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Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania
Received May 11, 2007; accepted June 26, 2007
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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2 modulates effectors, β5 associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate β5 complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 subunits and RGS7 to compete for interaction with β5. Among the subunits, β5 interacted preferentially with 2, followed by 7, and efficacy of phospholipase C-β2 activation correlated with amount of β5 complex formation. β5 also slightly preferred 2 over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), β5 interacted similarly with 2 and RGS7. Moreover, 2 interacted preferentially with β1 rather than β5. These results suggest that multiple coexpressed proteins influence β5 complex formation. Fluorescent β52 labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas β5RGS7 stained the cytoplasm diffusely. Coexpression of 
o targeted both β5 complexes to the plasma membrane, and q also targeted β52 to the plasma membrane. The constitutively activated o mutant, oR179C, produced greater targeting of β5RGS7 and less of β52 than did o. These results suggest that o may cycle between interactions with β52 or other β complexes when inactive, and β5RGS7 when active. Moreover, the ability of β52 to be targeted to the plasma membrane by subunits suggests that functional β52 complexes can form in intact cells and mediate signaling by G protein-coupled receptors.
; Jones et al., 2004). Also, unlike the other β subunits, β5 can associate with RGS proteins in the R7 family (RGS6, RGS7, RGS9, and RGS11), which interact with β5 via their G protein -like (GGL) domain (Jones et al., 2004). β5R7 complexes can activate the GTPase activity of o (Posner et al., 1999; Hooks et al., 2003) and accelerate both the activation and deactivation kinetics of GIRK channels (Kovoor et al., 2000; Drenan et al., 2006). Coexpression of β5 and RGS7 increases the expression levels of both proteins compared with when they are expressed individually (Witherow et al., 2000), and mice lacking β5 have reduced levels of R7 family RGS proteins (Chen et al., 2003), suggesting that these proteins are obligate dimers.
Whether β5 also interacts with G protein subunits in vivo is controversial. β52 can activate phospholipase C-β2 (Watson et al., 1994; Zhang et al., 1996; Lindorfer et al., 1998) and inhibit GIRK channels (Mirshahi et al., 2002; Lei et al., 2003) and N-type Ca+2 channels (Zhou et al., 2000). However, when purified from native tissues, β5 is associated with R7 family RGS proteins rather than subunits (Witherow et al., 2000). Complicating the issue, β52 dimers are unstable under nondenaturing buffer conditions (Jones and Garrison, 1999; Jones et al., 2004), which could explain why they have yet to be isolated.
Because G protein-coupled receptors and G protein subunits localize predominantly to the plasma membrane, complexes between β5 and either R7 family proteins or subunits would be expected to localize there as well to modulate signaling. Plasma membrane targeting of β5R7 complexes is promoted by association with both o (Takida et al., 2005) and R7BP (Drenan et al., 2006). Using BiFC, which involves the reconstitution of a fluorescent signal from nonfluorescent fragments of YFP or CFP when they are fused to interacting proteins (Kerppola, 2006), we previously visualized complexes between β5 and 1, 2, or 7 and found that they localized intracellularly rather than at the plasma membrane (Hynes et al., 2004b). This indicated that the β subunit could regulate targeting of β complexes, because these same subunits localized to the plasma membrane when associated with other β subunits. The β subunit, unlike the subunit, is not known to contain modifications that cause membrane targeting (Wedegaertner et al., 1995). However, one means by which β subunits could regulate targeting would be via association with subunits. Because o could target β5RGS7 to the plasma membrane (Takida et al., 2005), we hypothesized that coexpression of o and/or other subunits might lead to plasma membrane targeting of β5 complexes.
Here, using live cell-based assays, we address the issues of which proteins β5 forms complexes with, how complex formation and functionality are related, which subunits β5 complexes interact with, when in the GTPase cycle these interactions take place, and how the localization of β5 complexes is regulated. Using multicolor BiFC, we compare the abilities of seven subunits (1, 2, 5, 7, 10, 11, and 12) and RGS7 to compete for interaction with β5. Using a plasma membrane targeting assay, we compare the abilities of active and inactive o to target β52 and β5RGS7 to the plasma membrane and of o and q to target β52 to the plasma membrane. These studies demonstrate and quantify interactions that have not been detected using in vitro approaches and lead to a model for the roles of β5 complexes in regulating G protein signaling.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Subunit Constructs. YFP-N-β1 was produced as described previously (Hynes et al., 2004b). Cer-N-β1 and Cer-N-β5 were produced in the same manner as YFP-N-β1 using the human β1 and β5 cDNAs and Cer(1–158)pcDNAI/Amp, which was produced as described previously (Mervine et al., 2006) using monomeric Cerulean (Rizzo et al., 2004) (obtained from David Piston, Vanderbilt University, Nashville, TN), which contains S72A, Y145A, H148D, and A206K substitutions in ECFP. Cer-N-RGS7 was produced in the same manner as Cer-N-β1 and Cer-N-β5 using human RGS7-S2 (Guthrie cDNA Resource Center, Sayre, PA). For CFP-N-RGS7t, the procedure was the same except that the sequence amino terminal to the GGL domain was deleted by amplifying RGS7 residues 202 to 479 and the polymerase chain reaction product was subcloned into CFP(1–158)pcDNAI/Amp. Cer-N- constructs and CFP-C-β1 were produced as described previously (Mervine et al., 2006). YFP-N-2 was produced in the same manner as the Cer-N- constructs, using YFP(1–158)pcDNAI/Amp (Hynes et al., 2004b). CFP-C-β5 and CFP-C-2 were produced in the same manner as CFP-C-β1, using the human β5 and 2 cDNAs, respectively. Cer-C-β5 was produced in the same manner as CFP-C-β5, using Cer(159–238)pcDNAI/Amp.
mCherry-Mem was produced as described for mRFP-Mem (Mervine et al., 2006) except that mCherry (Shaner et al., 2004) (obtained from Roger Tsien, University of California, San Diego, CA) was used as the polymerase chain reaction template. pEYFP-Golgi, encoding a fusion protein consisting of EYFP and the amino-terminal 81 residues of human beta1,4-galactosyltransferase, which targets to the trans-medial region of the Golgi apparatus, was obtained from Clontech (Mountain View, CA). pEYFP-ER, encoding a fusion protein consisting of EYFP with the ER targeting sequence of calreticulin at the amino terminal end and the ER retrieval sequence, KDEL, at the carboxyl terminal end, was obtained from Clontech. pGM130-EGFP, encoding a fusion protein consisting of EGFP and GM130, a cis-Golgi matrix protein, was obtained from Graham Warren (Yale University, New Haven, CT).
