From the
Department of Molecular and Cellular Pharmacology and the Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136 and
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom
Received for publication, December 18, 2002 , and in revised form, March 17, 2003.
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ABSTRACT |
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INTRODUCTION |
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Although the majority of RGS proteins have been shown to be GAPs, some RGS proteins can also act via non-GAP mechanisms. For example, although RGS4 and GAIP can both act as GAPs for Gq, they also regulate signal transduction by blocking effector binding (12, 13). Additionally, RGS12 and RGS14 contain a unique motif that binds to G
-GDP and acts as a guanine nucleotide dissociation inhibitor (14, 15).
With the exception of the G5L·RGS9 complex in photoreceptors, which has been demonstrated to be a GAP for transducin in vivo (9), the function of G
5·RGS complexes is not clear. For the G
5·RGS7 dimer, there are seemingly contradicting lines of evidence. Using in vitro GAP assays, it was shown that G
5·RGS7 acts exclusively on the G
o subunit, and not on G
q or even G
i (16). Despite this ability to act as a GAP, immunoprecipitation and pull-down assays have been unable to detect this interaction in vitro or in vivo (10, 11, 16). Furthermore, in cell-based experiments, monomeric RGS7 (17, 18) or its dimer with G
5 (11) attenuated G
q-mediated signaling, suggesting that in vitro assays lack some factors present in cells. Here, we used fluorescence resonance energy transfer (FRET) to study direct protein-protein interactions between RGS7 and G
subunits in living cells.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TransfectionHEK 293 cells were cultured in Eagle's minimal essential medium with Glutamax-1 and Earle's salts (Invitrogen) supplemented with nonessential amino acids (1%), fetal calf serum (10%), and gentamycin (0.5%). CHO-K1 cells were maintained in Ham's F-12 medium (Invitrogen) supplemented with 10% fetal calf serum with penicillin/streptomycin. Cells were incubated in a humidified atmosphere (95% O2, 5% CO2, 37 °C) with the culture medium replaced every third day and were passaged when they reached 80% confluence. For single cell imaging, cells were plated onto 25-mm borosilicate glass coverslips coated with 0.1% poly-L-lysine (Sigma). After 2 days in culture, cells were transfected with the relevant DNA using Genejuice (Calbiochem) at a ratio of 3:1 (µl of Genejuice:µg of DNA) according to the manufacturer's instructions. Cells were harvested or used for imaging 24 h post-transfection.
ImmunoprecipitationTransiently transfected CHO-K1 cells were lysed 24 h after transfection and extracted with 1% Triton X-100. The supernatant after centrifugation (100,000 x g) was immunoprecipitated using anti-RGS7 antibodies as described previously (11). Detection of YFP and CFP fusion proteins was achieved using a polyclonal antibody #83722 from Clontech (1:10,000 dilution).
Confocal Imaging and Calcium RecordingsCells on coverslips were loaded with fluo-3 in Krebs-Henseleit buffer (10 mM Hepes, 4.2 mM NaHCO3, 10 mM glucose, 1.18 mM MgSO4·7H2O, 1.18 mM KH2PO4, 4.69 mM KCl, 118 mM NaCl, 1.29 mM CaCl2 at pH 7.4) by incubation with fluo-3 AM (2 µM, prepared in anhydrous Me2SO) for 45 min at 20 °C followed by a further 45-min incubation in Krebs-Henseleit buffer to allow de-esterification of the indicator. Cells on coverslips were then mounted onto the stage of an Olympus IX50 inverted microscope and maintained at 37 °C. Measurement of [Ca2+]i was performed as follows. Fluo-3 fluorescence was monitored using an Olympus FV500 or PerkinElmer Life Sciences Ultraview confocal microscope with images captured every second. On-line analysis was carried out using Fluoview software provided by the manufacturer with raw fluorescence data exported to Microsoft Excel and expressed as fluo-3 fluorescence/basal fluo-3 fluorescence for each cell. Traces represent the average fluo-3 fluorescence recordings made from at least 10 transfected and 10 untransfected cells per coverslip and are representative of data obtained from at least six coverslips. RGS7-CFP-transfected cells were identified before calcium imaging, and the resultant calcium signals were compared between transfected and untransfected cells from the same field of view.
FRET SpectroscopyCHO-K1 cells were transfected in 100-mm plates as described earlier using a total of 8 µg of DNA. For RGS7 fusions, 4 µg of DNA was used for each plate, and for all other constructs (except YFP or CFP alone) 2 µg of DNA was used. For YFP or CFP alone, 0.5 µg of DNA was used for transfection. In all cases, lacZ was used as a carrier DNA to bring the total amount up to 8 µg.
