Visualization of a Functional Galpha q-Green Fluorescent Protein Fusion in Living Cells

ASSOCIATION WITH THE PLASMA MEMBRANE IS DISRUPTED BY MUTATIONAL ACTIVATION AND BY ELIMINATION OF PALMITOYLATION SITES, BUT NOT BY ACTIVATION MEDIATED BY RECEPTORS OR AlF4-*,

Thomas E. HughesDagger , Hailin Zhang§, Diomedes E. Logothetis§, and Catherine H. Berlot||**

From the Dagger  Departments of Ophthalmology and Visual Sciences and the || Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026 and the § Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, New York 10029

Received for publication, August 21, 2000, and in revised form, October 16, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To investigate how G protein alpha  subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha q. alpha q-GFP stimulates phospholipase C in response to activated receptors and inhibits beta gamma -dependent activation of basal G protein-gated inwardly rectifying K+ currents as effectively as alpha q does. Association of alpha q-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha q palmitoylation sites, suggesting that alpha q must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF4- on the localization of alpha q-GFP in cells expressing both alpha q-GFP and a protein kinase Cgamma -red fluorescent protein fusion that translocates to the plasma membrane in response to activation of Gq. In cells that clearly exhibit protein kinase Cgamma -red fluorescent protein translocation responses, relocalization of alpha q-GFP is not observed. Thus, under conditions associated with palmitate turnover and beta gamma dissociation, alpha q-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha q receives an anchoring signal in addition to palmitoylation and association with beta gamma , or that during activation, one or both of these factors continues to retain alpha q in this location.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is good evidence that the in vivo localization and organization of G proteins and the proteins they interact with are important factors that regulate the specificity, kinetics, and magnitude of signaling pathways (1). Recent studies using fusions of GFP1 with G protein-coupled receptors, a G protein beta  subunit, and other proteins in the G protein signaling pathway, have provided new insights into how these proteins behave under basal and activated conditions (2-5). However, an important player in this pathway, the G protein alpha  subunit, has not yet been visualized in this way, presumably because the importance of both the amino and carboxyl termini for localization and function has precluded the fusion of GFP to these sites. Understanding the mechanism by which alpha  subunit localization is regulated requires tools such as GFP-tagged alpha  subunits that allow real-time visualization of alpha  subunits in living cells. Studies using conventional procedures such as cell fractionation and immunohistochemistry provide only a limited view of the process, are subject to artifacts, and have yielded conflicting results.

G protein alpha  subunits are peripheral membrane proteins that attach to the plasma membrane as a result of amino-terminal myristoylation and/or palmitoylation, and association with the beta gamma subunits. Whereas myristoylation occurs co-translationally and is stable throughout the lifetime of the alpha  subunit, palmitate is added post-translationally and turns over upon stimulation by activated receptors (6-10). Members of the alpha i family (alpha i1, alpha i2, alpha i3, alpha o, alpha z, and alpha t) are myristoylated and all except alpha t are also palmitoylated. alpha q and alpha s are palmitoylated, but not myristoylated. However, the existence of additional membrane-targetting signals associated with the amino termini of alpha q and alpha s has been proposed (11, 12).

For alpha q and alpha s, the roles of palmitoylation and association with beta gamma in regulating localization are controversial. Mutations of the palmitoylation sites have been reported either to cause these alpha  subunits to partition exclusively in the soluble rather than the particulate fraction (13) or to have little or no effect on the fractionation of these alpha  subunits (7, 11, 14-16). Mutation of alpha q residues important for beta gamma interaction has been reported to decrease palmitoylation and disrupt association with the plasma membrane (17), but mutational activation of alpha s, which should decrease association with beta gamma , has been reported to be associated with both decreased (6, 18) and normal (19) association with the plasma membrane.

Varying effects of receptor-mediated activation on the localization of alpha q and alpha s have been observed. beta -Adrenergic receptor stimulation has been reported to cause alpha s to move from the particulate to the soluble fraction (18, 20) and from the plasma membrane to the cytoplasm (18). However, in other studies, beta -adrenergic receptor-dependent relocation of alpha s was not observed (19, 21). Thyrotropin-releasing hormone induced patching of alpha 11 has been reported to occur within 10-60 min of hormone exposure, followed by internalization within 2-4 h, time scales that are slow compared with that of the activation cycle (22). alpha q/11 has also been reported to transiently translocate to the plasma membrane of adrenal glomerulosa cells in response to stimulation by angiotensin II for 1-5 min (23).