3FLAG-R7BP, consisting of the R7BP coding region subcloned into p3FLAG-CMV10 (Sigma-Aldrich, St. Louis, MO) was obtained from Kendall Blumer (Washington University, St. Louis, MO). The human phospholipase C-β2 cDNA in pRc/CMV (Invitrogen, Carlsbad, CA) was obtained from Ravi Iyengar (Mount Sinai School of Medicine, New York, NY).
The EE epitope (EYMPTE) was introduced into the rat o-1 cDNA by replacing Asp167 with Glu and Gln169 with Met and Arg179 in o-EE was replaced by Cys to produce oR179C-EE by oligonucleotide-directed in vitro mutagenesis using the Bio-Rad Muta-Gene kit. s-YFP was produced as described for s-CFP (Hynes et al., 2004a) except that EYFP (Clontech) containing a substitution of Met for Gln69 was substituted for ECFP. q-YFPpcDNAI/Amp was produced from q-GFP/pcDNAI/Amp (Hughes et al., 2001). EYFP (Clontech) containing a substitution of Met for Gln69 and including S-G-G-G-G-S linkers on each end was substituted for GFP containing the same linkers as a BamHI/SacI cassette. This substitution was performed after the other BamHI and SacI sites in q-GFP were removed by silent mutations using oligonucleotide-directed in vitro mutagenesis and q-GFP was subcloned as a NotI insert into a modified version of pGEM-HE (Hughes et al., 2001) containing no BamHI or SacI sites. The resultant q-YFP cDNA was then subcloned into pcDNAI/Amp as a NotI insert. To produce o-YFP, a BglII site in the 5' untranslated region of o-EE/pcDNAI/Amp was removed by digestion with T4 DNA polymerase and religation and then a unique BglII site was introduced in frame between Pro119 and Phe120 in the B/C loop of the helical domain, analogous to the YFP insertion site in q-YFP, using polymerase chain reactions that produced DNA fragments with overlapping ends that were combined subsequently in a fusion polymerase chain reaction. EYFP (Clontech, Moutain View, CA) containing a substitution of Met for Gln69 and including S-G-G-G-G-S linkers on each end was then subcloned into the BglII site as a BamHI cassette. All subunit constructs used in this study contain the EE epitope. Henceforth in the text the EE designation is omitted for simplicity. All of the above constructs were verified by DNA sequencing.
Imaging of Transfected Cells by Spinning Disc Confocal Microscopy. HEK-293 cells (American Type Culture Collection, Manassas, VA) were plated at a density of 2 x 105 cells per well on four-well chambered coverslips (Lab-Tek II; Nalge Nunc International, Rochester, NY). On the following day, the cells were transiently transfected using 0.25 µl of LipofectAMINE 2000 Reagent (Invitrogen). Plasmids were transfected as described in the legends to Figs. 1, 5, 7, 9, and 10. A membrane marker (YFP-Mem or mCherry-Mem) was included in all transfections.
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The criteria for selecting cells for imaging were visible expression of all transfected fluorescent constructs, a clear section of plasma membrane border with adjacent region of cytoplasm, and a defined nucleus. The background intensity was determined by averaging the intensity of a region of pixels outside the cell and was subtracted from each image. All image processing was performed using IPlab software.
Normalized Cytoplasmic Standard Deviation. The normalized cytoplasmic standard deviation is a measure of the variation in pixel intensities within the cytoplasmic area of the cell. Using a Cintiq pen-based display screen (Wacom, Vancouver, WA), a membrane border 6 pixels wide and centered on the plasma membrane was drawn around the cell using the image of the plasma membrane marker. A separate nuclear border was drawn just inside the nucleus excluding any intensity in the nuclear membrane. The standard deviation of the intensities of pixels inside the membrane border and outside the nuclear border was calculated and normalized by dividing by the average intensity of the cytoplasmic pixels to correct for differences in the intensities of fluorescent complexes.
Nuclear-to-Cytoplasmic Intensity Ratio. The nuclear-to-cytoplasmic intensity ratio is a measure of the distribution of the labeled protein between the nuclear and cytoplasmic compartments and was determined using the membrane and nuclear borders defined above. The nuclear intensity was calculated as the average intensity of pixels in the nucleus including the border. The cytoplasmic intensity was calculated as the average intensity of pixels inside the membrane border and outside the nuclear border. The ratio is the nuclear intensity divided by the cytoplasmic intensity.
Plasma Membrane Fraction. The plasma membrane fraction is a measurement of the distribution of a labeled protein between the plasma membrane and cytoplasm and the method of its determination has been described in detail previously (Mervine et al., 2006). In brief, the plasma membrane-to-cytoplasm intensity ratio of the protein of interest is compared with that of plasma membrane and cytoplasm markers. A value of 0 corresponds to a completely cytoplasmic distribution, and a value of 1 corresponds to a completely plasma membrane distribution.
Colocalization of β52 with ER and Golgi Markers. To visualize colocalization of β52 with the ER or Golgi apparatus, Cer-C-β5Cer-N-2 was coexpressed in HEK-293 cells with YFP-ER, YFP-trans-medial Golgi, or GFP-cis-Golgi markers as described in the legend to Fig. 2. 3D Z-stacks (16 slices, 0.6 µm/slice) of live cells were collected on a Leica TCS SP2 confocal microscope using 458 nm and 514 nm laser lines for excitation of CFP and YFP. 3D Z-sections were analyzed because the regions of a cell with clear ER or Golgi structures often occur at different levels of focus. The Z-sections displayed in Fig. 2 were selected to highlight the structure of the coexpressed marker. Two color laser TIRF images were collected on a Nikon TE200-E microscope equipped with Perfect Focus, TIRF-2 illuminator, and 440 nm and 514 nm laser lines for excitation of CFP and YFP. To insure image registration, a triple-pass dichroic (Z442/514/594; Chroma, Brattleboro, VT) was used with emission filters in a motorized filter wheel (Ludl, Hawthorne, NY). The TIRF micrometer was motorized to control the TIRF angle for each laser line. Data collection was automated using IPLab software.
