylation as the plasma membrane-localized acyl-protein thioesterase 1 (APT1). zDHHC9/14/18 reside in the Golgi/trans-Golgi subcellular compartment (Golgi complex), and trafficking of the ␤ 2 AR to the Golgi complex for S-palmitoylation and thence to the plasma membrane for depalmitoylation represents a previously unreported intracellular itinerary for the ␤ 2 AR initiated by agonist stimulation. Inhibition of depalmitoylation revealed that S-palmitoylation of Cys-265 stabilizes the receptor at the membrane. Further, we found that under conditions of sustained adrenergic stimulation (as in pathophysiologies that may result in down-regulation of ␤AR), the ␤ 2 AR S-palmitoylated at Cys-265 are selectively preserved, maintaining a pool of receptors that are primed for reactivation upon depalmitoylation.

Agonist-induced and Dynamic S-Palmitoylation of a Second
Site(s) within the ␤ 2 AR-To examine ␤ 2 AR S-palmitoylation, we employed the acyl-RAC method (resin-assisted capture of fatty acylated proteins) in which the thioester bond between palmitate and Cys is cleaved with neutral hydroxylamine and the resultant free thiol is coupled to thiopropyl-Sepharose for pulldown and subsequent analysis (6,7). This method directly assesses the presence of palmitate-modified Cys, and omission of hydroxylamine provides a rigorous control for false positives. In human embryonic kidney (HEK293) cells stably expressing FLAG-tagged wild-type human ␤ 2 AR, acyl-RAC in combination with Western blotting for FLAG revealed S-palmitoylation under basal conditions (Fig. 1A), consistent with previous results obtained with metabolic labeling by [ 3 H]palmitic acid (3,4). The acyl-RAC assay cannot by itself distinguish between one or more sites of S-palmitoylation within a given substrate. In HEK293 cells stably expressing FLAG-tagged ␤ 2 AR in which Cys-341 was mutated to Ala (␤ 2 AR-C341A), S-palmitoylation of ␤ 2 AR under basal conditions was greatly diminished, but we consistently observed a small, hydroxylamine-dependent signal (Fig. 1A), suggesting the possibility of an additional site(s) of S-palmitoylation. Because previous results suggested that agonist-induced activation resulted in the turnover of palmitate on the ␤ 2 AR (4), we examined the effects of activation of ␤ 2 AR-C341A by the prototypical ␤ 2 AR agonist isoproterenol (ISO). Stimulation with ISO (10 M) greatly enhanced S-palmitoylation of ␤ 2 AR-C341A (Fig. 1B). Enhancement of S-palmitoylation was evident following stimulation with 1 nM ISO, and a sigmoidal dose-response curve revealed progressive enhancement through 10 M ISO (Fig. 1C); 10 M ISO was employed in most subsequent assays. The fidelity of the acyl-RAC method as a reporter of S-palmitoylation under these conditions was verified by the finding that treatment with the inhibitor of S-palmitoylation, 2-bromopalmitate (2-BP, 100 M), largely eliminated hydroxylamine-dependent pulldown of ␤ 2 AR-C341A following stimulation with ISO (Fig. 1B). Thus, there is a second site(s) of S-palmitoylation within the ␤ 2 AR, and S-palmitoylation of that site(s) is predominantly dependent upon receptor activation. Enhanced S-palmitoylation of ␤ 2 AR-C341A was evident as soon as 5 min after exposure to ISO, and the population stoichiometry of S-palmitoylation increased linearly until it reached a plateau after 1 h of stimulation (Fig. 1, D and E). Quantification of Western blotting analysis following acyl-RAC revealed that the proportion of ␤ 2 AR modified by S-palmitoylation of Cys-265 plateaued at about 25%. It has been shown that sustained activation of the ␤ 2 AR results in ␤ 2 AR downregulation, which reflects at least in part ␤ 2 AR ubiquitination by Mdm2 (mouse double minute 2 homolog) (8). We observed that ␤ 2 AR-C341A was down-regulated with prolonged stimulation (Ͼ2 h) by ISO but that the abundance of S-palmitoylated ␤ 2 AR was not decreased (Fig. 1, E and F). Thus, the population of ␤ 2 AR that is S-palmitoylated at the second site(s) is apparently distinct from the population that is subject to ubiquitination, and further, down-regulation accompanying prolonged stimulation of ␤ 2 AR results in a progressive and substantial (ϳ85%) increase in the proportion (relative abundance) of ␤ 2 AR that is S-palmitoylated at the second site(s) (Fig. 1F).