To elucidate the factors responsible for targeting of alpha q to the plasma membrane and to determine whether the cellular localization of alpha q changes during the activation cycle, we have followed alpha q localization in vivo under basal and activated conditions using a functional alpha q-GFP fusion protein in which GFP is inserted into an internal loop of alpha q. The effects of mutational activation on targeting of alpha q-GFP have been tested by substituting cysteine for Arg183 of alpha q, which inhibits GTPase activity (24). We have also tested the effects of mutating the alpha q palmitoylation sites, Cys9 and Cys10 (13), on alpha q-GFP localization. To determine whether membrane-associated alpha q-GFP relocalizes upon activation, the effects of stimulation by hormone-activated receptors or AlF4- have been tested. Our results indicate that although the basal activation state and palmitoylation are important for initially targeting alpha q-GFP to the plasma membrane, activation of membrane-associated alpha q-GFP, which is thought to cause palmitate turnover and dissociation from beta gamma , does not cause relocalization. These results suggest that upon reaching the plasma membrane alpha q receives an anchoring signal in addition to palmitoylation and association with beta gamma , or that during activation, one or both of these factors continues to retain alpha q in this location.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of GFP and RFP Fusion Proteins-- alpha q-GFP was generated from murine alpha q (25) and "humanized" GFP with the S65T mutation (26) using polymerase chain reactions that produced DNA fragments with overlapping ends that were combined subsequently in a fusion polymerase chain reaction (27). GFP was inserted in between alpha q residues 124 and 125. A 6-residue linker sequence (SGGGGS) was inserted at both of the junctions between alpha q and GFP using oligonucleotide-directed in vitro mutagenesis (28) using the Bio-Rad Muta-Gene kit. alpha qRC-GFP and alpha q-C9S/C10S-GFP were generated from alpha q-GFP using oligonucleotide-directed in vitro mutagenesis. All alpha q and alpha q-GFP constructs contain an epitope, referred to as the EE epitope (29) that was generated by mutating alpha q residues SYLPTQ (171) to EYMPTE. PKCgamma -RFP was produced by replacing a KpnI/NotI fragment encoding eGFP in pPKCgamma -EGFP (CLONTECH) with a KpnI/NotI fragment encoding RFP (CLONTECH). For expression in Xenopus oocytes, alpha q and alpha q-GFP were subcloned as NotI fragments into pGEM-HE (30). Subcloning and mutagenesis procedures were confirmed by restriction enzyme analysis and DNA sequencing.

Cell Fractionation-- HEK-293 cells (ATCC, CRL-1573) (12.5 × 106 per 150-mm dish) were transfected with 37.5 µg of plasmid using DEAE-dextran (31). 48 h after transfection, the cells were lysed by 10 passages through a 27-gauge needle in 0.5 ml of an ice-cold buffer containing 50 mM Tris (pH 8.0), 2.5 mM MgCl2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 1 mM dithiothreitol, and 10% glycerol. Nuclei were pelleted (750 × g, 5 min, 4 °C) and the postnuclear supernatant was then fractionated (18,000 × g, 30 min, 4 °C) into membrane pellets and supernatants. The pellets were washed once and resuspended in 0.5 ml of the same buffer. Membrane proteins were quantified using the Lowry assay (32). Samples (10 µg of membrane proteins and normalized volumes of the supernatant fractions) were resolved by SDS-polyacrylamide electrophoresis (10%), transferred to nitrocellulose, and probed with the anti-EE monoclonal antibody as described (33). The antigen-antibody complexes were detected using an anti-mouse horseradish peroxidase-linked antibody according to the ECL Western blotting protocol.

Inositol Phosphate Formation Assay-- Recombinant alpha  subunits were transiently expressed in HEK-293 cells using DEAE-dextran (31). 106 cells per 60-mm dish were transfected with 1 µg of vector containing the porcine alpha 2a-adrenergic receptor (34) and varying amounts of vector alone or vector containing an alpha  subunit construct as indicated in the figure legends. 24 h after transfection, the cells were replated in 24-well plates and labeled with [3H]inositol (5 µCi/ml for 24 h). The assay for intracellular inositol phosphates was performed as described (33).