FRET spectroscopy was performed similar to previous experiments done in yeast (19). To accurately measure FRET, each experiment required four types of cells. First, untransfected cells are needed to account for the background autofluorescence. Also, the YFP and CFP fusion proteins need to be expressed alone. Finally, the YFP and CFP fusions must be co-transfected. The cells were harvested 24 h post-transfection by washing twice with PBS and then gently scraped from the plate using 1 ml of PBS and stored on ice. Using a fluorescence spectrophotometer (SLM Instruments), the cells were initially diluted to 2 ml, and the fluorescence spectra of the samples were recorded. First, the emission spectrum of untransfected cells from 450 to 550 nm was recorded upon excitation at 433 nm (CFP emission). Second, the cells containing YFP alone were scanned at 433 nm. The same kinds of cells were also scanned from 500 to 550 nm upon excitation at 488 nm (YFP emission). Next, the cells containing both the CFP and YFP fusions were scanned at both CFP and YFP emission. Last, the cells containing CFP only were scanned at CFP emission. To accurately measure changes in FRET between the samples, the amount of total CFP in the CFP only and the CFP + YFP cells was normalized. To achieve this, either the CFP alone or the CFP + YFP cells were diluted and rescanned until the fluorescence of the CFP peak at 480-nm emission was identical between the samples. In most cases, only slight dilutions with PBS were needed to achieve this normalization. After acquisition of the spectra, the data was transferred to a spreadsheet (Microsoft Excel) for further analysis.
Mathematical manipulation of the spectral data was performed by initially subtracting the spectra acquired via excitation at 433 nm of the untransfected cells from the CFP alone and CFP + YFP emission spectra. Second, the CFP alone spectrum was subtracted from the CFP + YFP spectrum. To determine the amount of fluorescence due to background excitation of YFP, the spectra of YFP alone and the CFP + YFP pair at YFP emission were normalized. This normalization factor was then applied to the CFP + YFP trace. Subsequently, this trace and the YFP-alone trace were zeroed at 480 nm by adding or subtracting a constant to the entire trace. The resulting traces were then compared with the analysis of the final FRET data.
FRET MicroscopyCHO cells were transfected as described earlier in 6-well plates containing a square glass coverslip (18-mm length, 0.15-mm thick). A total of 1 µg of DNA was used for each transfection. For RGS7·G5 FRET, 0.5 µg of RGS7-YFP and 0.5 µg of G
5-CFP was used. For transfections with G
, 0.4 µg of RGS7-YFP, 0.4 µg of G
CFP, and 0.2 µg of G
5 was used. Cells were washed twice with PBS 24 h after transfection and then fixed using 3% paraformaldehyde in PBS for 20 min at room temperature. After fixation, the cells were washed three times with PBS and then three times with water before being mounted onto slides using ProLong anti-fade reagent (Molecular Probes).
All imaging was performed using a fluorescence imaging work station consisting of a Nikon Eclipse TE2000-U microscope equipped with a 60x oil objective lens, cooled charge-coupled device CoolSnap HQ camera (Photometrics), z-step motor, dual filter wheels, and a Xenon 175-W light source, all controlled by MetaMorph 5.0r2 software (Universal Imaging Corp.). Filter sets used were YFP (excitation, 500/20 nm; emission, 535/30 nm), CFP (excitation, 436/20 nm; emission, 480/40 nm), and FRET (excitation, 436/20 nm; emission, 535/30 nm) along with an 86004BS dichroic mirror (all from Chroma Technology Corp.).
To assay for FRET, cells of low to moderate expression of both YFP and CFP fusion proteins were selected. First, the cell was imaged with the FRET and CFP filters using an exposure time to produce significant, but non-saturating fluorescence of each representative pixel within the cell. For lower expressing cells, 2 x 2 binning was used to increase the sensitivity of the camera. Next, the cell was imaged for YFP, again making sure to obtain a non-saturating level of fluorescence. After YFP imaging, the field was then photobleached using YFP excitation light. Fluorescence was monitored live until the YFP signal was undetectable using the identical conditions as used to visualize YFP before bleaching. Immediately after photobleaching, FRET and CFP images of the same field were captured using identical parameters as used to obtain these images before photobleaching.