Gradual acquisition of a steady state of S-palmitoylation while maintaining stimulation by ISO points to establishment of an equilibrium between S-palmitoylation and depalmitoylation. Active depalmitoylation was indicated by the finding that removal of ISO (following exposure for 2 h), coupled with the addition of the ␤ 2 AR antagonist propranolol (PROP, 100 M) to assure truncation of receptor activation, was followed by rapid loss of palmitate from ␤ 2 AR-C341A as revealed by acyl-RAC (t1 ⁄ 2 ϭ 17.65 min) (Fig. 1G).
Cys-265 Is the Second Site of ␤ 2 AR S-Palmitoylation-To identify the site(s) of agonist-induced palmitoylation, Cys residues within the ␤ 2 AR were mutated to Ala, and mutant or wildtype receptors were stably expressed in HEK293 cells. Whereas mutation of the canonical site of S-palmitoylation, Cys-341, had no apparent effect on ISO-induced S-palmitoylation as assessed by acyl-RAC, the combined mutation of Cys-341 and of Cys-265 within the third intracellular loop largely eliminated agonist-induced S-palmitoylation ( Fig. 2A). Thus, Cys-265 is the single second site of S-palmitoylation. Unlike Cys-341, the canonical C-terminal-tail site of palmitoylation, Cys-265, is not conserved in ␤ 1 AR or ␤ 3 AR (Fig. 2B) and is conserved within ␤ 2 AR more variably across vertebrate phylogeny than Cys-341 (Fig. 2C).   We then examined agonist-induced turnover of palmitate on the wild type and ␤ 2 AR-C265A employing metabolic labeling with the -alkynyl-palmitate analog 17-octadecynoic acid followed by bio-orthogonal click chemistry with azide-PEG3biotin (9 -11). Treatment with ISO resulted in enhanced incorporation of 17-octadecynoic acid into the wild type but not ␤ 2 AR-C265A (Fig. 2D). In addition, whereas exposure to 2-BP largely eliminated ligand-induced S-palmitoylation of ␤ 2 AR-C341A following stimulation with ISO as assessed by acyl-RAC (Fig. 1B), 2-BP had no significant effect on S-palmitoylation of ␤ 2 AR-C265A (Fig. 2E). Thus, S-palmitoylation of Cys-265 apparently accounts for at least the large part of ligand-induced dynamic turnover of palmitate on the ␤ 2 AR, whereas S-palmitoylation of Cys-341 is relatively stable.
Cys-265 Is Depalmitoylated by APT1-Agonist-induced S-palmitoylation of Cys-265 within the ␤ 2 AR is rapidly reversed upon removal of agonist (Fig. 1G), indicating the operation of a depalmitoylating mechanism. In mammalian cells, four thioesterases are well established as mediators of enzymatic depalmitoylation. The palmitoyl-protein thioesterases PPT1 (14,15) and PPT2 (16) operate at the lysosome, whereas the acyl-protein thioesterases APT1 (17) and APT2 (18), members of the serine hydrolase/lysophospholipase family that share a Ser/ His/Asp catalytic triad, have a broad subcellular distribution and are themselves S-palmitoylated and thereby localized to membranes including the plasma membrane (19). APT1/2 are therefore poised to mediate dynamic depalmitoylation in the context of cellular signaling, although the substrate specificities and regulation of APT1/2 are not well understood. We found by qPCR that all four protein thioesterases were expressed in HEK293 cells (Fig. 4A). To identify the enzyme(s) responsible for depalmitoylation of Cys-265, we first examined the effect of palmostatin B (PalmB), a catalytic inhibitor of APT1/2. We found that PalmB inhibited depalmitoylation of Cys-265 in ␤ 2 AR-C341A in a dose-dependent fashion after the termination of ISO-induced receptor activation (Fig. 4B), indicating that APT1 and/or APT2 mediated the depalmitoylation of Cys-265. We then employed siRNA-mediated knockdown of individual enzymes to isolate the activity responsible for depalmitoylation of Cys-265 as verified by qPCR (Fig. 4C) and determined that knockdown of APT1 suppressed the depalmitoylation, whereas knockdown of APT2, PPT1, or PPT2 had no significant effect (Fig. 4D). Thus, APT1 mediates depalmitoylation of Cys-265 within the ␤ 2 AR. However, it has recently been reported that additional members of the serine hydrolase enzyme superfamily exhibit PalmB-inhibitable protein depalmitoylating activity (20), and our results do not rule out a role for additional depalmitoylating enzymes in the regulation of Cys-265 S-palmitoylation. Confocal immunofluorescence microscopy of overexpressed but untagged APT1 demonstrated that, under these conditions, APT1 was localized pre-dominantly to the plasma membrane (Fig. 4E); in contrast to zDHHC9/14/18 (see below), we did not observe a perinuclear concentration that would suggest localization of APT1 to the Golgi complex.
