A Family of G Protein beta Subunits Translocate Reversibly from the Plasma Membrane to...

A Family of G Protein �� Subunits Translocate Reversiblyfrom the Plasma Membrane to Endomembranes onReceptor Activation*□S

Received for publication, February 8, 2007, and in revised form, April 13, 2007 Published, JBC Papers in Press, June 20, 2007, DOI 10.1074/jbc.M701191200

Deepak Kumar Saini‡, Vani Kalyanaraman‡, Mariangela Chisari‡, and Narasimhan Gautam‡§1

From the ‡Department of Anesthesiology and §Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

The present model of G protein activation by G protein-cou-pled receptors exclusively localizes their activation and functionto the plasmamembrane (PM).Observation of the spatiotempo-ral response of G protein subunits in a living cell to receptoractivation showed that 6 of the 12 members of the G protein �subunit family translocate specifically from the PM to endo-membranes. The � subunits translocate as �� complexes,whereas the � subunit is retained on the PM. Depending on the� subunit, translocation occurs predominantly to the Golgicomplex or the endoplasmic reticulum. The rate of transloca-tion also varies with the � subunit type. Different � subunits,thus, confer distinct spatiotemporal properties to translocation.A striking relationship exists between the amino acid sequencesof various � subunits and their translocation properties. � sub-units with similar translocation properties are more closelyrelated to each other. Consistent with this relationship, intro-ducing residues conserved in translocating subunits into a non-translocating subunit results in a gain of function. Inhibitors ofvesicle-mediated trafficking and palmitoylation suggest thattranslocation is diffusion-mediated and controlled by acylationsimilar to the shuttling of G protein subunits (Chisari, M., Saini,D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem.282, 24092–24098). These results suggest that the continualtesting of cytosolic surfaces of cell membranes by G proteinsubunits facilitates an activated cell surface receptor to directpotentially active G protein �� subunits to intracellularmembranes.

GPCR2 stimulation results in the activation of G protein �and �� subunit complexes which modulate the function ofdownstream effector molecules that function on the cytosolicsurface of the PM (1–4). The classic model of GPCR action,thus, restricts the activation of G proteins and consequently

their effectors to the two-dimensional plane of the PM (1–4).Intracellular effects have been thought to occur through secondmessengers released through the activation of effector mole-cules such adenylyl cyclase, phospholipase C�, and ion-con-ducting channels. Direct communication of a GPCRwith intra-cellular membranes through the G protein subunits has notbeen anticipated. We have used live cell imaging methods toexamine the spatiotemporal dynamics of the localization ofcomponents of a GPCR-mediated signaling pathway when thepathway is activated and deactivated.In mammalian cells similar to GPCRs the G protein subunits

are also families of proteins. Based on the presence of distinctgenes, there are 16 � subunits, 5 � subunit, and 12 � subunittypes. The � subunit types appear to possess distinctly differentproperties (3, 4). Although evidence exists for the differentialactivity of �� subunit types in terms of their role in receptoractivation of a G protein and modulation of effector function,these differences have been subtle and quantitative (5–12). Onepotential reason for a lack of evidence for qualitative differencesin the properties of these diverse proteins is that most assaysused so far tomeasure G protein function have used techniquesthat lead to disruption of cells. The possibility that these pro-teins are involved in spatially distinct functions has, thus,remained unexplored.Here we examined the entire family of � subunit types com-

plexedwith different� subunit types for potential translocationin response to GPCR activation in various cell lines. There havebeen previous indications from our laboratory that the �1�11and �1�5 subunit complexes translocate away from the PM onreceptor activation (13). To identify the mechanistic basis, thetranslocation properties were examined in the presence ofinhibitors of vesicle-mediated trafficking, an acylation inhibi-tor, and after introducing mutations into the � subunit C-ter-minal domain previously shown to interact with receptors (14).We identified potential �� complexes capable of translocationin a live cell by examining fluorescence resonance energy trans-fer (FRET) between fluorescent protein-tagged subunits. Weexamined the impact of cell lines of different origins and vari-ous � subunit types and receptors on the translocation of ��complexes. Although it has been known that a large and diversefamily of � subunits with highly conserved structures exists (12,15, 16), striking differences in their signaling properties havenot been found. Results here show that the � subunits controlthe spatiotemporally distinct translocation of a large variety of�� complexes from the PM to endomembranes, thus making it

* This work was supported by National Institutes of Health Grant GM 69027and an American Heart Association post-doctoral fellowship (to M. C.). Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 6 and Notes.

1 To whom correspondence should be addressed: Box 8054, Washington Uni-versity School of Medicine, St. Louis, MO 63110. Tel.: 314-362 8568; Fax:314-362-8571; E-mail: [email protected].

2 The abbreviations used are: GPCR, G protein-coupled receptor; PM, plasmamembrane; FRET, fluorescence resonance energy transfer; CHO, Chinesehamster ovary; ER, endoplasmic reticulum; CFP, cyan fluorescent protein;YFP, yellow fluorescent protein; 2BP, 2-bromopalmitate; mCh, mCherry.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 33, pp. 24099 –24108, August 17, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

AUGUST 17, 2007 • VOLUME 282 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 24099

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possible for GPCRs on the cell surface to direct an active com-ponent of a G protein to intracellular membranes.