Measurement of beta gamma -Dependent Activation of Basal G Protein-gated Inwardly Rectifying K+ Currents in Xenopus Oocytes-- A human homolog of GIRK4, referred to as GIRK4*, in which threonine is substituted for Ser143, was used as described previously (35). Briefly, plasmids expressing GIRK4*, alpha q, or alpha q-GFP were linearized with Nhel and cRNAs were transcribed in vitro using the "message machine" kit (Ambion). RNAs were electrophoresed on formaldehyde gels and concentrations were estimated from two dilutions using RNA marker (Life Technologies, Inc.) as a standard.

Xenopus oocytes were surgically extracted, dissociated, and defolliculated by collagenase treatment, and microinjected with 50 nl of a water solution containing 1 ng of the desired cRNA. Oocytes were incubated for 3 days at 19 °C. Whole oocyte currents were then measured by conventional two-microelectrode voltage clamp with a GeneClamp 500 amplifier (Axon Instruments). Agarose-cushion microelectrodes were used with resistance between 0.1 and 1.0 MOmega . Oocytes were constantly superfused with a high potassium solution having (in mM): 91 KCl, 1 NaCl, 1 MgCl2, and 5 KOH/HEPES (pH 7.4). To block currents, the oocyte chamber was perfused with solutions of the same composition with 3 mM BaCl2. Typically oocytes were held at 0 mV (EK) and currents were constantly monitored by 500-ms pulses to a command potential of -80 mV for 200 ms followed by a step to +80 mV for another 200 ms and the cycle was repeated every 2 s. Current amplitudes were measured at the end of the 200-ms pulse at each potential.

Imaging of GFP and RFP Fusion Proteins-- 0.5 × 106 HEK-293 cells were plated onto 35-mm tissue culture dishes containing a glass coverslip (MatTek Corp., Ashland, MA) and transiently transfected using LipofectAMINE 2000 Reagent (Life Technologies). Cells were imaged 48-72 h after transfection. The cells were imaged using an inverted Zeiss microscope or a confocal microscope (Bio-Rad MRC600). The inverted microscope was fitted with computer controlled (IPLabs, Scanalytics) filter wheels (Ludl electronics) on the excitation and emission paths to sequentially image the GFP and RFP signals (excitation and emission filters from HQ fluorescein isothiocyanate and Texas Red filter sets, dichroic mirror from fluorescein isothiocyanate/Texas Red Pinkel filter set; Chroma, Brattelboro, VT). For the confocal microscopy, factory supplied fluorescein isothiocyanate and Texas Red excitation/emission optics were used to image the GFP and RFP signals separately. The pixel densities across the membrane were fit with IGOR software (Wavemetrics, Oswego, OR).

Immunohistochemistry-- The cells were fixed in ice-cold, PBS-buffered paraformaldehyde. The cells were subsequently rinsed with PBS, and then incubated with 10% normal goat serum in PBS with 0.05% Triton X-100 for 30 min. The primary antibodies (a rabbit anti-GFP antibody from CLONTECH, a rabbit anti-peptide antibody to a COOH-terminal sequence of alpha q/alpha 11 from PerkinElmer Life Sciences, or a rabbit anti-peptide antibody to an internal sequence of alpha q/alpha 11 corresponding to residues 277-294 of alpha q from Sigma) were diluted with PBS and 10% normal goat serum with 0.05% Triton X-100 to a final concentration of 1:1000, 1:250, or 1:250, respectively, and added for an overnight incubation. The following day, the cells were washed with three changes of PBS, incubated for 60 min in a 1:500 dilution of a Texas Red-conjugated goat anti-rabbit secondary serum (Jackson ImmunoResearch) and then washed three times in PBS. All experiments involved examining plates processed in parallel that were not exposed to the primary antibody. The signal to noise in each experiment was determined by comparing the signals produced in the presence and absence of the primary antibody.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha q and alpha q-GFP Exhibit Similar Functional Properties-- To produce a functional alpha q-GFP fusion protein, GFP was inserted in between alpha q residues 124 and 125 in the alpha B/alpha C loop of the helical domain (Fig. 1). The rationale for this fusion strategy was that the helical domain of alpha q does not specify activation of PLC (33), and the insertion site corresponds to a region in GPA1, a G protein alpha  subunit in Saccharomyces cerevisae, in which there is an insertion of ~100 residues relative to other alpha  subunits (36, 37). This design avoided modifying the amino or carboxyl termini of alpha q, which are important for interaction with receptors, PLC, and beta gamma , and for membrane attachment (11, 38). A 6-residue linker sequence (Ser-Gly-Gly-Gly-Gly-Ser) was placed at each of the junctions between alpha q and GFP. To enable comparison of the expression levels of alpha q and alpha q-GFP, both constructs contain an epitope, referred to as the EE-epitope (29), located in the alpha E/alpha F loop of the helical domain. This epitope does not interfere with receptor-mediated stimulation of PLC by alpha q (39).