S-Palmitoylation of Cys-265 within the ␤ 2 AR Is Associated with a Previously Undescribed Intracellular Itinerary-The zDHHC palmitoyl transferases are integral membrane proteins that are associated with internal membranes as well as the plasma membrane, and it has been shown that zDHHC9/14/18 are localized to the Golgi complex (21,22). It is well established that activation of ␤ 2 AR is followed by internalization and routing of the receptor into trafficking pathways that involve its association with endosomes, which may be followed by recycling to the plasma membrane or either proteasomal or lysosomal degradation (23). In view of the known subcellular localization of zDHHC9/14/18 to the Golgi complex, our results suggest the possibility that a population of the ␤ 2 AR traffics to the Golgi complex following agonist-induced internalization, which is followed by a return to the plasma membrane that would be required for depalmitoylation by plasma membraneresident APT1, thus representing a previously undescribed intracellular itinerary for this receptor.
To examine this possibility, we employed confocal immunofluorescence microscopic analysis in HEK293 cells stably expressing FLAG-tagged wild-type ␤ 2 AR and transiently transfected with HA-tagged zDHHC9, -14, or -18. Under basal conditions, ␤ 2 AR (FLAG) immunofluorescence was localized to the plasma membrane, whereas zDHHC9 (HA) immunofluorescence was localized to the Golgi complex, as demonstrated by co-localization with the Golgi complex marker Golgi-RFP (Fig. 5A). Following stimulation with ISO (10 M, 1 h), ␤ 2 AR immunofluorescence was observed in a perinuclear distribution where it co-localized with zDHHC9/14/18 immunofluorescence, consistent with localization to the Golgi complex (Fig.  5B). Labeling of FLAG-tagged ␤ 2 AR with anti-FLAG Ab prior to ISO stimulation directly demonstrated trafficking of plasma membrane-localized ␤ 2 AR to the Golgi complex, identified by Golgi-RFP ("antibody feeding" assay) (Fig. 5C). Disruption of the Golgi complex with brefeldin A (2 h) resulted in a dose-dependent decrease in subsequent ISO-induced (10 M, 1 h) S-palmitoylation of Cys-265 within ␤ 2 AR-C341A (Fig. 5D), which was associated with delocalization of zDHHC9/14/18 from a perinuclear concentration to a diffuse cytoplasmic distribution (Fig. 5E). Thus, a population of the ␤ 2 AR traffics to the Golgi complex upon ligand-induced activation and internalization, where they are S-palmitoylated by zDHHC9/14/18 and from whence they must traffic to the plasma membrane for depalmitoylation by plasma membrane-localized APT1.
We also determined, by employing cell-surface biotin labeling followed by acyl-RAC, pulldown of biotinylated proteins with streptavidin, and Western blotting for the ␤ 2 AR, that at least some proportion of the ␤ 2 AR S-palmitoylated at Cys-265 following ISO stimulation (10 M, 1 h) is localized to the plasma membrane following transit from the Golgi complex (Fig. 5F).
PKA-mediated Phosphorylation of Ser-261/Ser-262 Is Necessary but Not Sufficient for Cys-265 S-Palmitoylation-Ligandinduced activation of the ␤ 2 AR activates G s and thereby adenylyl cyclase, which generates cAMP, thus activating PKA. PKA then phosphorylates Ser residues at two locations within the ␤ 2 AR: Ser-345/Ser-346 within the C-terminal tail and Ser-261/ Ser-262 within the third intracellular loop (24). PKA-mediated phosphorylation has been associated with agonist-induced desensitization of the ␤ 2 AR (24) and with the regulation of coupling to G i versus G s (25), but distinct functions for PKA-mediated phosphorylation of Ser within the third intracellular loop versus the C-terminal tail remain poorly delineated. Ser-345/ Ser-346 and Ser-261/Ser-262 are located adjacent to the canonical and newly discovered sites of S-palmitoylation, respectively. Because previous reports based on mutation of Cys-341 have suggested the possibility of cross-talk between S-palmitoylation of Cys-341 and phosphorylation of Cys-345/Cys-346 (26), we examined the possibility that agonist-induced S-palmitoylation of Cys-265 might be coupled functionally to phosphorylation of Ser-261/Ser-262.