MATERIALS AND METHODS

Chemicals and Expression Constructs—All chemicals werepurchased from Sigma unless otherwise indicated. �o-cyan flu-orescent protein (CFP), yellow fluorescent protein (YFP)-�1and CFP- or YFP-tagged �5 and �11 constructs have beendescribed previously (13). The citrine mutant of YFP and thenon-oligomerizing forms of both CFP and YFP (17) were engi-neered by mutagenesis and used in all experiments. The �1, �2,�3, �4, �7, �8 (�olf), �9 (�cone), �10, �12, �13, �1, �2, �3, and �4cDNAs were introduced downstream of YFP or mCherry (18)(from R. Tsien) in the pCDNA 3.1 or pDest vectors (Invitro-gen). All constructs were checked by determining the nucleo-tide sequence. Red fluorescent protein DsRed2-KDEL markerand DsRed monomer galactosyl transferase (Clontech). Trans-ferrin-Texas Red was fromMolecular Probes.Cells and Transfection—Native CHO cells and those stably

expressing theM2muscarinic receptor (M2-CHO) (19) and theM3 muscarinic receptor (M3-CHO) (20) have been describedpreviously. CHO cells were grown in CHO IIIa medium(Invitrogen) containing dialyzed fetal bovine serum (AtlantaBiologicals), methotrexate (for M2-CHO, M3-CHO), penicil-lin, streptomycin, glutamine, and Fungizone. HeLa and J774.1cell lines were obtained from American Type Culture Collec-tion and were grown in specific media recommended by theATCC. All the transfections were performed using Lipo-fectamine 2000 (Invitrogen). M2-CHO cells stably expressing�o-CFP, �1, and YFP-�11 were generated using standard proce-dures, screened with flow cytometry, and cultured in CHO-IIIamedium containing methotrexate and Geneticin (G418).Treatment of Cells—To disrupt Golgi in the cells, 10 �M

brefeldin A was used in the culture medium 6–10 h before cellimaging. For treatment with nocodazole, transfected or stablecells were cooled on ice for 5 min, then nocodazole (5 �g/ml)was added, and cells were further incubated for 15 min on icefollowed by incubation at 37 °C for 5min. As a control, 5 �g/mltransferrin-Texas Red conjugate was then added to the cells for15 min at 37 °C after which the cells were visualized to monitorvesicular trafficking. The imaging was done in the presence ofnocodazole. For monensin treatment the cells were treatedwith 50�Mmonensin for 1 h at 37 °C before imaging. For cyclo-heximide treatment the cells were treated with 50 �g/ml cyclo-heximide for 6–8 h at 37 °C before imaging. For treatment with2-bromopalmitate (2BP), the media was removed from thecells, and fresh Hanks’ buffer saline solution containing 50 �M2BP (stock prepared inMe2SO (21)) was added to the cells, andthe cells were incubated for a further 30 min at 37 °C beforeimaging.Imaging of Live Cells—Cells were cultured on acid-washed

glass coverslips and transiently transfected with appropriatecombinations of different G protein subunits and GPCRs asdescribed in the text and figure legends. After 16–24 h post-transfection the cells were processed for imaging as follows.The coverslips were washed with Hanks’ buffer saline solutionsupplemented with 10 mM HEPES, pH 7.4, and mounted on animaging chamber with an internal volume of 25 �l (RC-30

chamber, Warner Instruments). For live cell imaging, a fluiddelivery system including a programmable valve controller andTeflon valves (10-ms open/closure time) (Automate Scientific)was used to deliver the buffer with or without agonist or antag-onist through the chamber at a rate of 0.5 ml/min with a regu-lated flow controller. The cells were visualized with a ZeissAxioskop fluorescent microscope using a 63� oil immersionobjective (1.4 NA) and 100-watt mercury lamp with aHamamatsu CCD Orca-ER camera. The shutter and emissionand excitation filter wheels were controlled by a Sutter Lambda10–2 optical filter changer (Sutter Instrument Co.) run byMetaMorph 6.3.7 (Molecular Devices) software. The filter andbeam splitter combinations (Chroma Technology) were as fol-lows; for CFP, D436/10 excitation, D470/30 emission; for YFP,D500/20 excitation, D535/30 emission; for mCherry or DsRedD580/20 excitation, D630/60 emission, a polychroic beamsplitter (Chroma86002BS) and 25 or 10%neutral density filters.In those cases where both CFP and YFP fusions were co-ex-pressed in cells, the cells expressing relatively similar levels ofthe two fusions were selected based on emission intensities.Images were acquired at 20- or 10-s intervals, and the exposuretimes were between 0.1 and 1.0 s. Details of the FRET analysisare described in Chisari et al. (66). FRET analysis was per-formed not only with YFP- and CFP-tagged proteins but alsomCherry (mCh) and YFP combinations as described. Details ofthe experiments performed with confocal microscopy are pro-vided in Chisari et al. (66).