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Fig. 1.   Model of alpha q-GFP. The structure of GFP (54) is green. The alpha  subunit structure is that of alpha t·GTPgamma S (55). The helical domain is pink and the GTPase domain is light blue. GTPgamma S is yellow. The SGGGGS linkers between GFP and the alpha  subunit are shown schematically in dark blue. This figure was drawn using MidasPlus, developed by the Computer Graphics Laboratory at University of California, San Francisco.

Cell fractionation and immunoblotting with an anti-EE monoclonal antibody showed that alpha q and alpha q-GFP were expressed in transiently transfected HEK-293 cells at similar levels and exhibited similar distribution patterns (Fig. 2). In addition to associating with the membrane pellet, both alpha q and alpha q-GFP were isolated in the cytoplasmic fractions. To investigate whether the activation state of alpha q affects targeting to the plasma membrane, GFP was inserted into alpha qRC, in which substitution of cysteine for Arg183 causes constitutive activation of alpha q by reducing its GTPase activity (24). For both alpha q and alpha q-GFP, the RC mutation caused a minor increase in the relative amount of protein that fractionated in the supernatant compared with the amount in the membrane pellet. To test the role of palmitoylation in membrane targeting, GFP was also inserted into alpha q-C9S/C10S, in which serines were substituted for the alpha q palmitoylation sites (13). The C9S/C10S mutations also increased the relative amount of both alpha q and alpha q-GFP in the supernatant. The effect of the C9S/C10S mutations was more pronounced than that of the RC mutation, but neither alpha q-C9S/C10S nor alpha q-C9S/C10S-GFP was completely soluble.



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Fig. 2.   Expression of alpha q and alpha q-GFP constructs in HEK-293 cells. HEK-293 cells were transfected with 3 µg/106 cells of plasmid encoding the indicated constructs. The cells were fractionated into membrane pellets (P), and supernatants (S), which were separated using SDS-polyacrylamide gel electophoresis and immunoblotted as described under "Experimental Procedures." The fractionation patterns of alpha q, alpha q-R183C, and alpha q-C9S/C10S are similar or identical to those of the corresponding GFP-fusion proteins. The R183C mutation modestly increases the relative amount of protein in the supernatant, while the C9S/C10S mutations have a more pronounced effect. Similar results were obtained in two additional experiments.

alpha q-GFP and alpha q exhibited indistinguishable abilities to stimulate PLC in response to stimulation by the alpha 2a-adrenergic receptor in transiently transfected HEK-293 cells (Fig. 3). Although these cells express alpha q endogenously, inositol phosphate production in response to stimulation with the alpha 2-adrenergic agonist, UK-14,304, was minimal in cells that were transfected solely with plasmid encoding the alpha 2a-adrenergic receptor. Co-transfection of these cells with plasmid encoding either alpha q or alpha q-GFP resulted in agonist-dependent increases in inositol phosphate production. The relationship between the magnitude of the response and the amount of transfected plasmid was the same for the alpha q- and alpha q-GFP-expressing plasmids. Thus, the ability of alpha q to interact with receptors and effectors is not impaired by the GFP insertion.



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Fig. 3.   alpha q and alpha q-GFP exhibit identical abilities to stimulate PLC in response to receptor stimulation. For each data point, 106 HEK-293 cells were transfected with 1 µg of plasmid encoding the alpha 2a-adrenergic receptor and the indicated amounts of plasmid encoding alpha q (circles) or alpha q-GFP (squares). Inositol phosphate (IP) formation was measured in the presence (filled symbols) or absence (open symbols) of 10 µM UK-14,304. The relationship between the amount of IP formation and the amount of transfected plasmid is the same for the alpha q- and alpha q-GFP-encoding plasmids. Values represent the means of triplicate determinations ± S.D. from a single experiment, which is representative of 2 such experiments.