After acquisition of the images, the data was interpreted by comparing the CFP levels before and after YFP photobleaching (20, 21). FRET was identified as an increase in CFP fluorescence after photobleaching due to the inability of the photobleached YFP to accept emission from CFP. Simply, the CFP image obtained after photobleaching was subtracted by the CFP image before photobleaching. Because the amount of energy transferred from CFP to YFP represents a small portion of the overall CFP energy, the change in intensity is quite subtle. Thus, for representing the subtracted image, the scale on which the subtracted image is displayed had to be adjusted to 20% of the original scale.
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RESULTS |
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First, co-immunoprecipitation experiments carried out with RGS7 and G5 fusion proteins show that presence of YFP (or CFP) on either RGS7 or G
5 does not affect the ability of the proteins to dimerize (Fig. 1C). It should be noted that YFP and CFP differ only in a few amino acid mutations that allow for the differences in fluorescent properties between the proteins. Thus, localization and functionality of the CFP construct is assumed to be identical to the YFP fusion protein. Second, similarly to wild-type RGS7, RGS7CFP was able to inhibit muscarinic M3 receptor-mediated Ca2+ mobilization in HEK 293 cells (Fig. 2A). The addition of G
5 did not influence the ability of RGS7 to attenuate M3 receptor-mediated Ca2+ release in cells (Fig. 2, B and C). This is in apparent contrast to the previous report in which G
5 appeared to enhance RGS7 function (11). In those experiments, however, G
5 had a profound effect on the level of RGS7, most likely via protein stabilization. Here, the addition of the fluorescent tag on RGS7 stabilizes the protein, and co-transfection with G
5 does not noticeably increase the levels of RGS7CFP (data not shown). Thus, these experiments likely are more truly representative of the effect of the G
5 moiety within the RGS7/G
5 dimer.
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Localization of Fusion Proteins in CellsAfter transfection in HEK 293 cells, confocal microscopy was used to visualize CFP fusions of the proteins of interest. RGS7CFP was expressed primarily in the cytosol (Fig. 3A). In native tissue such as the brain a significant fraction of RGS7 is known to be associated with the membrane (10, 11, 22, 23). However, in HEK 293 cells and other non-neuronal cell lines, we have observed cytosolic staining, consistent with the observation in Fig. 3A. The pattern of immunostaining of wild-type RGS7 transfected in CHO cells as well as endogenous RGS7 in PC12 cells was consistent with the distribution of the YFP-tagged RGS7 (data not shown). GoCFP-CT was primarily associated with the membrane, suggesting that the presence of CFP did not affect the ability of G
o to be myristoylated and associate with the membrane (Fig. 3C). In contrast, G
qCFP-CT was largely cytosolic (Fig. 3D). We reasoned that the location of tag on the C terminus must inhibit its ability to be properly targeted. To compare FRET between G
o and G
q, we needed to place the fluorescent tag in the same place in both constructs; thus, despite its improper targeting, we used this construct for FRET spectroscopy experiments. However, to show that the interaction also occurs with G
q on the plasma membrane, we used a G
qCFP construct in which CFP is placed between the
B/
C loop of the G
q helical domain, similar to that described by Hughes et al. (24). Expression of this construct in cells showed that G
qCFP was indeed localized to the plasma membrane (Fig. 3E). We found localization of the G
fusion proteins to be highly dependent on the level of expression. Cells expressing low amounts of the fluorescent proteins localized on the plasma membrane, as shown in Figs. 3, C and E. However, many cells in the overall population overexpressed the protein to a larger extent, in which case the protein could be found throughout the cytoplasm (data not shown).
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Interaction between RGS7 and G5 in Living CellsTo validate FRET as a viable technique to study G protein subunits and RGSs in transfected cells, we initially tested the established interaction between G
5 and RGS7. Fluorescence spectra of untransfected cells, cells expressing RGS7YFP, G
5CFP alone, and co-expressing both were measured as described under "Experimental Procedures." Fig. 4A shows the raw data obtained from excitation at 433 nm. To correct for the inherent background fluorescence of cells, the spectrum from the untransfected cells was subtracted from the other traces (Fig. 4B). Subsequently, the G
5CFP trace was subtracted from the RGS7YFP + G
5CFP trace to correct for any CFP fluorescence in the cells containing both fluorophores (Fig. 4C). Next, to normalize the amount of acceptor (RGS7YFP) present between samples, the amount of YFP in each sample was determined by YFP emission upon excitation of the samples at 488 nm (Fig. 4D). This shows the total amount of YFP in the cells, and these values are normalized so all the curves overlap (Fig. 4E). The constant used for normalization obtained in Fig. 4E was then applied to the RGS7YFP + G
5CFP trace in Fig. 4C to obtain the final data (Fig. 4F). The final data show FRET occurring between RGS7 and G
5, as seen by the increase in YFP fluorescence at
528 nm in cells containing the CFP-YFP FRET pair. A negative control between RGS7YFP and CFP showed no nonspecific energy transfer from CFP to the RGS7YFP fusion (data not shown).