RESULTS

Receptor Induced �� Subunit Translocation—G protein sig-naling has been thought to be exclusively localized to the PMwith heterotrimers being activated by transmembrane recep-tors and the activated subunits acting on PM-associated effec-tors. We used CHO cells stably expressing M2 receptors (M2-CHO) transiently transfected with �o, �1, and different �subunits tagged with YFP to evaluate the effect of receptor acti-vation on various G protein � subunits. We have previouslydemonstrated that � and � subunits with an N-terminal fluo-rescent protein fusion localize normally on the PM and supportnormal activation of the G protein by a receptor (22). Images ofthe cells were captured at defined intervals of time, whereasthey were sequentially exposed first to a muscarinic receptoragonist, carbachol, and then to an antagonist, atropine. Emis-sion intensities from the intracellular membranes were plottedas a function of time to quantitate potential translocation of thefluorescent protein-tagged � subunit. The � subunits whichtranslocated were initially clearly localized to the PM (Fig. 1).When variousmembers of the � subunit family were examined,it was observed that six different � subunit types translocated tospecific intracellular membranes in a receptor-mediated man-ner. On antagonist addition they translocated back to the PM.The �1, �5, �9, and �10 translocated to a focused intracellularregion, which was similar to the cellular localization of thetranslocated �11 subunit previously reported (13) (Fig. 1A, toppanel, Fig. 1B, and supplemental Figs. 1 and 2). In striking con-trast, the �13 subunit translocated to endomembranes with amore diffuse spatial distribution (Fig. 1C, top panel). Corre-sponding to the changes in endomembrane fluorescence inten-

Translocation of a Family of G Protein �� Subunits

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sity in response to the agonist and antagonist, correspondingchanges in the opposing direction were observed in the PM(supplemental Fig. 3). In addition, a plot of the intensity of theGolgi marker, galactosyl transferase-DsRed, after exposure toagonist and antagonist did not show significant changes like the

intensity plot for YFP-�9 (supplemental Fig. 4). These resultsconfirmed that the increase in endomembrane fluorescenceintensity is not due to a change in Golgi size or shape.The G protein � subunits are modified with a prenyl moiety.

�1, �9, and �11 are farnesylated, whereas all other � subunits are

FIGURE 1. Receptor-mediated translocation of G protein �� complexes. M2-CHO cells transfected with �o, mCherry-�1, and YFP-tagged � subunits asindicated were used. Images of YFP-� subunits from transfected cells were captured every 10 or 20 s. Cells were exposed to 100 �M carbachol (Agonist) followedby 100 �M atropine (Antagonist) at the indicated time points. YFP intensity changes over time in Golgi or ER were plotted. Plots are of similar scales forcomparison. Representative images are shown. Mean pixel intensity measurements for plots were from regions enclosed in black circles or squares, indicatedin agonist-treated images. The decrease in fluorescence intensity over time is due to photobleaching. A, translocation of YFP-�9 to Golgi (n � 20). Shown areconfocal images of M2-CHO cells expressing �o, �1, YFP-�9, and galactosyl transferase (GalT)-DsRed monomer (DsRed) after exposure to carbachol (n � 8). B,translocation of YFP-�10 to the Golgi (n � 10). C, translocation of YFP-�13 to ER (n � 20). Cells were treated with brefeldin A to eliminate the Golgi. Shown areconfocal images of carbachol-exposed M2-CHO cells expressing �o, �1, YFP-�13, and KDEL-DsRed, an ER marker (n � 10). Note that the PM distribution of �13compared with the ER marker is visible (green peripheral signal) in the overlay. D, cladogram shows the relationship between primary structures and translo-cation properties. The predominant endomembrane targets after translocation of � subunit types are shown.




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geranylgeranylated (23). The translocation process by itself isnot dependent on the type of prenyl moiety attached to a �subunit type because �5, �10, and �13 are capable of transloca-tion, although they are geranylgeranylated.Endomembranes as Target Membranes for Translocating ��

Complexes—To identify the membranes to which the �9 and�13 subunits translocate, markers tagged with fluorescent pro-teins specific to the Golgi and the ER were used. A trans-Golgimarker, galactosyl transferase-tagged with monomeric DsRed(24, 25) and YFP-�9 were coexpressed in M2-CHO cells andimaged in a confocal microscope after M2 activation. Overlay-ing the images of galactosyl transferase-DsRed monomer andYFP-�9 showed that YFP-�9 localized specifically to the endo-membranes (predominantly Golgi complex) after translocation(Fig. 1A, bottom panel). To further confirm this localization, wetreated the transfected M2-CHO cells with brefeldin A, whichis known to disrupt the Golgi complex (26). When these cellswere examined after agonist treatment, translocation of YFP-�9to the Golgi region could no longer be detected (not shown).These results indicated that �9 translocates primarily to theGolgi region.To identify the membranes to which the G protein �1 �13

complex translocated, we coexpressed the ER retention KDELsequence of calreticulin, a specific marker for the ER taggedwithDsRed (27–29)withYFP-�13 inM2-CHOcells and imagedthe cells after M2 activation. To examine whether �13 translo-cates to the Golgi also apart from the ER, we treated the trans-fectedM2-CHO cells with brefeldin A. On agonist addition �13translocated to endomembranes. Overlaying the images ofKDEL-DsRed and translocated YFP-�13 showed that YFP-�13was localized to the ER (Fig. 1C, bottom panel). When antago-nist was added, �13 reverse-translocated back to the PM. The�13 subunit was initially localized to the PMand ER (Fig. 1C, toppanel). When cells expressing �10 were similarly examinedwith a Golgi marker, the target of the translocation was pre-dominantly the Golgi complex (not shown).We examined whether reverse translocation on the addition