As for alpha q, the RC mutation caused constitutive activation of alpha q-GFP (Fig. 4). Stimulation of cells co-transfected with plasmid encoding the alpha 2a-adrenergic receptor and plasmid encoding either alpha qRC or alpha qRC-GFP with UK-14,304 resulted in further elevation of inositol phosphate levels, indicating that the RC mutation inhibits, but does not abolish GTP hydrolysis. In contrast to alpha q-GFP, which stimulated PLC to the same extent as alpha q did (Figs. 3 and 4), alpha qRC-GFP was less effective at PLC stimulation than alpha qRC was. Since the RC mutation has been reported to destabilize alpha s (20), it is possible that combining this mutation with the insertion of GFP results in decreased stability of alpha qRC-GFP compared with that of alpha q-GFP or alpha qRC.



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Fig. 4.   Substitution of Arg183 in alpha q with cysteine (the RC mutation) causes constitutive activation of alpha q-GFP. 106 HEK-293 cells were transfected with 3 µg of vector containing the indicated construct and 1 µg of vector containing the alpha 2a-adrenergic receptor. Inositol phosphate (IP) formation was measured in the presence (light gray bars) or absence (dark gray bars) of 10 µM UK-14,304. The RC mutation increases the basal activity of both alpha q and alpha q-GFP. Further increases in activity in response to UK-14,304 indicate that this mutation inhibits, but does not abolish GTPase activity. Values represent the means of triplicate determinations ± S.D. from a single experiment, which is representative of three such experiments.

alpha q-GFP and alpha q also exhibited identical abilities to interact with beta gamma , as demonstrated by inhibition of beta gamma -dependent activity of the G protein-gated inwardly rectifying K+ channel, GIRK4 (Fig. 5). Agonist-independent (basal) potassium currents through GIRK4 homotetrameric channels expressed in Xenopus oocytes can be inhibited by coexpression of alpha i subunits (35, 40). Since GIRK channels bind to and are activated by only the beta gamma subunits of G proteins (41, 42), this inhibition serves as a functional assay of alpha  subunit binding to beta gamma subunits. Coexpression of alpha q displayed the same inhibitory effect on beta gamma -stimulated basal GIRK4 currents. Coexpression of alpha q-GFP with GIRK4 channels reduced K+ currents to the same extent as alpha q did, indicating that GFP-tagged alpha q can bind to beta gamma and prevent beta gamma -mediated activation of the channel as efficiently as alpha q can.



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Fig. 5.   alpha q and alpha q-GFP exhibit identical abilities to inhibit beta gamma -dependent basal activity of GIRK4. K+ currents were recorded from a GIRK4 mutant, GIRK4(S143T), referred to as GIRK4*, that conducts large currents through homotetrameric channels expressed in Xenopus oocytes (40). The addition of Ba2+ selectively blocks K+ currents resulting from heterologous expression of GIRK4* currents (A-C). Coexpression of alpha q or alpha q-GFP with GIRK4* significantly and similarly reduces agonist-independent (basal) currents compared with those in oocytes expressing only GIRK4*. Summary data in D represent averages in each group from five oocytes isolated from the same frog. Similar results were obtained from experiments in oocytes isolated from a different frog.

Imaging of alpha q-GFP, alpha qRC-GFP, and alpha qC9S/C10S in Living Cells and After Extraction with 1% Triton X-100-- Imaging of transiently expressed alpha q-GFP in live HEK-293 cells with confocal microscopy demonstrated signal in the plasma membrane and the cytoplasm (Fig. 6A). In some cells there were distinct patches of alpha q-GFP in the plasma membrane. There was some variability in the ratio of signal at the membrane compared with that in the cytoplasm, but there was no correlation with the level of expression. Bright cells had the same variability as did the dim ones.



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Fig. 6.   Confocal imaging of alpha q-GFP and alpha q-GFP mutants in living cells. HEK-293 cells were transiently transfected with: A, alpha q-GFP; B, alpha qRC-GFP; or C, alpha qC9S/C10S-GFP. The cells were imaged with a ×100 lens, numerical aperture of 1.2. alpha q-GFP exhibits distinct signal in the plasma membrane, as well as in the cytoplasm and the nucleus. The RC mutation greatly reduces the relative amount of signal in the plasma membrane compared with that in the cytoplasm, while the C9S/C10S mutations eliminate all detectable plasma membrane signal. Bar = 10 µm.