Treatment of HEK293 cells stably expressing FLAG-tagged ␤ 2 AR-C341A with the PKA inhibitor H-89 (20 M, 15 min) abrogated ISO-induced ␤ 2 AR S-palmitoylation (Fig. 6A). Inhi-bition by H-89 of ISO-induced Ser-262 phosphorylation was confirmed (Fig. 6A) by Western blotting with a monoclonal antibody specific for phospho-Ser-262 (27). Treatment with H-89 had no detectable effect on ␤ 2 AR internalization under these conditions (Fig. 6B). However, augmenting the cAMP levels by treatment of cells with the adenylyl cyclase activator forskolin, or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), failed to induce S-palmitoylation, although the phosphorylation of Ser-262 was greatly enhanced (Fig. 6C). Therefore, PKA phosphorylation is necessary but not sufficient to induce receptor S-palmitoylation. Unlike stimulation by ISO, treatment with forskolin or IBMX did not induce receptor internalization (Fig. 6D). Thus, both PKA phosphorylation and internalization are required to induce receptor S-palmitoylation at Cys-265, consistent with the requirement that the receptor must traffic to the Golgi complex where zDHHC9/14/18 reside.
Treatment with H-89, which suppresses S-palmitoylation of Cys-265, would suppress ISO-induced PKA-mediated phos-  (Fig. 6E). We considered two potential explanations for the obligate role of Ser-261/Ser-262 phosphorylation: phosphorylation might be required to route the ␤ 2 AR to the Golgi complex; and/or phosphorylation of Ser-261/Ser-262, closely adjacent to Cys-265, might exert an allosteric effect on S-palmitoylation per se. We could detect no effect of the S261A/S262A mutation on ISOinduced trafficking of the ␤ 2 AR to the Golgi complex as assessed by co-localization with DHHC9 (Fig. 6F) or by an antibody feeding assay (as in Fig. 5C), which directly demonstrated ISO-induced trafficking of plasma membrane-localized ␤ 2 AR bearing a S261A/S262A mutation to the Golgi complex, as shown by co-localization with Golgi-RFP (Fig. 6G). We therefore concluded that an allosteric mechanism is likely. We also determined that activation of ␤ 2 AR-C341A by the endogenous ␤ 2 AR ligand epinephrine (10 M, 2 h; parameters employed for all compounds tested) and by the synthetic ␤ 2 AR agonist fenoterol significantly enhanced S-palmitoylation of Cys-265, whereas exposure to norepinephrine or the synthetic agonist salbutamol or procaterol was much less efficacious (Fig.  7A). Although we found that, under these conditions, cAMP production was induced to a similar degree by these agonists (Fig. 7B), it has been shown that receptor internalization induced by stimulation with norepinephrine and salbutamol is substantially less efficacious vis à vis ISO or fenoterol (29,30), which supports the dual determination of Cys-265 S-palmitoylation. In addition, exposure to any of a panel of 14 compounds classified as ␤ 2 AR antagonists or partial, inverse, or neutral ago- . S-Palmitoylation of ␤ 2 AR Cys-265 is associated with a previously undescribed intracellular itinerary. A, confocal immunofluorescence microscopic analysis in HEK293 cells stably expressing FLAG-tagged wild-type ␤ 2 AR (green) and transiently transfected with HA-tagged zDHHC9 (blue) as well as the Golgi complex marker Golgi-RFP (red) under basal conditions. ␤ 2 AR is localized predominantly to the plasma membrane, whereas HA-zDHHC9 is localized to the Golgi complex. B, in HEK293 cells stably expressing FLAG-␤ 2 AR (red), transiently transfected with HA-tagged zDHHC9, -14, or -18 (green), and stimulated with ISO (10 M, 1 h), ␤ 2 AR are present at a perinuclear location (consistent with localization to the Golgi complex) where they co-localize with zDHHC9, -14, or -18. Merged images are shown at a relatively low and a higher magnification in the top (white boxes) and bottom rows, respectively. Scale bar ϭ 10 m. C, in HEK293 cells stably expressing FLAG-tagged wild-type ␤ 2 AR (green) and Golgi RFP, cell-surface FLAG-␤ 2 AR was labeled with anti-FLAG M2 antibody in the absence or presence of 10 M ISO for 30 min, and surface antibody was removed by acid wash. Hoechst 33342 was employed for nuclear staining (blue). This antibody feeding assay reveals directly that a population of ␤ 2 AR traffics from the plasma membrane to the Golgi complex upon ISO stimulation. D, treatment with brefeldin A (BFA) (50 g/ml, 2 h) suppresses ISO-induced S-palmitoylation of ␤ 2 AR Cys-265 in a dose-dependent fashion. n ϭ 3; *, p Ͻ 0.05 versus ISO without brefeldin A by ANOVA. E, treatment with brefeldin A (50 g/ml, 2 h) results in delocalization of HA-zDHHC9 from a perinuclear concentration to a diffuse cytoplasmic distribution. Scale bar ϭ 10 m. F, cell-surface biotin labeling followed by acyl-RAC and then pulldown of biotinylated and S-palmitoylated proteins with streptavidin followed by Western blotting for the ␤ 2 AR demonstrates that at least some proportion of ␤ 2 AR S-palmitoylated at Cys-265 following ISO stimulation (10 M, 1 h) returns to the plasma membrane following transit from the Golgi complex. Data shown are representative of two separate experiments.