of an antagonist subsequent to agonist addition was due to aneffect independent of the receptor. Instead of antagonist addi-tion, agonist was withdrawn by introducing buffer. The �9, �11,and �13 subunits reverse-translocated in these cells, similar tothe cells treated with antagonist, although the t1⁄2 for reversalwas comparatively slower (not shown). The slower kineticswould be anticipated because the antagonist would be able toshift the receptor to its inactive statemore rapidly by displacingagonist binding directly. This experiment indicated that reversetranslocation is the outcome of switching the activated state ofthe receptor to its inactive state.Subfamilies of � Subunits with Distinct Kinetics of Trans-

location—We measured the rate of translocation of the var-ious � subunits by measuring the proportion of the fluores-cent protein-tagged � subunit that translocated from PM to

intracellular membranes at various time points. When therate of translocation of the different � subunits was compared,distinct differenceswere seen. �10 and �5 translocated relativelyslowly (t1⁄2 � 39–85 s) (Fig. 1B, plot, and supplemental Fig. 2),whereas the other subunits, �1, �9, �11, and �13, translocatedrapidly (t1⁄2 � 6–13 s) (Fig. 1, A and C, supplemental Fig. 1). Toexamine the relationship between the translocating and non-translocating � subunits, the human � subunit amino acidsequences were aligned and analyzed for their evolutionaryrelationship. The resulting phylogenetic tree shows that the �subunits that translocate in a receptor induced fashion, �1, �5,�9,�10,�11, and�13, showa strikingly closer relationship to eachother compared with other � subunits (Fig. 1D and Fig. 2A).The two classes of � subunit types based on the rate of trans-location, relatively slow and rapid, also show a closer rela-tionship within a subfamily (Fig. 1D). Furthermore, �13 isdistinct compared with the other rapidly translocating �subunits. These results suggest that translocation is depend-ent on the amino acid sequences of the � subunits. This is aninference that is consistent with the nature of the prenylmoietynot having any discernible effect on translocation (below). Thedifferential translocations of the �� complexes are, thus, bothqualitative and quantitative. Such differences in the propertiesof G protein �� subunits have not been noted before.The G Protein � Subunit Mediates Translocation of a ��

Complex—The � subunit types, �o, �i, or �q, coexpressed witha translocating � subunit did not translocate (supplemental Fig.5A). This result suggested that translocation is restricted to the�� complex because the � subunit is known to be tightly boundto the � subunit (12, 15, 16). To confirm that the � subunitstranslocate as �� complexes, untagged � subunits wereexpressed with the �1 subunit tagged with YFP or mCh (18) inM2-CHO cells. In the presence of agonist, translocation of theYFP-�1 or mCh protein was observed (supplemental Fig. 5B).This result confirmed (i) � subunit translocation occurs as a ��complex, (ii) that the translocation process is directly influ-enced only by the type of � subunit, and (iii) the � subunitfluorescent protein tag does not have an effect on the translo-cation. To rule out the possibility that� subunit translocation isa result of interaction with a cellular protein other than the �subunit, which is capable of translocation, we evaluated recep-tor-mediated translocation in M2-CHO cells transfected withthe fluorescent protein-tagged �1 subunit without a translocat-ing � subunit. In such cells significant translocation of the �1subunit was not detected on receptor activation (data notshown). This result indicated that the � subunits directly medi-ate translocation of the �� complex. Furthermore, YFP ormCh-�1 not only cotranslocated with CFP- or YFP-taggedtranslocation-competent � subunits, but more important,acceptor (YFP or mCh) photobleaching indicated measurableFRET between YFP or mCh-�1 and CFP or YFP-� subunits as

FIGURE 2. Translocation of � subunit chimeras and mutants. A, the � subunit family is divided as in the cladogram (Fig. 1D) into four groups based on thespatiotemporal properties of translocation. Amino acids mostly conserved within the C-terminal domain of the subunits are highlighted. B, translocation of awild type and mutant �3 subunit. Mutated residues are highlighted in both the wild type and mutant �3. Transfected cells were assayed for translocation, andthe data are plotted as described in Fig. 1. Shown are representative data (n � 4). C, translocation of chimeric molecules of �9 and �13. Transfected cells wereassayed for translocation, and the data are plotted as described in Fig. 1. The images of cells after translocation were overlaid with corresponding images of thesame cells expressing Golgi or ER marker. Representative data are shown (n � 5).




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described below. These results confirm that translocationoccurs as a �� complex.Mutation of Residues at the C-terminal Domain of a Non-

translocating Subunit Results in the Gain of TranslocatingAbility—The ability of the G protein � subunit to influencereceptor-mediated translocation was reminiscent of the role ofthe � subunit in receptor interaction of a G protein that we firstshowed in the rhodopsin-transducin system and later withligand binding receptors (5, 6, 19, 30). It raised the possibilitythat receptor contact with the � subunit is a critical mediator oftranslocation. Previous evidence from the rhodopsin and mus-carinic receptor systems has suggested that an 11 amino aciddomain at the C terminus of the processed � subunit contactsreceptors (19, 30–33). Two-dimensional NMR has shown thata peptide encompassing this domain directly interacts withactivated but not inactive rhodopsin (14, 34). To examine thispossibility we compared the amino acid sequences of thehuman � subunits. Fig. 2A shows an alignment of the � sub-unit primary structures. In Fig. 2A residues in this C-terminaldomain conserved within the subfamily of subunits that trans-locate rapidly and the subfamily of subunits that do not trans-locate are highlighted. The conservation of these residueswithin the subfamilies with distinct translocation propertiessuggested that they may play a role in regulating translocation.We examined a hypothesis that the affinity of this C-terminaldomain of a � subunit for a receptor determines whether itwill translocate. To examine whether conserved residues thatare strikingly different between the non-translocating and