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2 image to generate a 3D subtracted image illustrating the remaining protein distribution that was not associated with the marker. The percentage subtracted was the amount that minimized the standard deviation of pixel intensities in the subtracted image, in the cytoplasm excluding the Golgi region for the ER marker, and in the cytoplasm region that included the Golgi region for the Golgi marker. The standard deviation minimum indicated that pixel intensity variations resulting from visible ER or Golgi structures had been optimally subtracted. Measurement of Fluorescence in Cell Populations. HEK-293 cells (1.6 x 106 per 60-mm dish) were transfected with plasmids as described in the legends to Figs. 3, 4, 6, 7, and 8 using 6 µl of Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions. Two days later, cells were washed once in 4 ml of HBSS + CaCl2 media (20 mM HEPES, pH 7.2, 118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, and 1 mM CaCl2). Two milliliters of HBSS + EDTA media (20 mM HEPES, pH 7.2, 118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, and 0.5 mM EDTA) were then added to the dish, and the cells were scraped off with a rubber policeman and resuspended in a 1-cm square glass cuvette with a magnetic stir bar.
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).
In multicolor BiFC experiments, the IC50 for inhibition of association of YFP-N-2 with CFP-C-β5 by Cer-N- subunits or Cer-N-RGS7 was defined as micrograms of Cer-N- subunit or Cer-N-RGS7 plasmid that produced a 50% decrease in the intensity of CFP-C-β5YFP-N-2. To determine IC50 values, the data were fit, using Kaleidograph (Abelbeck/Synergy Software, Reading, PA), to Y = (100)/(1 + (X/a)b), where X is micrograms of of transfected Cer-N- or Cer-N-RGS7 plasmid, Y is the percentage of maximal fluorescence produced by CFP-C-β5YFP-N-2, a is the half-maximal inhibitory concentration (IC50) of the Cer-N- subunit or Cer-N-RGS7, and b is the slope factor. The IC50 for inhibition of association of YFP-N-β1 with CFP-C-2 by Cer-N-β subunits was determined in the same manner.
Immunoblots. The expression levels of Cer-N-proteins were determined in HEK-293 cells (1.6 x 106 per 60-mm dish) that were transfected as described in the legends to Figs. 3, 6, 7, and 8 using 6 µl of Lipofectamine 2000 Reagent. Two days after transfection, total cell lysates (7.5 or 15 µg) were resolved on NuPAGE Bis-Tris 4 to 12% gels (Invitrogen) and transferred to nitrocellulose. The expression levels of the Cer-N- subunits were determined for Fig. 3 by probing with a polyclonal antibody to residues 3 to 17 of GFP (Anti-GFP, N-terminal; Sigma-Aldrich, St. Louis, MO), and the expression levels of Cer-N-2 and Cer-N-RGS7 were determined for Figs. 6 and 7 by probing with a polyclonal antibody to full-length GFP (Rockland Immunochemicals, Gilbertsville, PA). The antigen-antibody complexes were detected according to the ECL Western blotting protocol (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Chemiluminescence was imaged using a Lumi-Imager (Roche Applied Science, Indianapolis, IN). The expression levels of Cer-N-β1 and Cer-N-β5 were determined for Fig. 8 by probing with a polyclonal antibody to full-length GFP (Rockland Immunochemicals). The antigen-antibody complexes were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). Chemiluminescence was imaged using a FluorChem SP Imaging System (Alpha Innotech, San Leandro, California). Bands in the images were quantified using IPLab software.
The expression levels of EE-tagged subunits were determined for Fig. 9 using membranes prepared as described previously (Medina et al., 1996) 2 days after transfection of HEK-293 cells (4.45 x 106/100-mm dish) with 4.45 µg of each subunit plasmid using 5.56 µl of LipofectAMINE 2000 Reagent. 50 µg of membrane proteins were resolved by SDS-polyacrylamide electrophoresis (10%), transferred to nitrocellulose, and probed with a monoclonal antibody to the EE epitope. The antigen-antibody complexes were detected according to the ECL Western blotting protocol and chemiluminescence was imaged using a Lumi-Imager. Bands in the images were quantified using IPLab software.
Assay for Inositol Phosphate Accumulation in Transiently Transfected Cells. HEK-293 cells (1.6 x 106 per 60-mm dish) were transfected with plasmids as described in the legend to Fig. 4 using 6 µl of Lipofectamine 2000 Reagent according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were replated in 24-well plates and labeled with [3H]inositol (GE Healthcare). After an additional 24 h, inositol phosphate levels were determined in the presence of 5 mM LiCl as described previously (Medina et al., 1996).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Subunits Exhibited Distinct Localization Patterns. We investigated the ability of β5 to form complexes with 1, 2, 5, 7, 10, 11, and 12 in HEK-293 cells. β5 (Wang et al., 1999a) and each of these subunits, with the exception of 1 (Wang et al., 1997), have been detected at the protein level in these cells. β5 complexes were imaged using BiFC, which involves the production of fluorescence by two nonfluorescent fragments of CFP or YFP when they are brought together by interactions between proteins fused to each fragment. In contrast to fluorescence resonance energy transfer (FRET), in which the intensity of the signal depends on the distance between and relative orientation of two fluorophores, BiFC is based on the formation of a fluorescent complex from nonfluorescent constituents and does not require that the interacting proteins position the fluorescent protein fragments in a specific orientation or within a fixed distance from each other (Kerppola, 2006). However, steric constraints can prevent the association of the fluorescent protein fragments within a complex, in which case inserting peptide linkers between the fragments and the interacting proteins may enable association of the fluorescent fragments (Kerppola, 2006). We applied the BiFC approach previously to image β1 complexes, which were demonstrated to be functional by their abilities to potentiate activation of adenylyl cyclase by s in COS-7 cells (Hynes et al., 2004b) and to internalize in response to stimulation of the β2-adrenergic receptor in HEK-293 cells (Hynes et al., 2004a). For live cell imaging, we fused a carboxyl-terminal fragment (residues 159–238) of cerulean, an engineered form of ECFP that is 2.5-fold brighter than ECFP (Rizzo et al., 2004), to β5 to produce Cer-C-β5, and an aminoterminal cerulean fragment (residues 1–158) to the subunits, producing Cer-N- subunits.