Altered Membrane Stability and Signaling Consequent upon S-Palmitoylation of Cys-265 within ␤ 2 AR-Our results indicate that a population of ␤ 2 AR undergoes a tightly regulated, dynamic cycle of S-palmitoylation/depalmitoylation that entails traffic to the Golgi complex (zDHHC9/14/18) and a return to the plasma membrane (APT1). We used PalmB in combination with ISO stimulation to enhance the population of plasma membrane-localized, Cys-265 S-palmitoylated ␤ 2 AR to elucidate the effects of Cys-265 S-palmitoylation on ␤ 2 AR disposition and ligand-induced signaling.
We first examined trafficking of the wild type versus ␤ 2 AR-C265A induced by stimulation with ISO followed by truncation of stimulation by PROP in the presence or absence of PalmB. As assessed by fluorescence-activated cell sorting, the steady-state proportion of receptors internalized as a result of ISO-stimulation (10 M, 60 min) did not differ significantly in cells expressing the wild type or ␤ 2 AR-C265A and also was unaffected by PalmB (Fig. 8A, 0 min). The time course of re-establishment of plasma membrane-localized receptors during PROP treatment also did not differ (Fig. 8A). However, the abundance of plasma membrane-localized wild-type receptors following truncation of ISO stimulation was substantially (ϳ50%) enhanced by treatment with PalmB, an effect that was eliminated by mutation of Cys-265 (Fig. 8A). Because the abundance of the plasma membrane-localized receptor under these conditions will reflect competing mechanisms of membrane addition and removal of the receptor following the addition of PROP (including ligandindependent (basal) turnover (33)), this result indicates that ␤ 2 AR is stabilized at the plasma membrane by S-palmitoylation of Cys-265. In F and G, merged images are shown at a relatively low (white boxes) and a higher magnification in the top and bottom rows, respectively. Hoechst 33342 was employed for nuclear staining (blue). Scale bars ϭ 10 m. G, labeling of FLAG-tagged ␤ 2 AR with anti-FLAG Ab prior to ISO stimulation directly demonstrates trafficking of plasma membranelocalized ␤ 2 AR to the Golgi complex, identified by Golgi-RFP (antibody feeding assay). SEPTEMBER 16, 2016 • VOLUME 291 • NUMBER 38

JOURNAL OF BIOLOGICAL CHEMISTRY 20239
To investigate the possible changes in signaling through ␤ 2 AR S-palmitoylated at Cys-265 that would support the ascription of a distinct trafficking itinerary, we employed stimulation with ISO (1 M, 1 h) in combination with PalmB treatment (50 M) to maintain the population of plasma membrane-localized ␤ 2 AR S-palmitoylated at Cys-265 followed by truncation of ISO-induced activation by treatment with PROP (1 M, 1 h) in the presence of PalmB and restimulation with ISO (10 M, 5 min). We first verified that Cys-265-S-palmitoylated and plasma membrane-localized ␤ 2 AR were preserved in the presence of PalmB during PROP treatment following the initial ISO stimulation (Fig. 8B), employing cell-surface biotin labeling followed by acyl-RAC, pulldown of biotinylated proteins with streptavidin, and Western blotting for the ␤ 2 AR (as in Fig. 5D). Ligand-induced activation of ␤ 2 AR elicits signaling via G protein (resulting in altered production of cAMP via G s/i ) and activation of MAPK-based transduction via both G i and ␤-arrestin (34). We found that after cAMP returned to basal levels during exposure to PROP, cAMP production induced by restimulation with ISO (10 M, 5min) of wild-type ␤ 2 AR was significantly suppressed in the presence of PalmB (Fig. 8C). Suppression of ISO-induced cAMP production by PalmB was eliminated by mutation of Cys-265 (Fig. 8C). MAPKbased signaling as assessed by ERK phosphorylation was also modestly suppressed by treatment with PalmB, dependent upon Cys-265 (Fig. 8, D and E). Inhibition of both cAMP production and ERK phosphorylation by S-palmitoylation of Cys-265 is consistent with a direct effect on ligand-induced activation. However, we do not exclude alternative explanations for diminished activity including the effects of S-palmitoylation on the subcellular disposition of the receptor that result in diminished access to ligand or altered G s/i coupling. Note that the suppression of signaling under these conditions is evident, despite the fact that the abundance of plasma membrane-localized ␤ 2 AR is enhanced in the presence of PalmB (Fig. 8A).