translocating subunits played a rolein the translocation, we mutatedfive residues in the �3 subunit thatdoes not translocate (Fig. 2B). The�3 mutant was expressed inM2-CHO cells, and receptor-in-duced translocation was examined.Results in Fig. 2B show that the �3mutant has gained the ability totranslocate due to the alterations inthe C-terminal domain. This resultshows that C-terminal residues ofthe � subunit are critical for translo-cation, consistent with a model thatnon-translocating � subunits have ahigh affinity for the activatedreceptor, whereas translocating �subunits have distinctly loweraffinity (32).Translocation to Different Endo-

membranes Is Not Dependent on theC-terminal Domain of the � Subunitor the Type of Prenyl Moiety—�13 istargeted predominantly to the ER,unlike �1, �9, and �11, which are tar-geted to the Golgi, although theyalso translocate relatively rapidly.We examined whether the C-termi-nal domains of the G protein � sub-units are involved in the differential

targeting of the translocating � subunit types. Because thisdomain is also post-translationally modified with different pre-nyl moieties (farnesyl or geranylgeranyl), these experimentsalso examined whether the prenyl moiety influences differen-tial targeting. TheC-terminal 14 residues of�9were substitutedwith the corresponding sequence of the �13 subunit (Fig. 2, AandC). This domain has been shown to be involved in receptorinteraction previously (14, 19, 30, 31). Although �9 is farnesyl-ated, the C-terminal residues of �13 (-CTIL; Fig. 3A) will ensurethat the chimera is geranylgeranylated in the cell as demon-strated before for such � subunit mutants using high perform-ance liquid chromatograph separation and mass spectrometry(35, 36). When the YFP-�9–�13 chimera was expressed inM2-CHO cells and its receptor-mediated translocation wasexamined, it translocated predominantly to the Golgi complex(Fig. 2C). In a similar experiment, a YFP-�13 chimera made upof the C-terminal 14 residues of �9 which will be farnesylated,was targeted predominantly to the ER (Fig. 2C). These resultsindicated that the targeting of the � subunits to different mem-branes is not influenced by the C-terminal domain of the �subunit. The results also show that the prenyl moiety does notplay a role in the differential targeting.Effect of Different � Subunit Types, Receptors, and Cell Types

on Translocation—The � subunits of G proteins are the mostdiverse among the G protein subunit families and are known tobe specific in their interaction with GPCRs (4). To evaluate theeffect of different � subunits on receptor-mediated transloca-tion of �� complexes, we examined translocation in the pres-

FIGURE 3. Effect of different � subunits on translocation of different � subunits. Plots of emission inten-sities from the intracellular membranes (Golgi or ER) as a function of time in CHO cells expressing M3 receptor,�q, mCh-�1, and YFP-tagged � subunits as indicated to quantitate potential translocation. Transfected cellswere assayed for translocation, and the data are plotted as described in Fig. 1. Representative data are shown(n � 4). A, translocation plot for YFP-�9 subunit. B, translocation plot for YFP-�13 subunit. C, translocation plotfor YFP-�10 subunit.




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ence of �q, which couples with M3 receptors (20). M3-CHOcells showed receptor-induced translocation of different � sub-units (Fig. 3). Similarly different GPCRs also induced �� trans-location (Fig. 4). Translocation occurred in a variety of cell lines(Fig. 5, A and B). �9 and �11 translocation was also detected inCOS 7 cells, and �11 was detected in human lung epithelial cells(not shown). These results showed that receptor-induced ��translocation is a general phenomenon. We then examinedwhether receptors endogenous to a cell induce translocation of�13, which is targeted to the ER. �2-adrenergic receptorsendogenous to HeLa cells (11) induced the translocation of�1�13 complexes (Fig. 5C), and hence, translocation is not aconsequence of heterologous expression of a receptor.Identification of the �� Complexes Capable of Translocation

in a Live Cell—To identify the � subunit types capable of form-ing a complex with the translocating � subunits in a live cell, weused two approaches.We examined the cotranslocation of YFPormCh-� subunit types in the presence of various translocating� subunit types tagged with YFP or CFP. Secondly, we exam-ined FRET between the various members of the translocatingfamily of � subunits and the four members of the � subunitfamily known to function as complexes with � subunits. mCh-or YFP-tagged � subunits were used as acceptors, and YFP- or

CFP-tagged � subunits were used asdonors. Acceptor photobleachingexperiments were used to examinefor the presence of a FRET signal aswe and others have done (22,37–39). We obtained a measurableFRET signal between some combi-nations of � and � subunits but notall, indicating which combinationsbound effectively with each other(supplemental Fig. 6 and Table 1).The cotranslocation of mCh-� sub-unit types was completely consist-ent with the results of the FRETexperiments; only those � subunitsthat provided FRET with a � sub-unit type induced co-translocationof that � subunit type. Togetherthese experiments showed that �1and �3 subunits form a complexwith all the translocating� subunits,whereas �2 and �4 form a complexwith only �5, �9, and �13 subunits(Table 1). We and others haveshown selective association of Gprotein � and � subunit types, andthese results both emphasize suchselectivity and also expand theexamination of association betweenmembers of these families to theentire family of potential �� com-plexes (40–44). Furthermore, theresults show that even if a cell con-tains � subunits that are capable oftranslocating, translocation of a ��

complex can occur only if the appropriate � subunit isexpressed in that cell.Translocation Process Is Controlled byAcylation and Is Likely