Each of the Cer-C-β5Cer-N- complexes produced a fluorescent signal (Fig. 1, A–G). However, Cer-C-β5 and Cer-N-2, which produced one of the brightest signals when coexpressed, were not fluorescent when expressed individually (data not shown). The localization patterns of the β5 complexes varied depending on the associated subunit (Fig. 1), in agreement with a previous study of a subset of these β5 complexes (Hynes et al., 2004b), and in contrast to the corresponding β1 complexes, which localized predominantly to the plasma membrane (Mervine et al., 2006). The β5 complexes exhibited very little plasma membrane signal and varied in their distribution between the cytoplasm and the nucleus (Fig. 1, A–G, and J). β51, β55, β510, and β511 exhibited relatively high ratios of nuclear to cytoplasmic signal (0.63–0.88) (Fig. 1A, C, E, F, and J), whereas β52, β57, and β512 exhibited lower ratios of nuclear-to-cytoplasmic signal (0.36–0.45) (Fig. 1B, D, G, and J). For comparison, s, which, when over-expressed without exogenous β, labels the cytoplasm diffusely (Mervine et al., 2006) (Fig. 1H), yielded a ratio of nuclear-to-cytoplasmic signal (0.34) similar to that of β52 and β57 (Fig. 1J), suggesting that this amount of nuclear signal represents background labeling by proteins that are excluded from the nucleus. In contrast, mCherry, which diffuses freely between the nucleus and cytoplasm (Fig. 1I), exhibited a nuclear-to-cytoplasmic signal ratio of 0.97 (Fig. 1J).
The β5 complexes also varied in the degree to which their cytoplasmic signals were diffuse or associated with discrete intracellular structures (Fig. 1, A–G). This aspect of the cytoplasmic β5 signals was quantified by determining normalized cytoplasmic standard deviations of pixel intensity as described under Materials and Methods. This measurement indicates the extent to which labeled proteins in the cytoplasm are distributed evenly as free soluble proteins (low standard deviation), as opposed to being localized on discrete vesicles, membranes, or other structures that would increase the range of pixel intensities significantly (high standard deviation). Lower standard deviations were associated with the diffuse labeling patterns of s-YFP and mCherry (Fig. 1, H, I, and K). This analysis showed that β51 exhibited by far the lowest standard deviation and was comparable with s-YFP and mCherry (Fig. 1K). In contrast, β52 and β57 had the greatest standard deviation. The other β5 complexes had values that were closer to those of β52 and β57 than to that of β51. The diffuse nature of the β51 signal may be due, in part, to the fact that 1 is farnesylated, rather than geranylgeranylated (Wedegaertner et al., 1995). However, because 11 is also farnesylated and β511 exhibited much more discrete staining than did β51, additional differences between 1 and the other subunits seem to be important in determining the nature of the signal. In summary, these results show that the partitioning of β5 complexes between the cytoplasm and the nucleus and the nature of their distribution in the cytoplasm (diffuse or discrete) are determined by the subunit component.
The discrete cytoplasmic labeling observed with some of the β5 complexes, notably β52, seemed to reside on a number of intracellular structures, primarily the ER, Golgi apparatus, and nuclear membrane. To define the localization of these complexes more precisely, 3D stacks of images of β52 coexpressed with markers for the ER or the Golgi apparatus were collected on a laser-scanning confocal microscope (Fig. 2, A and C). Colocalization of β52 with both the ER and trans-medial Golgi apparatus was observed in the merge images (Fig. 2, A and C). Colocalization with a cis-Golgi marker was similar to that seen with the trans-medial Golgi marker (data not shown). To visualize β52 distribution not associated with the coexpressed markers, the marker images were subtracted from the β52 images as described under Materials and Methods. After subtraction of the ER images from the β52 images, significant intensity remained in the perinuclear region, as well as some diffuse cytoplasmic and nuclear membrane intensity (Fig. 2A). The perinuclear intensity of β52 was due primarily to the Golgi apparatus, with clear colocalization in the merge image and very little signal visible above the surrounding intensity of the ER and diffuse cytoplasm staining in the subtracted image (Fig. 2C). Colocalization of β52 with the ER marker was also observed using a two-color laser TIRF microscope, where the densely packed folds of the ER membranes seen in the confocal cross-sections were visualized distinctly (Fig. 2B).
β5 Interacted Preferentially with 2 Compared with 1, 5, 7, 10, 11, and 12. The intensities of CFP-C-β5Cer-N- complexes were quantified in HEK-293 cell populations using a spectrofluorometer. In the presence of an excess of CFP-C-β5, the intensities of the CFP-C-β5Cer-N- complexes varied over a 100-fold range, with CFP-C-β5Cer-N-2 and CFP-C-β5Cer-N-1 being the most and least intense, respectively (Fig. 3A). This range was much greater than the 3-fold range seen previously when the intensities of the corresponding CFP-C-β1Cer-N- complexes were compared under the same conditions (Mervine et al., 2006). The expression levels of the Cer-N- subunits, when coexpressed with excess CFP-C-β5, were compared by immunoblotting total cell lysates with an antibody to the amino terminus of GFP (Fig. 3B). There was a larger range in expression levels than when the same Cer-N- subunits were coexpressed with an excess of CFP-C-β1 (Mervine et al., 2006), suggesting that β1 and β5 differ in their abilities to stabilize these subunits. However, the range in Cer-N- expression levels (Fig. 3B) was narrower than the range in intensities of the CFP-C-β5Cer-N- complexes (Fig. 3A). CFP-C-β5Cer-N-2 exhibited by far the highest ratio of CFP-C-β5Cer-N- intensity to Cer-N- expression level, followed by CFP-C-β5Cer-N-7 (Fig. 3C). The CFP-C-β5Cer-N- intensity-to-Cer-N- expression ratio of CFP-C-β5Cer-N-2 was 18.6-fold greater than that of CFP-C-β5Cer-N-11, which had the lowest ratio (Fig. 3C). In contrast, the intensity-to-Cer-N- expression ratios of the corresponding CFP-C-β1Cer-N- complexes varied by 2-fold or less (Mervine et al., 2006).