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In addition to spectroscopy, FRET was also visualized in individual cells using epifluorescence microscopy. In these assays, FRET was detected by comparing the overall CFP levels in cells before and after acceptor (YFP) photobleaching. CHO-K1 cells transfected with both RGS7YFP and G5CFP and visualized using CFP and YFP filter sets are shown in Fig. 5. After photobleaching of RGS7YFP, an increase in G
5CFP fluorescence was observed, indicative of FRET occurring between the proteins, confirming again that the proteins interact in cells.
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RGS7 Directly Interacts with Gq in CellsThe G
5·RGS7 dimer has been implicated in regulation of both G
o- and G
q-mediated signaling. To determine whether either of these proteins interacts directly with RGS7 in cells, FRET spectroscopy between RGS7 and G
was performed. Because FRET is highly dependent on the distance of the donor and acceptor fluorophores, we used G
proteins tagged in the same place (the C terminus) on the protein to accurately compare them to each other. FRET spectroscopy analyzing these interactions is shown in Fig. 6. FRET was more strongly detected between RGS7 and G
qCFP-CT than with G
oCFP-CT. This result indicates that RGS7 and G
q are proximal enough to each other to undergo FRET, almost assuredly due to their direct interaction in cells. We also were unable to detect FRET spectra in cells with G
qCFP-M, apparently because it is localized only at the membrane (Fig. 3E) or because the middle position of the tag makes it a less sensitive probe.
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RGS proteins are generally considered to have the highest affinity for G proteins in the transition state of GTP hydrolysis, a state mimicked by the addition of aluminum-magnesium-fluoride (20 mM sodium fluoride, 20 mM magnesium chloride, and 10 µM aluminum chloride). Surprisingly, the addition of aluminum-magnesium-fluoride did not alter the fluorescence spectra of any of the traces and, therefore, did not change the FRET signal between the two proteins (data not shown). We hypothesized that receptor-G protein coupling might be necessary for increased RGS affinity to G
. To test this idea, we transfected the constructs into CHO cells stably expressing the muscarinic type 1 (CHO-M1) or type 4 receptor (CHO-M4) and measured FRET in the presence and absence of muscarinic agonist. However, no effect of receptor presence or activation was observed (data not shown).
After the determination that Gq directly interacts with RGS7 in cells based on spectroscopy, we investigated this interaction using microscopy. In cells that do not express overwhelmingly high amounts of proteins, G
qCFP-M is localized primarily on the plasma membrane, whereas RGS7YFP is localized throughout the cell. FRET between RGS7 and G
q is observed essentially only on the plasma membrane (Fig. 7, AF). Interestingly, in FRET experiments performed with G
qCFP-CT, FRET was detected throughout the cell, suggesting that membrane localization of G
q is not required for the interaction with RGS7 (data not shown). It appeared that in cells with G
qCFP-CT the overall FRET signal was higher than in cells with G
qCFP-M, where it was membrane-delimited. Conversely, for the RGS7-G
o interaction, no discernable FRET was observed (Fig. 7, GL). The results of FRET microscopy confirm that RGS7 directly interacts with G
q in cells.
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DISCUSSION |
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This controversy between the GAP and cellular assays can be reconciled by two considerations. First, it is possible that G5·RGS7 acts on G
q via a non-GAP mechanism, similarly to that described for RGS4 and GAIP (12, 13). Second, the interaction of G
5·RGS7 with the G protein may require the presence of other components of the pathway, such as effector or receptor. It is known that the photoreceptor RGS protein, RGS9, acts as a GAP
5 times more effectively in the presence of the G protein effector
subunit of cGMP phosphodiesterase (9, 25). Similarly, GRK2 GAP activity is dependent on the presence of a G protein-coupled receptor (26). Indeed, the recent comprehensive study by Hooks et al. (27) shows that in the presence of receptors incorporated into lipid vesicles, RGS7 and other G protein
-like RGS proteins have activity to G
i subunits as well as G
o. Thus, the lack of RGS7 GAP activity toward G
q could be due to the lack of effector, receptor, or other necessary components that are present in a cell. These arguments could also explain the lack of a detectable interaction of the G
5·RGS7 dimer with other proteins through pull-downs and co-immunoprecipitation, as signaling complexes could be disrupted upon cell lysis (4, 11, 16).