Diffusion-mediated—Evaluation of the kinetics of translocationand reverse translocation of �1, �9, �11, and �13 indicated thatthese subunits respond swiftly to both receptor-dependent ago-nist activation and antagonist inactivation as shown above (Fig.1 and supplemental Fig. 1). This rapidity of translocation inboth directions suggested that the translocation process ismostlikely diffusion-mediated. To examine whether the transloca-tion was vesicle-mediated or diffusive, we observed the recep-tor-induced translocation of the �11 subunit at 16 °C. At thistemperature, vesicle-mediated transport is inhibited com-pletely (45). Translocation induced by M2 receptor activationwas unaffected by lowering the temperature (data not shown),indicating that the translocation is most likely diffusion-medi-ated. To further confirm that the translocation is independentof vesicle-mediated trafficking, translocation of YFP-�11 wasexamined inM2-CHO cells after treating themwith amicrotu-bule disrupting agent, nocodazole, which blocks vesicular traf-ficking occurring through Golgi, and also monensin, whichblocks the trafficking of proteins from the Golgi to PM (26).Each of these treatments did not have an effect on �� translo-

FIGURE 4. Effect of different receptors on translocation of � subunits. Plots of emission intensities from theintracellular membranes (Golgi) as a function of time in CHO cells expressing different receptors as indicated toquantitate potential translocation of YFP-tagged � subunits as indicated. Transfected cells were assayed fortranslocation, and the data are plotted as described in Fig. 1. Representative data are shown (n � 4). A, trans-location plots in CHO cells transfected with �2 adrenergic receptor (�2AR) tagged with CFP, �o, mCh-�1 andYFP-�9 subunits. Transfected cells were exposed to 100 �M norepinephrine, and images were captured at theindicated time points followed by exposure to 20 �M yohimbine. B, translocation plots in CHO cells transfectedwith D2 dopamine receptor tagged with CFP, �o, mCh-�1 and YFP-�13 subunits. Transfected cells were exposedto 10 �M quinpirole, and images were captured at indicated time points followed by exposure to 100 nM

sulpiride. C, translocation plots in CHO cells transfected with �2 adrenergic receptor tagged with CFP, �o,mCh-�1 and YFP-�10 subunits. Transfected cells were exposed to 100 �M norepinephrine, and images werecaptured at the indicated time points followed by exposure to 20 �M yohimbine.




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cation, confirming the earlier observation that the transloca-tion process is most likely diffusion-mediated (not shown).Receptor-mediated translocation of the �� subunits was alsounaffected by inhibition of protein synthesis with cyclohexi-mide (not shown). This result showed that the translocationprocess does not require newly synthesized proteins. Further-more, we observed antagonist-mediated reverse translocationof �� in the presence of monensin, showing that receptor-gov-erned reverse translocation of the �� complex to the PM isindependent of forward vesicular trafficking of proteins fromGolgi to PM. Although the trafficking pathway that determinesG protein localization on the PMoriginating from theGolgi hasbeen shown to be both vesicle-mediated and non-vesicle-me-diated (46, 47), the results here show that, similar to forward

translocation, reverse transloca-tion is also likely to be entirely dif-fusion-mediated. These findingsare consistent with a prediction thattranslocating membrane-bindingproteins are likely to move rapidlyback and forth through the cytosoldiffusively (48).We determined that shuttling of

G protein subunits between the PMand endomembranes is likely regu-lated by an acylation/deacylationcycle because it is inhibited by 2BP(66), a well characterized inhibitorof palmitoyl transferases (49).When M2-CHO cells stably ex-pressing �o-CFP, �1, and YFP-�11were exposed to 2BP and receptor-induced translocation was exam-ined, translocation was significantlyinhibited in most cells (Fig. 6). Inaddition, 2BP inhibited ��11 trans-location by a different receptor, M3,in cells coexpressing �q (not shown).AFRETsignalwasdetected in thePMof the cells stably expressing �o-CFP,�1, andYFP-�11, and FRETwas abro-gated on receptor activation with anagonist showing that the G proteinpresent on the PM in 2BP-treatedcells was capable of getting activated(66). This result showed that activa-

tion of theG protein is not sufficient for translocation and an acy-lation dependent mechanism underlies this process.

DISCUSSION

Overall these results indicate that free�� subunits generatedas a result of G protein activation by a GPCR are directed toendomembranes from the PM. This spatial dislocation of acti-vated G protein molecules uncovers an unanticipated processin cellular regulation mediated by G proteins. As shown in theChisari et al. (66), the inactive heterotrimericGproteins shuttlebetween the PM and endomembranes. Because heterotrimersare inactive, the shuttlingwill not have on effect ondownstreameffectors. The striking effect of 2BP on both shuttling and trans-location and the possible diffusion-dependent movement inboth cases suggests that the shuttling process is harnessed forreceptor-mediated translocation of �� subunits. Thus, inactiveheterotrimer shuttling is converted to the translocation of apotentially active �� complex to specific endomembranes atdifferent rates.Two broad roles for the translocation of �� subunits can be

hypothesized. One possibility is that the translocation willreduce the�� available to act on effectors and to support recep-tor activation of an � subunit. We predicted that the � subtypeconstitution of a cell type can, thus, regulate signal amplifica-tion in a cell (13). Consistent with this prediction, a recentreport shows that translocation of ��1, which is specific to rod