Imaging of alpha qRC-GFP-expressing HEK-293 cells (Fig. 6B) demonstrated a significantly higher ratio of cytoplasmic to plasma membrane signal than in alpha q-GFP-expressing cells (Fig. 6A). As with alpha q-GFP, there was expression level independent cell-to-cell variability in the localization pattern. The C9S/C10S mutations had a more dramatic effect on localization than the RC mutation in that they completely eliminated detectable plasma membrane labeling by alpha q-GFP (Fig. 6C). These results suggest that targeting of alpha q-GFP to the plasma membrane requires both association with beta gamma , which would be impaired by the RC mutation, and palmitoylation of Cys9 and Cys10.

To more clearly visualize the plasma membrane-associated populations of the alpha q-GFP constructs, we treated cells expressing these constructs with 1% Triton X-100 to extract the soluble proteins. Surprisingly, although alpha qRC-GFP and alpha q-C9S/C10S-GFP exhibited quite similar distribution patterns in living cells (Fig. 6), treatment of cells expressing one or the other of these constructs with 1% Triton X-100 produced very different results. Treatment of alpha qRC-GFP-expressing HEK-293 cells (Fig. 7C) with 1% Triton X-100 revealed a signal in the plasma membrane (Fig. 7D) that was similar to that seen in Triton-treated alpha q-GFP-expressing cells (Fig. 7B). Therefore, although the RC mutation decreases the percentage of alpha q-GFP that is associated with the plasma membrane, there is residual alpha qRC-GFP signal at this location that is masked in living cells because it is of similar intensity to the cytoplasmic signal. However, in contrast to the results obtained with cells expressing alpha q-GFP or alpha qRC-GFP, after treatment of alpha q-C9S/C10S-GFP-expressing cells (Fig. 7E) with 1% Triton X-100, no staining of the plasma membrane was seen (Fig. 7F). Thus, Cys9 and Cys10, the alpha q palmitoylation sites, are essential for plasma membrane association.



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Fig. 7.   Imaging of alpha q-GFP constructs in living cells and after extraction with 1% Triton X-100. Transiently transfected HEK-293 cells were imaged on an inverted Zeiss microscope before and after the addition of ice-cold 1% Triton X-100. The cells were transfected with plasmid encoding alpha q-GFP (A and B), alpha qRC-GFP (C and D), or alpha q-C9S/C10S-GFP (E and F). A, C, and E are images of the living cells, while B, D, and F are images derived from the same cells 2.5 min after the addition of detergent. Although there is much less detectable plasma membrane signal due to alpha qRC-GFP than to alpha q-GFP in living cells, the signals in the Triton-treated cells are similar. However, although the signals due to alpha qRC-GFP and alpha q-C9S/C10S-GFP are quite similar in living cells, there is no detectable signal after Triton treatment of cells transfected with alpha q-C9S/C10S-GFP. 2-s acquisition times were used for all images, but considerable variation in signal strength required adjusting the brightness/contrast of the final images by plotting restricted ranges of the pixel values as follows: A, 400-2000; B, 400-1000; C, 400-3000; D, 400-1500; E, 300-2500; F, 300-1250. Bar = 30 µm.

Imaging of alpha q-GFP in Living and Fixed Cells-- In previous immunohistochemistry studies, alpha q was found either predominantly in the plasma membrane (22), in the plasma and Golgi membranes (43), or associated with the cytoskeleton (23, 44). Reasoning that preparation of specimens for immunohistochemistry might affect the apparent localization pattern of alpha q and therefore contribute to these diverse observations, we investigated whether the alpha q-GFP signal was altered during fixation and immunostaining (Fig. 8). The alpha q-GFP signal observed in living cells (Fig. 8A) did not change after fixation with 2% paraformaldehyde, extraction with 0.05% Triton X-100, and treatment with an antibody to GFP followed by a Texas Red-conjugated secondary antibody (Fig. 8B). Furthermore, the Texas Red signal (Fig. 8C) was virtually identical to the intrinsic GFP signal. Therefore, standard fixation procedures and detergent permeabilization do not appear to selectively mask the alpha q-GFP signal or extract it from particular cellular compartments.



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Fig. 8.   Imaging of alpha q-GFP in living cells and after fixation. Transiently transfected HEK-293 cells were imaged on an inverted Zeiss microscope, and the immunohistochemical labeling was done on the microscope such that the same cells could be imaged after each step. A, GFP signal before fixation. B, GFP signal after fixation with paraformaldehyde, Triton X-100 extraction, and antibody treatments. C, Texas Red anti-GFP antibody signal. The GFP signal is the same before and after immunohistochemical labeling and is accurately represented by the anti-GFP antibody signal. Bar = 25 µm.