Given that cells coexpress multiple isoforms of β and subunits, the predominance of particular β complexes will be influenced both by the relative expression levels and the association preferences of the expressed β and subunits. Although CFP-C-β5Cer-N-2 was clearly the most intense complex when CFP-C-β5 was not limiting, we sought to determine whether this was also the preferred complex when different subunits were coexpressed with a limiting amount of β5 and whether differences between the less preferred subunits would be revealed under these conditions. Multicolor BiFC makes it possible to simultaneously image multiple complexes and quantify the abilities of different proteins to compete for a limiting amount of a common binding partner, because the amino terminal fragment of the fluorescent protein determines the spectral properties of the complex (Grinberg et al., 2004). We found previously that the intensities of CFP-C-β1Cer-N- complexes were similar when CFP-C-β1 was not limiting, but when the intensities of coexpressed CFP-C-β1Cer-N- (cyan) and CFP-C-β1YFP-N-2 (yellow) complexes were compared under conditions in which CFP-C-β1 was limiting, the Cer-N- subunits exhibited an approximately 4.5-fold range in their abilities to compete with YFP-N-2 for association with CFP-C-β1 (Mervine et al., 2006).
The abilities of the Cer-N- subunits to compete with YFP-N-2 for association with limiting amounts of CFP-C-β5 were compared by determining the amounts of each Cer-N- subunit that decreased the intensity of CFP-C-β5YFP-N-2 by 50%. Cer-N-2 competed 18-fold more effectively than Cer-N-10 (the least effective competitor) and 2.7-fold more effectively than the next best competitor, Cer-N-7 (Fig. 3D, Table 1). When the amounts of transfected Cer-N- plasmids were normalized to their relative expression levels in the presence of limiting amounts of CFP-C-β5, Cer-N-2 was still the most effective Cer-N- subunit, competing 23-fold more effectively than Cer-N-11, the least effective subunit (Fig. 3E, Table 1). When expression levels were corrected for, Cer-N-1 became almost as effective in competition as Cer-N-7. Cer-N-2 was 4-fold more effective than Cer-N-7 and 6.5-fold more effective than Cer-N-1(Fig. 3E, Table 1). This range in the abilities of the Cer-N- subunits to compete for limiting amounts of CFP-C-β5 was much greater than that of their abilities to compete for CFP-C-β1 (Mervine et al., 2006) and the relative efficacies of the Cer-N- subunits were different, as described under Discussion.
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Efficacy of Phospholipase C-β2 Activation by β5 Combinations Was Correlated with the Amount of Complex Formation. Previous comparisons of the abilities of β5 complexes to modulate effectors demonstrated that cells expressing β52 exhibited greater phospholipase C-β2 activity than did cells expressing β51, β53, β54, β55, or β57 (Watson et al., 1994; Watson et al., 1996) and N-type Ca+2 channel inhibition was obtained in cells expressing β52 but not β51 or β53 (Zhou et al., 2000). These results indicated either that β52 was more effective than the other β5 complexes at modulating these effectors or that β52 complexes formed preferentially relative to the other β5 combinations. To distinguish between these two alternatives, we compared phospholipase C-β2 activity in cells expressing CFP-C-β5 and different Cer-N- subunits with the relative amounts of CFP-C-β5Cer-N- complex formation detected as BiFC. Four of the CFP-C-β5Cer-N- complexes (those containing Cer-N-2, Cer-N-5, Cer-N-7, or Cer-N-12) activated coexpressed phospholipase C-β2, whereas the other three complexes (those containing Cer-N-1, Cer-N-10, or Cer-N-11) produced no activation above that seen in cells transfected with empty vector (Fig. 4A). CFP-C-β5Cer-N-2 and CFP-C-β5Cer-N-7 exhibited the greatest activity. No activity was obtained when CFP-C-β5 was expressed without a Cer-N- subunit or when any of the Cer-N- subunits was expressed without CFP-C-β5 (Fig. 4A). The three CFP-C-β5Cer-N- complexes that did not stimulate phospholipase C-β2 exhibited only minimal fluorescence, indicating that lack of activity was due to ineffective complex formation (Fig. 4B). For the 4 CFP-C-β5Cer-N- complexes that activated phospholipase C-β2, the ratios of CFP-C-β5Cer-N--stimulated activity (Fig. 4A) to amount of complex formation (Fig. 4B) were similar (Fig. 4C), indicating that the different efficacies of the CFP-C-β5Cer-N- combinations were due primarily to different amounts of complex formation. This is the first time that β function in intact cells has been correlated directly with the amount of β complex formation.
β52 and β5RGS7 Complexes in the Same Cell Could Be Imaged Simultaneously using BiFC. The above studies demonstrated that β5 associates preferentially with 2 compared with the other subunits tested. To determine the association preference of β5 for subunits versus R7 family RGS proteins and to compare the localization patterns of these β5 complexes, we expressed fluorescent β52 and β5RGS7 complexes in the same cells. CFP-C-β5 was coexpressed with Cer-N-RGS7 and YFP-N-2 to produce CFP-C-β5Cer-N-RGS7 (cyan) and CFP-C-β5YFP-N-2 (yellow). Both complexes exhibited minimal localization to the nucleus (Fig. 5, A–C and E), compared with β51, β55, β510, and β511 (Fig. 1J). However, CFP-C-β5Cer-N-RGS7t, containing a truncated form of RGS7 in which the DEP domain was deleted, localized preferentially in the nucleus (Fig. 5, D and E), in agreement with a previous study of RGS6 splice variants that demonstrated that the DEP domain can function as a cytoplasmic retention signal (Chatterjee et al., 2003). In contrast to the distribution of CFP-C-β5YFP-N-2 on discrete structures in the cytoplasm (Fig. 5, A and C), the cytoplasmic signals of CFP-C-β5Cer-N-RGS7 and CFP-C-β5Cer-N-RGS7t were diffuse (Fig. 5, B and D). The different types of cytoplasmic signals were confirmed and quantified by the higher normalized standard deviation of cytoplasmic pixel intensity of CFP-C-β5YFP-N-2 compared with CFP-C-β5Cer-N-RGS7 and CFP-C-β5Cer-N-RGS7t (Fig. 5F). These results indicate that both subunits and R7 family RGS proteins can dictate the localization pattern of β5. Fluorescence was not obtained when CFP-C-β5, Cer-N-RGS7, or YFP-2 were expressed alone (data not shown), and minimal fluorescence was obtained when CFP-C-β1 and Cer-N-RGS7 were coexpressed (Fig. 6D), consistent with previous reports that β1 and R7 family RGS proteins do not interact (Snow et al., 1998; Posner et al., 1999).