FIGURE 5. Translocation of different � subunits in different cell lines. Plots of emission intensities from theintracellular membranes (Golgi or ER) as a function of time. Different cell lines (as indicated) were transfectedwith M3 receptor, �q, �1, and YFP-tagged � subunits as indicated to quantitate potential translocation. Trans-fected cells were assayed for translocation, and the data are plotted as described in Fig. 1. Representative dataare shown (n � 4). A, translocation plots of �13 in HeLa cells transfected with M3 receptor, �q, mCh-�1, andYFP-�13 subunits. B, translocation plots of �11 in J774 cells transfected with M2 receptor, �o, mCh-�1, andYFP-�11 subunits. C, translocation plots of �13 in HeLa cells transfected with �o, mCh-�1, and YFP-�13 subunitsstimulated through the endogenous �2 adrenergic receptor (�2AR).

TABLE 1Interaction between G protein � subunits and translocating �subunits (n > 4)Cotransl, cotranslocation; ND, not determined.

�1 �2 �3 �4

Cotransl FRET Cotransl FRET Cotransl FRET Cotransl FRET

�1 � � � ND � � � ND�5 � ND � � � � � ��9 � � � ND � � � ��10 � � � ND � � � ��11 � � � � � ND � ��13 � � � ND � � � �




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photoreceptors, helps photoreceptors adapt to light by reduc-ing the Gt available for activation by rhodopsin (50). Themech-anisms at the basis of the translocation are, however, quite dis-tinct from the translocation detected here. The translocation of�1 occurs along with the �t rod subunit, is comparatively veryslow (t1⁄2), and has been shown to be dependent on the nature ofthe prenyl moiety. Translocation occurs only when the �1 sub-unit is modified with farnesyl but not when it is modified withgeranylgeranyl. A related possibility is that�� translocation canregulate unwanted cross-talk. In a variety of cell types, G pro-tein activation leads to activation of a G�-mediated pathwaybut not the ��-mediated pathway. In heart cells Gs activatesadenylyl cyclase (through the �s subunit) but not K� channels,although �� is released and is known to be capable of acting onthe channel (51). A second potential function for the translo-cated �� subunits is regulation of unknown effectors in theGolgi or ER. Recent reports show that Ras isoforms are presentin endomembranes and are capable of getting activated at thatlocation (52). There is extensive evidence of biologically signif-icant cross-talk between GPCR and receptor-tyrosine kinase-mediated pathways (53). It is known that one mechanism forthe activation of Ras and MAPK by the G proteins is throughthe �� complex (54). Although there are suggestions that ��regulation of Ras activity may occur through transactivation ofa receptor-tyrosine kinase (55) or through direct action on Rasthrough Ras guanine nucleotide exchange factors (56), themechanisms at the basis of GPCR and receptor-tyrosine kinasepathways is still unclear, especially in intact cells. The GPCR-mediated translocation of a variety of �� complexes to endo-membranes provides a mechanism for regulating Ras activitydirectly or indirectly. Additionally and importantly, the trans-located �� may act on proteins that regulate trafficking of pro-teins through the endomembrane system. G protein subunitshave been shown to be associated with the Golgi complex, andevidence has been presented to show that they are capable ofregulating trafficking of proteins through the Golgi (57–62),but there has been little direct evidence indicating that thesefunctions are executed in a regulated manner (61). It has notbeen clear whether the G protein subunits identified remaintransiently in the endomembranes on their way to the plasmamembrane or are resident proteins of the endomembrane sys-

tem. Mechanisms that would allow these subunits to associatewith endomembranes and function there in a controllableman-ner have not been found. Such a mechanism is provided by theconstant testing of the surfaces of the PM and intracellularmembranes by a G protein (66), facilitating the translocation ofa potentially active �� complex on receptor stimulation. Thelarge family of � subunits with diverse sequences confers spa-tiotemporal complexity to this unanticipated signaling mecha-nism. �� complexes are targeted differentially to intracellularmembranes, ��13 predominantly to the ER and the others pre-dominantly to theGolgi. The temporal kinetics of translocationdiffers; ��1, ��9, ��11, and ��13 are rapid, ��5 and ��10 areslow, and the remaining �� subunits do not translocate.Although the existence of a large family of mammalian G pro-tein � subunits has been known for a long time (2, 63, 64), suchstriking distinctions in properties among members of the fam-ily have not previously been identified. The ability to alter anon-translocating subunit to a translocating subunit bydirected mutagenesis in a domain previously shown to interactwith receptors further emphasizes the importance of the differ-ences in the primary structures. It is also consistent with thehigh degree of evolutionary conservation of the � subunit-typeamino acid sequences (12, 65). To our knowledge this is theonly instance of activation-dependent reversible translocationof a family of proteins between the PM and endomembranes.

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Supplementary Fig 1.

A. Translocation of γ1 subunit. B. Translocation of γ11 subunit.

Images of M2-CHO cells transfected with αo, mCherry-β1 and YFP-γ subunits as indicated. Agonist and antagonist treatment were as above. Images of YFP emission were captured at 10 sec intervals. Emission intensities of YFP-γ subunit from the Golgi (within black circle) were plotted as a function of time. Representative (γ1: n=4 and γ11: n=7). The decrease in fluorescence intensity over time is due to photobleaching.