To study the possible interaction between the G5·RGS7 dimer and G
subunits in cells, we used FRET spectroscopy and microscopy. We found that G
5·RGS7 interacts directly with G
q, supporting previous work of G
5·RGS7 inhibition of G
q signaling. Our inability to detect the interaction between G
5·RGS7 and G
o could be explained by the possibility that the orientation of the CFP tag on G
o is too distal from the YFP tag on RGS7 to detect their interaction. Additionally, the G
5·RGS7 interaction with G
o might be dependent upon phosphorylation and the subsequent binding of 14-3-3 to RGS7 (28). It is also possible that the high protein concentration required in the GAP assay could drive the interaction to occur in vitro (16), and in transfected cells the protein concentration is not high enough.
The G5·RGS7 Interaction with G
q Occurs in a Resting CellAccording to the currently accepted model, G
binds to RGS domains in the transition state of GTP hydrolysis. In our experiments, the G
q-RGS7 interaction was observed in a resting cell, arguing that it occurs with GDP-bound G
q. Although G
-GDP is not typically the preferred state for interaction with RGS proteins, such interactions have been previously reported. Some RGS proteins have a TPR motif, which binds to G
-GDP and acts as a guanine nucleotide dissociation inhibitor (14, 15). Similarly, G protein
-like-RGS proteins have the G
5/G protein
-like moiety, which should structurally resemble G
and bind G
-GDP. On the other hand, binding of monomeric RGS7 in vitro to G
o-GDP has been detected in a pull-down assay, where it was as high as 50% that in the presence of aluminum-magnesium-fluoride (5). The interaction between G
5L·RGS9 and G
t-GDP has also been shown (29). Earlier surface plasmon resonance studies of RGS16 and G
t revealed that in the presence of
subunit of cGMP phosphodiesterase the affinity of RGS16 to G
t-GDP-aluminum-magnesium-fluoride is only 2-fold higher than to G
t-GDP (30), also illustrating measurable interaction of some RGS proteins to G
-GDP. Finally, constitutively active G
subunits interact with RGS proteins, again suggesting that the transition state is not required for RGS binding (31, 32, 33). In a yeast two-hybrid assay, the interaction of RGS-GAIP was equally potent with wild-type G
i3 and constitutively active G
i3 (34), suggesting that the requirement for the transition state is not mandatory.
The RGS7-Gq interaction we observed in the resting cells may represent a low affinity interaction of RGS7 with the GDP-bound state of G
q or a small fraction of G
q bound to GTP due to basal receptor activity. Upon agonist stimulation we expected to see changes in the FRET signal due an increase proportion of GTP-bound G protein. Why did our FRET recordings not detect these changes? One possibility is that agonist-induced desensitization was so rapid (35) that the change in the interaction between G
and RGS7 occurs faster than we can record the FRET data. Another explanation is that receptor stimulation does affect the functional interaction between G
and RGS7 but does not result in the change of the distance between the fluorophore groups, which is required for the change in FRET. This idea is consistent with a model in which the receptor, G protein, and RGS do not physically dissociate but, rather, go through a series of conformational changes while progressing through the functional cycle. Such a model helps explain the rapid kinetics of many G protein cascades and the sub-second time constant of RGS-G
interactions (36). At this point it is not clear whether the dynamics of RGS-G
binding was not detected due the technical limitations of CFP-YFP FRET method or due to the true properties of G
q-G
5·RGS7 complex in cells. However, this work shows the feasibility of this approach for studying these proteins, and in accord with the previous data of RGS7 inhibition of G
q-mediated calcium mobilization, indicates a direct protein-protein interaction between RGS7 and G
q.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: University of Miami School of Medicine R-189, 1600 NW 10th Ave., Miami, FL 33136. Tel.: 305-243-3430; Fax: 305-243-4555; E-mail: v.slepak{at}miami.edu.
1 The abbreviations used are: RGS, regulator of G protein signaling; GAP, GTPase-activating protein; FRET, fluorescence resonance energy transfer; YFP, yellow fluorescence protein; CFP, cyan fluorescence proteins; HEK, human embryonic kidney; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.
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ACKNOWLEDGMENTS |
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REFERENCES |
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