Discussion
We report here that a population of ␤ 2 AR, a canonical heptahelical transmembrane receptor, embarks on a previously unreported intracellular itinerary following ligand-induced activation that is associated with phosphorylation of Ser-261/ Ser-262 within the third intracellular loop, and that takes them to the Golgi complex, where they are S-palmitoylated at Cys-265 within the third intracellular loop by zDHHC9/14/18 (a distinct subfamily of the zDHHC family of palmitoyl transferases), and from whence they traffic to the plasma membrane, where they are depalmitoylated by APT1. To our knowledge, trafficking from the plasma membrane to the Golgi complex has not been reported for any ␤AR.
Dynamic S-Palmitoylation of ␤ 2 AR-Earlier demonstrations of the ligand-induced dynamic turnover of palmitate on ␤ 2 AR were interpreted on the assumption that Cys-341 was the sole site of S-palmitoylation (4), but turnover of palmitate on ␤ 2 AR lacking Cys-341 has not been examined previously. Our results demonstrate that the ligand-induced dynamic turnover of palmitate on ␤ 2 AR reflects predominantly S-palmitoylation/ depalmitoylatiom of the previously unreported site of S-palmitoylation, Cys-265. In this regard, it is of note that, as reported recently, ␤ 1 AR is S-palmitoylated at Cys-392/Cys-393, equivalent to Cys-341 within ␤ 2 AR, and also at a more distal site within the C-terminal tail, Cys-414, and that (basal) turnover at the distal site is much greater than at the proximal site (35); that is, S-palmitoylation of the canonical GPCR consensus Cys within ␤ 1 AR is relatively stable, as we have shown for ␤ 2 AR. Determinants of Cys-265 S-Palmitoylaytion-S-Palmitoylation of Cys-265 is predominantly dependent upon ligand-induced activation of ␤ 2 AR and both the consequent phosphorylation of Ser-261/Ser-262 and receptor internalization. Because mutation of Ser-261/Ser-262 does not detectably affect ligand-induced trafficking of ␤ 2 AR to the Golgi complex, the effect of the phosphorylation of Ser-261/Ser-262 on the S-palmitoylation of Cys-265 is likely allosteric. This is the first example of which we are aware of allosteric regulation of zDHHC-mediated S-palmitoylation. Although most previous analyses describe the regulation of ␤ 2 AR function by phosphorylation of Ser-261/Ser-262 and Ser-345/Ser-346 in combination (24,25), we have demonstrated a role for Ser-261/Ser-262 phosphorylation specifically: the regulation of Cys-265 palmitoylation. It is of interest that S-palmitoylation of H-Ras (the first mammalian protein shown to be endogenously S-palmitoylated) targets Cys-181/Cys-184 (36), mediated at least in part by zDHHC9 (21), and that Ser-183 is reportedly a site of phosphorylation within H-Ras (37).
Enzymatic Regulation of Cys-265 S-Palmitoylation and Receptor Subtype Specificity-Enzymatic regulation of S-palmitoylation/depalmitoylation in mammals is mediated respectively by the 23 members of the zDHHC family and the four depalmitoylating enzymes, PPT1/2 and APT1/2. Elucidation of the substrate specificities of the zDHHC family and PPT/APT enzymes remains incomplete. Our results demonstrate that S-palmitoylation of Cys-265 is mediated by zDHHC9/14/18, which constitute a distinct subfamily of the 23 mammalian zDHHC, and that depalmitoylation is mediated selectively by APT1. Thus, our results indicate marked specificity of the enzymatic mechanisms of S-palmitoylation/depalmitoylation that operate under physiological conditions. Both ␤ 2 AR and ␤ 1 AR are S-palmitoylated at a canonical site within the C-terminal tail, but only ␤ 2 AR is S-palmitoylated at Cys-265 within the third intracellular loop. Functional differences between ␤ 2 AR and ␤ 1 AR are well known (38,39) and include the fact that, in contrast to ␤ 1 AR, ␤ 2 AR are preferentially localized under basal conditions to caveolae and/or lipid rafts from which they transit upon agonist-induced stimulation (39). It is also well known that S-palmitoylation is associated with the localization of modified substrates to caveolae and/or membrane rafts (40). Structural analysis indicates that Cys-265 resides in a juxtamembrane position (33), consistent with plasma membrane anchoring in caveolae/lipid rafts by S-palmitoylation of Cys-265. Thus, perhaps differential coupling by ␤ 2 AR versus ␤ 1 AR to G i (38) (also preferentially localized to caveolae/lipid rafts by virtue of S-palmitoylation of G i ␣ (41)) may be facilitated by S-palmitoylation of Cys-265 within ␤ 2 AR under potential conditions in which S-palmitoylation of Cys-265 is sustained.