Basal Agonist Antagonist

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Supplementary Fig 2. Translocation of the γ5 subunit.

Plots of emission intensities from the Golgi of YFP-γ5 as a function of time in M2-CHO and M3-CHO cells as indicated. Experiments were performed as above. Representative (n=4). The decrease in fluorescence intensity over time is due to photobleaching.

A. Translocation plots of YFP-γ5 in M2-CHO cells cotransfected with αo and mCherry-β1. B. Translocation plots of YFP-γ5 in M3-CHO cells cotransfected with αq and mCherry-β1.

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Supplementary Fig 3.Translocation of βγ subunit monitored by changes in PM fluorescence Plots of emission intensities from PM of YFP-tagged γ subunits as indicated as a function of time in M2-CHO cells. The representative plots shown above demonstrate that fluorescence intensity changes in the PM shows a clear inverse correlation with that in the endomembranes (Fig. 1) in cells observed for translocation as showed in a previous publication (J. Biol. Chem (2004) 279, 51541). A. Translocation plots of YFP−γ11 in M2-CHO cells cotransfected with αo and mCherry−β1. B. Translocation plots of YFP−β1 in M2-CHO cells cotransfected with αo and γ9. Representative (n>10).

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Plots of emission intensities from the Golgi of YFP-γ9 and GalT-DsRed-monomer as a function of time in M2-CHO cells also transfected with αo and β1 subunits. Experiments were performed as above. Representative (n=8). The ratio of YFP-γ9 intensity and DsRed-GalT was determined in M2-CHO cells before and after agonist and antagonist addition. All other details were as in the main text and figure legends. The plot did not show a significant difference compared to the plots of translocation measured directly indicating translocation of the γ subunit was being measured in all plots shown earlier rather than changes in the shape/size of the Golgi.

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(A) α subunit localization is unaffected by receptor activation. Images of M2-CHO cells stably expressing αo-CFP, β1 and YFP-γ11 subunits. Transfected cells were assayed for translocation and the data was plotted as described in Fig. 1. Images of CFP emission were captured at 20 sec intervals. Representative (n>10). (B) β subunit cotranslocates. Images of M2–CHO cells transfected with αo, YFP−β1 and untagged γ9 subunit. Transfected cells were assayed for translocation and the data was plotted as described in Fig. 1. Each experiment was performed for n>8. The decrease in fluorescence intensity over time is due to photobleaching.


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G protein β and γ subunits tagged with corresponding donor/acceptor fluorescent protein pairs – CFP/YFP or YFP/mCherry were used for FRET analysis by acceptor photobleaching. The following pairs were expressed independently in M2-CHO cells and examined for FRET (i) mCherry-β1 and YFP-γ11 (ii) YFP-β2 and CFP-γ11. FRET efficiency was determined by monitoring gain in donor emission by photobleaching acceptor. Means ± SEM (n=5). Details are in the methods section. The values represented are not corrected for photobleaching of donor by acceptor bleaching.

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Notes 1. The variation in photobleaching is due to different time intervals between images. In Fig. 1B there is less bleaching because of fewer images being acquired at longer intervals and the resulting reduction in exposure of YFP (citrine) to light. In Fig. 1A and C more images were acquired because of the shorter time interval. Although citrine is less prone to bleaching compared to earlier generations of YFP, it is still more susceptible compared to CFP. The bleaching however does not affect the interpretation of the results. 2. The average fractional increase in Golgi was calculated as suggested and was found to vary in the range of 10 – 50 %. This variability can be due to the following (i) differences in the expression of tagged proteins between cells, (ii) differences in the initial PM versus Golgi ratio of the cell which depends on the cell state and (iii) differences due to the image plane along the Z axis. Such variations appear to be common in imaging protein activity in single live cells (e.g., Science (2004) 306, 1370-1373) because of the endemic differences between various cell characteristics. 3. We have consistently seen the presence of G proteins in the endomembranes on transient transfections and stable transfectants. We have not observed any specific correlation between levels of expression and endomembrane distribution of G proteins. In a stable cell line (M2-CHO αo-CFP β1 YFP-γ11-- described in the manuscript) the level of expression of the β subunit is expected to be 50,000 molecules per cell compared to the same number of endogenous β subunits as we have shown before in another stable line (M2-CHO αo-CFP β1) (Azpiazu I and Gautam N (2004) J Biol Chem 279, 27709). As γ subunits are degraded in the absence of β subunits, the transfected cells are likely to increase the level of βγ molecules only two fold. αo-CFP is also expressed at about 50,000 molecules per cell. The transient transfectant cells that we select for assays in general have fluorescent intensities that are comparable to the stable transfectants and therefore express G protein subunits at about the same levels.

Deepak Kumar Saini, Vani Kalyanaraman, Mariangela Chisari and Narasimhan GautamMembrane to Endomembranes on Receptor Activation

Subunits Translocate Reversibly from the PlasmaγβA Family of G Protein

doi: 10.1074/jbc.M701191200 originally published online June 20, 20072007, 282:24099-24108.J. Biol. Chem.

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A Family of G Protein beta Subunits Translocate Reversibly from the Plasma Membrane to...

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