β52 and β5RGS7 Complexes Formed with Equal Efficiency When Excess β5 Was Coexpressed with Either 2 or RGS7. In the presence of excess cotransfected CFP-C-β5 plasmid, linear relationships between the amounts of transfected Cer-N-2 and Cer-N-RGS7 plasmids and the intensities of CFP-C-β5Cer-N-2 and CFP-C-β5Cer-N-RGS7 complexes, respectively, were obtained (Fig. 6A). The intensity of CFP-C-β5Cer-N-2 was 19-fold greater than that of CFP-C-β5Cer-N-RGS7, based on the slopes of linear fits to the data. To determine whether this difference was due to a greater ability of CFP-C-β5 to form fluorescent complexes with Cer-N-2 compared with Cer-N-RGS7 or to differences in expression of the Cer-N fusion proteins, the expression levels of Cer-N-2 and Cer-N-RGS7, when coexpressed with excess CFP-C-β5, were determined using immunoblots. The slope of the linear fit to the Cer-N-2 data was 22-fold greater than that for Cer-N-RGS7 (Fig. 6B). The ratios of CFP-C-β5Cer-N-2 intensity to Cer-N-2 expression level and of CFP-C-β5Cer-N-RGS7 intensity to Cer-N-RGS7 expression level were used to normalize the β5-interacting abilities of 2 and RGS7 to their expression levels. As shown in Fig. 6C, these ratios were the same, indicating that 2 and RGS7 exhibit the same ability to interact with an excess of β5 when tested one at a time. In contrast, minimal fluorescence intensity was obtained with CFP-C-β1Cer-N-RGS7. When Cer-N-2 and Cer-N-RGS7 were coexpressed with an excess of CFP-C-β1, the intensity of CFP-C-β1Cer-N-2 was 254-fold greater than that of CFP-C-β1Cer-N-RGS7 (Fig. 6D). Under these conditions, the expression level of Cer-N-2 was 43-fold greater than that of Cer-N-RGS7 (Fig. 6E). The decreased expression of Cer-N-RGS7 relative to Cer-N-2 when coexpressed with CFP-C-β1 rather than CFP-C-β5 suggests that, despite the Cer-N tag, the stability of Cer-N-RGS7 is at least partially dependent on interaction with β5, consistent with losses of R7 family RGS proteins in β5 knockout mice (Chen et al., 2003). In addition, the ratio of CFP-C-β1Cer-N-2 intensity to Cer-N-2 expression level was 6-fold greater than that of CFP-C-β1Cer-N-RGS7 intensity to Cer-N-RGS7 expression level (Fig. 6F).
β5 Exhibited a Slight Preference for 2 over RGS7 That Was Eliminated in the Presence of R7BP. To determine whether preferential association of β5 with 2 or RGS7 would be revealed when a limiting amount of β5 was coexpressed with both 2 and RGS7 at the same time, we compared the abilities of Cer-N-RGS7 and Cer-N-2 to compete with YFP-N-2 for binding to CFP-C-β5. The amount of yellow fluorescence obtained from CFP-C-β5YFP-N-2 was measured when a range of amounts of either Cer-N-RGS7 or Cer-N-2 plasmid was coexpressed. The amount of Cer-N-RGS7 plasmid required to reduce the CFP-C-β5YFP-N-2 intensity by 50% was 8-fold higher than that of Cer-N-2 (Fig. 7A). When the amounts of Cer-N-RGS7 and Cer-N-2 plasmids used were normalized to their relative expression levels in the presence of limiting amounts of CFP-C-β5, three times as much Cer-N-RGS7 compared with Cer-N-2 was required to reduce the intensity of CFP-C-β5YFP-N-2 by 50% (Fig. 7B). Thus, β52 and β5RGS7 complexes can form simultaneously in intact cells, and β5 exhibits a slight preference for 2 over RGS7 when coexpressed with both potential binding partners.
β5RGS7 complexes can be targeted to the plasma membrane by R7BP, which, like β5 and RGS7, is highly expressed in the nervous system (Drenan et al., 2006). To determine whether R7BP can influence the formation of β52 and β5RGS7 complexes, we investigated whether coexpression of R7BP, at levels which targeted β5RGS7 to the plasma membrane (Fig. 7, C–E), affected competition between RGS7 and 2 for β5. We found that coexpression of R7BP decreased the preference of β5 for 2 over RGS7. In the presence of R7BP, the amount of Cer-N-RGS7 plasmid required to reduce the CFP-C-β5YFP-N-2 intensity by 50% was 5-fold higher than that of Cer-N-2 (Fig. 7F). When the amounts of Cer-N-RGS7 and Cer-N-2 plasmids used were normalized to their relative expression levels in the presence of R7BP and limiting amounts of CFP-C-β5, approximately the same amounts of Cer-N-RGS7 and Cer-N-2 reduced the intensity of CFP-C-β5YFP-N-2 by 50% (Fig. 7G).
2 Exhibited a Preference for β1 over β5 When the Three Proteins Were Coexpressed. The above studies compared the preferences of β5 for different interaction partners and demonstrated that 2 was preferred over the other six subunits tested and over RGS7 in the absence of R7BP. Because the prevalence of particular β5 complexes will reflect the interaction preferences of both β5 and its potential interaction partners, we investigated the interaction preferences of 2. The intensities of Cer-N-β1CFP-C-2 and Cer-N-β5CFP-C-2 complexes were compared as well as the abilities of Cer-N-β1 and Cer-N-β5 to compete with YFP-N-β1 for interaction with CFP-C-2.
In the presence of an excess of cotransfected CFP-C-2 plasmid, linear relationships between the amounts of transfected Cer-N-β1 and Cer-N-β5 plasmids and the intensities of Cer-N-β1CFP-C-2 and Cer-N-β5CFP-C-2 complexes, respectively, were obtained, and the intensities of the complexes were similar (Fig. 8A). The relationships between the expression levels of Cer-N-β1 and Cer-N-β5 and the amounts of transfected plasmid under these expression conditions were also linear and similar (Fig. 8B), resulting in similar ratios of Cer-N-βCFP-C-2 intensities to Cer-N-β subunit expression levels (Fig. 8C).
When the abilities of Cer-N-β1 and Cer-N-β5 to compete with YFP-N-β1 for interaction with CFP-C-2 were compared, the amount of Cer-N-β5 plasmid required to reduce the YFP-N-β1CFP-C-2 intensity by 50% was 4.2-fold higher than of that of Cer-N-β1 (Fig. 8D). The expression level of Cer-N-β1 was 0.98-fold of that of Cer-N-β5 in the presence of limiting amounts of CFP-C-2 (S.E. = 0.10, n = 3). This preferential interaction of 2 with β1 rather than β5 might be expected to work against the preference of β5 for 2 over RGS7 in cells coexpressing β1, β5, 2, and RGS7 by diverting some of the available 2 away from β5.