Effect on Signal Transduction of S-Palmitoylation of Cys-265-It should be emphasized that our experimental conditions (Fig.  8, PalmB effects on cAMP production and ERK phosphorylation following stimulation with ISO, truncation of stimulation with PROP, and restimulation with ISO), which revealed the suppression of transduction by S-palmitoylation, were not designed to assess the effects of S-palmitoylation on physiological signaling per se but rather to examine changes in second messengers and downstream effectors as evidence for trafficking to the plasma membrane of a unique micropopulation of receptors that is increasingly represented at the cell surface during sustained stimulation. Indeed, it is quite conceivable that the principal role of the S-palmitoylation of Cys-265 is to regulate signaling at the Golgi complex (see below), which remains to be tested in future experiments. With that said, our results indicate that inhibition of the depalmitoylation of Cys-265 within the ␤ 2 AR is associated with diminished ligand-induced signaling by both G protein and ␤-arrestin under our experimental conditions. This suppression might result from a direct effect of S-palmitoylation on receptor activation and/or altered membrane compartmentalization of the ␤ 2 AR, which alters accessibility to ligand and/or coupling to downstream effectors. However, other components of the ␤ 2 AR signalsome are modified by S-palmitoylation, including G protein ␣-subunits (41,42), and our results do not rule out the effects of PalmB on the depalmitoylation of relevant elements other than ␤ 2 AR itself. It is of note that Cys-265 is closely adjacent to Glu-268 at the cytoplasmic end of transmembrane domain VI, which provides one side of the "ionic lock" that constrains ␤ 2 AR (43). Consequent conformational change upon release from the ionic lock is thought to be a key step in receptor activation (43,44). Stabilization of the conformation maintained by the ionic lock by S-palmitoylation of Cys-265, perhaps due to membrane anchoring, would be consistent with inhibition of signaling by both G protein and ␤-arrestin. Thus, our findings suggest that depalmitoylation at the plasma membrane may sustain the ligand-induced trafficking cycle.
S-Palmitoylation of Cys-265 in Cellular Context-In vivo, ␤ 2 AR are subject to basal stimulation, modulated up and down with "sympathetic tone." Our findings suggest that, under pathophysiological conditions of heightened and sustained adrenergic stimulation that result in ␤ 2 AR desensitization and down-regulation, the relative abundance of ␤ 2 AR that are S-palmitoylated at Cys-265 will be greatly enhanced. It is well established that down-regulation of ␤ 1 AR, and to a lesser extent ␤ 2 AR, results from heightened and sustained adrenergic stimulation that is a concomitant of heart failure (45) and other conditions including severe burns (46), representing a principal homeostatic mechanism to counterpose adrenergic drive. Our findings indicate that enhancement of the relative abundance of the population of ␤ 2 AR S-palmitoylated at Cys-265 during sustained stimulation represents a signature of selective preservation of ␤ 2 AR.
Finally, our finding that a population of ␤ 2 AR follows a novel intracellular itinerary via the Golgi complex that entails S-palmitoylation of Cys-265 (distinct from the well characterized routes of endosomal recycling to the plasma membrane or routing into degradative pathways) suggests that Golgi complex-localized ␤ 2 AR may play a distinct functional role. It has recently been shown that internalized ␤ 2 AR mediate intracellular signal transduction from an endosomal compartment (47), and our results are consistent with the proposition that Golgi complex-localized ␤ 2 AR represent a distinct intracellular signaling platform (operative selectively for ␤ 2 AR versus ␤ 1 AR) regulated by S-palmitoylation of Cys-265. In this regard, it is of note that at least one prominent component of the ␤ 2 AR signalsome, endothelial nitric-oxide synthase, is localized to and active at both the plasma membrane and Golgi complex (48).
tor at room temperature for 2 h. The resin was washed four times with binding buffer and eluted in 30 l of SDS sample buffer containing 1% 2-mercaptoethanol at 42°C for 10 min prior to SDS-PAGE and Western blotting.