β52 and β5RGS7 Were Targeted Preferentially to the Plasma Membrane by Inactive and Activated o, Respectively. Although β12 and β22 localize to the plasma membrane, β52 accumulates on intracellular membranes, including the ER and the Golgi apparatus (Hynes et al., 2004b) (Figs. 1, 2, and 5). The ability of the β subunit to influence targeting of the subunit was surprising, because the β subunit is not known to have a membrane-targeting signal, such as the prenyl group on the subunit (Wedegaertner et al., 1995). Because o was reported recently to target β5RGS7 to the plasma membrane (Takida et al., 2005), we hypothesized that plasma membrane targeting of β52 also might require o, which is not expressed in HEK-293 cells (Wang et al., 1999b). Indeed, we found that coexpressed o did target both Cer-C-β5Cer-N-2 and Cer-C-β5Cer-N-RGS7 to the plasma membrane (Fig. 9, A–F). Because subunits in the inactive rather than the activated state have a higher affinity for β complexes, whereas RGS proteins interact preferentially with the activated form of subunits, we investigated how targeting of β52 and β5RGS7 were affected by an o mutant, oR179C, that is constitutively activated as a result of decreased GTPase activity. In agreement with the expected preferences of β52 and β5RGS7, oR179C was less effective than o at targeting β52 and more effective than o at targeting β5RGS7 (Fig. 9, A–F). The expression levels of o and oR179C, determined by immunoblotting membrane preparations using an antibody to the EE epitope included in both constructs, were similar (Fig. 9G). These results suggest that the inactive form of o interacts preferentially with β52, whereas activated o interacts preferentially with β5RGS7.
o and q Exhibited Similar Abilities to Target β52 to the Plasma Membrane. Our observation that o can target β52 to the plasma membranes of live cells is consistent with a previous in vitro study demonstrating that o can bind to β52 and prevent activation of phospholipase C-β2 (Yoshikawa et al., 2000) but contrasts with two other studies in reconstituted systems suggesting that β52 interacts with q but not other subunits (Fletcher et al., 1998; Lindorfer et al., 1998). To investigate the subunit specificity of β52 in live cells, we compared the abilities of o and q to target β52 to the plasma membrane using fluorescent versions of these subunits in which YFP was inserted at the homologous location in each. This made it possible to compare Cer-C-β5Cer-N-2 targeting by equivalent amounts of plasma membrane-associated o-YFP and q-YFP. This was important because previously we found that a significant amount of q-GFP localized to the cytoplasm in addition to the plasma membrane (Hughes et al., 2001). Although Cer-C-β5Cer-N-2 expressed in the absence of an -YFP subunit localized intracellularly (Fig. 10, A and B), both o-YFP (Fig. 10, C and D) and q-YFP (Fig. 10, E and F) targeted Cer-C-β5Cer-N-2 to the plasma membrane. When coexpressed with either o-YFP or q-YFP, the fraction of Cer-C-β5Cer-N-2 that associated with the plasma membrane varied linearly as a function of the ratio of o-YFP or q-YFP intensity in the plasma membrane to total cell intensity of Cer-C-β5Cer-N-2 (Fig. 10G). Moreover, the 2 -YFP constructs exhibited the same efficacy at targeting Cer-C-β5Cer-N-2 to the plasma membrane.
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2 relative to six other subunits, and also exhibits a modest preference for 2 over RGS7. These results shed some light on the quandary that despite the ability of β52 to modulate effectors (Watson et al., 1994; Zhang et al., 1996; Lindorfer et al., 1998; Zhou et al., 2000; Mirshahi et al., 2002; Lei et al., 2003), β5 is only associated with R7 family RGS proteins (Witherow et al., 2000) when purified from tissues. The instability of β52 dimers under nondenaturing buffer conditions (Jones and Garrison, 1999; Jones et al., 2004) may explain why they have not been isolated and illustrates the advantage of using BiFC in intact cells to identify protein interaction partners. The ability of β52 to be targeted to the plasma membrane by subunits supports the conclusion that functional β52 complexes can form in intact cells and mediate signaling by G protein-coupled receptors. The relative amounts of β5 versus β5R7 complexes in vivo will be influenced by the expression levels of potential β5 binding partners in addition to the association preferences of β5. Moreover, in the presence of coexpressed R7BP, β5 exhibited similar preferences for 2 and RGS7. Furthermore, 2 interacted preferentially with β1 rather than β5. Taken together, these results suggest that multiple coexpressed proteins affect β5 complex formation.
The interaction preferences of β5 identified using BiFC probably reflect association preferences, because BiFC generally seems to be irreversible (Kerppola, 2006). Efforts are under way in a number of laboratories to develop a reversible form of BiFC, which would enable dynamic analysis of reversible protein-protein interactions. Nevertheless, because monomeric β subunits and R7 family proteins are degraded rapidly in living cells (Wang et al., 1999a; Witherow et al., 2000; Chen et al., 2003), it seems unlikely that β5 exchanges binding partners in vivo. Association preferences identified using BiFC may reflect protein affinities, but other factors, such as accessibility or association with endogenous proteins, could also regulate complex formation in vivo. Comparisons of the affinities of β5 for different subunits may be possible in vitro, because purified β5 and 2 can be separated and then reassembled as a functional complex (Yoshikawa et al., 2000). However, it is not clear that RGS7 folds properly when expressed alone (Posner et al., 1999) or can be dissociated from β5 in a functional form. Alternatively, an elegant means of corroborating BiFC results is a recently described proximity ligation in situ assay that enables visualization of endogenous protein complexes in fixed samples (Söderberg et al., 2006).
Competition between R7 family RGS proteins and subunits for association with β5 and between β1 and β5 for 2 is likely to be of functional significance. Coexpression of RGS6 or RGS11 with β5 and 2 was found previously to impair β52-mediated inhibition of N-type Ca+2 channels (Zhou et al., 2000). In addition, GIRK channels are activated by β complexes containing β1–4 and inhibited by β5 complexes (Mirshahi et al., 2002; Lei et al., 2003). Our results suggest that β5 can inhibit GIRK channels both by means of competition between β5 and other β complexes for GIRK channel binding and via competition between β5 and other β subunits for subunit interaction.