Detection of S-Palmitoylation by Metabolic Labeling and
Click Chemistry-Metabolic labeling and click chemistry were carried out essentially as described (10) with minor modifications. HEK293 cells stably expressing wild-type or Cys265Ala FLAG-␤ 2 AR were cultured in 100-mm dishes and incubated with 30 M 17-octadecynoic acid (Cayman Chemical) and 5 g/ml cerulenin in labeling medium (minimum Eagle's medium containing 5% dialyzed FCS (Gemini BioProduct)) for 1 h followed by stimulation with 10 M ISO for 1 h. The cells were then washed and solubilized by sonication in ice-cold PBS containing 50 M PalmB and protease inhibitor mixture. To concentrate the membrane fraction, cell lysates were centrifuged at 100,000 ϫ g for 45 min at 4°C. The pellets were resuspended in lysis buffer and subjected to methanol-chloroform precipitation to remove excess probe and other probe-incorporated metabolites. The pellets were then resuspended in 1.2% SDS in PBS. After measuring the protein concentration, lysate containing 300 g of total protein was adjusted to a reaction volume of 300 l containing 100 M azide-PEG3-biotin (Sigma), 1 M neutralized tris(2-carboxyethyl)phosphine (TCEP), 100 M tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine dissolved in DMSO/tert-butanol (20:80%), and 1 M CuSO 4 (final reagent volume adjusted with PBS) and incubated for 1 h at room temperature in the dark. The proteins were then precipitated with chloroform-methanol, and the resultant pellets were dissolved in 120 l of 1% SDS in PBS. 20 l of solubilized proteins was retained to assess total input, and the remaining 100 l was incubated with 30 l of streptavidin-agarose in 1 ml of lysis buffer for 1 h at room temperature. After washing, the samples serving as specificity controls were incubated with 1 M hydroxylamine, pH 7.4, for 1 h at room temperature. The biotinylated proteins were eluted with 30 mM D-biotin in 2% SDS at 42°C. For Western blotting analysis, the eluted proteins were resolved by SDS-PAGE and visualized with anti-␤ 2 AR antibody. Western blotting signals were analyzed as done for acyl-RAC. The values obtained for S-palmitoylated ␤ 2 AR were normalized with respect to total ␤ 2 AR.
Detection of Phosphorylation of ␤ 2 AR Ser-262-HEK293 cells stably expressing FLAG-␤ 2 AR-C341A were harvested and lysed in 300 l of lysis buffer containing protease inhibitor mixture and PhosSTOP phosphatase inhibitor mixture (Roche Applied Science). FLAG-␤ 2 AR-C341A was immunoprecipitated using anti-FLAG M2 affinity gel for 30 min at 4°C and washed three times with lysis buffer. FLAG-␤ 2 AR-C341A was eluted in 30 l of SDS sample buffer containing 1% 2-mercaptoethanol at 42°C for 10 min followed by Western blotting. Phosphorylation of Ser-262 was visualized specifically with an anti-pS262 antibody (47,48).
Immunofluorescence Staining and Confocal Imaging-Cells in glass-bottom dishes were fixed with 4% paraformaldehyde/ PBS, permeabilized with 0.1% Tween 20 in PBS for 10 min, and incubated in 10% normal goat serum/PBS containing 0.1% Tween 20 for 30 min. After blocking, the cells were incubated with the appropriate primary antibody for 2 h at room temperature or overnight at 4°C followed by the respective fluorescence-tagged secondary antibody. Immunostaining was assessed by confocal immunofluorescence microscopy (Carl Zeiss, LSM700). Antibody Feeding Assay-Antibody feeding assays were performed essentially as described (35). Briefly, cells were fed 1 g/ml anti-FLAG M2 antibody in the presence or absence of 10 M ISO at 37°C for 30 min. After washing with PBS, cell-sur-face antibody was removed by an acid wash (0.5 M NaCl and 0.5% acetic acid, pH 1.0) for 1 min. To visualize internalized antibody, cells were washed with PBS, fixed, permeabilized with 0.1% Tween-20, and incubated with the respective fluorescence-tagged secondary antibody.
Western Blotting Analysis, Data Presentation, and Statistics-Western blotting signals were detected using the ChemiDoc XRS system (Bio-Rad) or ODYSSEY CLx (LI-COR Biosciences), and densitometric analysis was performed with Quantity One or Image Studio software, respectively. All comparisons were made between bands on a single blot. Densitometric values for S-palmitoylated ␤ 2 AR were normalized with respect to total ␤ 2 AR or in the analysis of cellular abundance of S-palmitoylated ␤ 2 AR (Fig. 1E) with respect to tubulin (which was unaffected by treatment).
All Western blotting data shown are from single blots at a single exposure. Within-blot cuts, for clarity of presentation, are indicated by dotted lines. All quantified data are presented as mean Ϯ S.D. Comparisons between groups were made by Student's t test or with one-way ANOVA and Tukey's post hoc test as appropriate. p Ͻ 0.05 was considered significant.