Our observation that amounts of β5 complex formation correlated with efficacies of phospholipase C-β2 activation suggests that previous observations of the greater functionality of β52 compared with other β5 complexes (Watson et al., 1994, 1996; Zhou et al., 2000) resulted from more efficient formation of β52 dimers compared with the other combinations. Comparisons of the BiFC intensities and activities of β complexes should be a widely applicable means of elucidating the functional importance of specific β combinations in living cells.
The range of interaction preferences of β5 for the seven subunits studied was greater than that of β1 (Mervine et al., 2006). There were also differences in the relative order of effectiveness of the subunits in competing for β5 versus β1. In the case of β1, 12 was the most effective competitor, followed by 2 and 7, whereas 1 was the weakest. In contrast, 2 was the most effective competitor for β5, followed by 7 and 1, whereas 12 was one of the weaker competitors. In a given cell, the β complexes that predominate will reflect both the interaction preferences and expression levels of each expressed β and subunit.
Our imaging results indicate that the interaction partner of β5 plays an important role in the targeting of β5 complexes. Some complexes containing β5 (β51, β55, β510, and β511) exhibited significant localization to the nucleus in addition to the cytoplasm, whereas others (β52, β57, β512, and β5RGS7) localized predominantly to the cytoplasm. There is evidence for transcriptional regulation by RGS proteins (Burchett, 2003), but the potential functions of β5 complexes in the nucleus remain to be elucidated. The type of signal exhibited by cytoplasmic β5 complexes was also determined by the interacting partner, ranging from being diffuse (1 and RGS7) to discrete (2, 5, 7, 10, 11, 12).
β5 also influences the localization of β5 complexes, because complexes containing β1 and each of the seven subunits studied here localized to the plasma membrane (Mervine et al., 2006). β Subunits, unlike prenylated subunits, lack an identified membrane-targeting signal (Wedegaertner et al., 1995). Instead, the membrane-targeting signal of β5 seems to be subunit interaction, because coexpression with an subunit targeted both β52 and β5RGS7 to the plasma membrane. A significant amount of β52 colocalized with either the ER or the Golgi apparatus, suggesting that interaction with subunits can occur at these locations, resulting in plasma membrane targeting. Similar results and conclusions were reported previously for β1 complexes (Michaelson et al., 2002; Takida and Wedegaertner, 2003), whereas we observed relatively minor effects of subunits on β1 targeting (Mervine et al., 2006). Differences in the observed dependence of heterologously expressed β1 complexes on coexpressed subunits for plasma membrane targeting are most likely due to differences in the expression levels of the transfected β1 complexes relative to endogenous subunits.
Our targeting assay results address the issue of which subunit(s) β52 interacts with. β52 was targeted to the plasma membrane by o and q to the same extent. Two previous studies suggested that β52 interacts with q, but not other subunits, including o. For instance, β52 coupled the M1 muscarinic and ETB receptors to Gq, but failed to couple the ETB receptor to Gi1 in a reconstituted system (Lindorfer et al., 1998). In another study using purified proteins or membrane extracts, β52 interacted exclusively with q and not i1, i2, o, or s (Fletcher et al., 1998). However, a third study demonstrated that o could bind to β52 and prevent activation of phospholipase C-β2 (Yoshikawa et al., 2000). The lack of plasma membrane targeting of β52 in the absence of a coexpressed subunit suggests that the endogenous q expressed in HEK-293 cells is insufficient for targeting heterologously expressed β52, just as endogenous β is unable to target overexpressed s (Mervine et al., 2006) (Fig. 1H).
Subcellular localization of β5R7 complexes seems to be regulated by many factors. o-mediated targeting of β5RGS7 involves o-promoted palmitoylation of RGS7 (Takida et al., 2005). The preferential interaction between β5RGS7 and active rather than inactive o (Takida et al., 2005) (Fig. 9) suggests plasma membrane localization of β5R7 complexes could be induced upon G protein activation. Subcellular localization of β5RGS7 is also regulated by association with R7BP (Drenan et al., 2006) and may also be influenced by interaction with 14-3-3 proteins (Benzing et al., 2002). In addition, the DEP domains of certain RGS proteins can target them to specific G protein-coupled receptors (Kovoor et al., 2005; Ballon et al., 2006). Relationships between this multitude of factors that potentially can regulate β5R7 localization and function remain to be elucidated.
Taken together with previously reported findings, our data are consistent with a model in which β52 and β5RGS7 interact preferentially with the inactive and activated forms of o, respectively. When o was reconstituted into phospholipid vesicles with β12 and the M2 muscarinic receptor, β5RGS7 activated the GTPase activity of o upon carbachol stimulation (Hooks et al., 2003). A plausible scenario is that β52, like other β complexes, associates with inactive subunits and plays a role in mediating specific interactions with G protein-coupled receptors. In this scheme, receptor-mediated activation of β52 complexes leads to modulation of effectors such as phospholipase C-β2, GIRK channels, and N-type Ca+2 channels, whereas activation of oβ complexes enables β5RGS7 to bind to and activate the GTPase activity of o to regulate the kinetics of effector modulation by Go. In addition, because some RGS proteins can interact directly with receptors (Kovoor et al., 2005; Ballon et al., 2006), the signaling pathways of certain receptors may be mediated entirely by oβ5RGS7 complexes without the involvement of β dimers.
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Acknowledgements |
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subunits, Kendall Blumer (Washington University, St. Louis, MO) for the 3FLAG-R7BP plasmid, and Ravi Iyengar (Mount Sinai School of Medicine, New York, NY) for the human phospholipase C-β2 plasmid. |
Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: GGL, G protein -like; RGS, regulators of G protein signaling; BiFC, bimolecular fluorescence complementation; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; GFP, green fluorescent protein; ECFP, enhanced cyan fluorescent protein; HEK, human embryonic kidney; ER, endoplasmic reticulum; Cer, monomeric cerulean protein; R7BP, R7 family binding protein; 3D, three-dimensional; TIRF, total internal reflection fluorescence; GIRK, G protein-coupled inwardly rectifying potassium channel.
Address correspondence to: Catherine Berlot, Weis Center for Research, Geisinger Clinic, 100 North Academy Avenue, Danville, PA 17822-2623. E-mail: chberlot{at}geisinger.edu
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References |
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