The substantial amount of cytosolic alpha q-GFP potentially could have been caused by the insertion of GFP. However, based on a comparison of the immunolocalization patterns of alpha q-GFP and alpha q using an antibody to a COOH-terminal alpha q/alpha 11 peptide, this does not appear to be the case. The cytosolic signal seen with GFP in alpha q-GFP-expressing cells (Fig. 9A) was clearly labeled by the anti-alpha q antibody in the same cells (Fig. 9B) and in cells expressing alpha q (Fig. 9C). The alpha q-GFP seen at the membrane with GFP (Fig. 9A) was under-represented by the Texas Red anti-alpha q antibody signal (Fig. 9B). It is possible that association of alpha q with receptors limits access of the anti-peptide antibody at the membrane, since the carboxyl termini of alpha  subunits are involved in receptor interaction. A second antibody to an internal alpha q epitope gave similar results (data not shown).



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Fig. 9.   Imaging of alpha q-GFP and alpha q after fixation. Transiently transfected HEK-293 cells were imaged on an inverted Zeiss microscope after fixation and immunohistochemical labeling. A, GFP signal in HEK-293 cells transfected with plasmid encoding alpha q-GFP, and B, Texas Red anti-alpha q peptide antibody signal in the same cells. The signals in A and B are similar, but plasma-membrane-associated alpha q-GFP is less apparent in B. C, Texas Red anti-alpha q peptide antibody signal in HEK-293 cells transfected with plasmid encoding alpha q. The substantial amount of cytosolic signal in both B and C indicates that the insertion of GFP is not responsible for the presence of cytosolic alpha q-GFP. Variation in signal strength required adjusting the brightness/contrast of the final images by plotting restricted ranges of the pixel values as follows: A, 0-3200; B, 0-2600; C, 0-800. Bar = 10 µm.

A Change in the Cellular Localization of alpha q-GFP Is Not Observed upon Hormonal Stimulation-- The effects of the RC and C9S/C10S mutations on the localization of alpha q-GFP suggested that targeting of alpha q to the plasma membrane requires association with beta gamma and palmitoylation of Cys9 and Cys10. Therefore, we investigated whether receptor-mediated activation, predicted to cause dissociation of alpha q from beta gamma and palmitate turnover, leads to release of alpha q-GFP from the plasma membrane. To be able to monitor activation of alpha q-GFP, we co-transfected HEK-293 cells with plasmids encoding alpha q-GFP, the alpha 2a-adrenergic receptor, and PKCgamma -RFP. PKC-GFP fusion proteins transiently associate with the plasma membrane in response to stimulation of Gq-coupled receptors (3). Thus, the PKCgamma -RFP signal served to confirm signaling through Gq on a cell by cell basis.

Stimulation of these cells with 10 µM UK-14,304 caused PKCgamma -RFP to transiently associate with the plasma membrane (Fig. 10, A and B). In any given field, not all of the cells exhibited a PKC translocation response, but in those that did, no change in the localization of alpha q-GFP was observed (Fig. 10, C and D). Simultaneous images of PKCgamma -RFP and alpha q-GFP were collected every 2 s for 800 s, which produced a three-dimensional stack of images with time in the Z dimension. The PKCgamma -RFP and alpha q-GFP signals at the membrane can be followed more carefully over time by looking at a 90o rotation of a small region of the image stack (boxes, Fig. 10, A and C). Such projections are shown in Fig. 10, B and D. Each projection extends 400 frames from top to bottom and in Fig. 10B the dramatic transient movement of PKCgamma -RFP to the plasma membrane is clear. Fig. 10D shows that the alpha q-GFP signal remained in the plasma membrane throughout the duration of the experiment. Video 1 is a movie showing the entire sequence of PKCgamma -RFP and alpha q-GFP images in this experiment.



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Fig. 10.   A change in the cellular localization of alpha q-GFP is not observed upon hormonal stimulation. HEK-293 cells were co-transfected with vectors encoding alpha q-GFP, PKCgamma -RFP, and the alpha 2a-adrenergic receptor. The cells were heated to 30 °C and imaged with confocal microscopy. 400 images were collected at 2-s intervals, and the cells were stimulated with 10 µM UK-14,304 in between frames 3 and 5. This produced a three-dimensional stack of images with time in the Z dimension. A, images of PKCgamma -RFP at selected time points indicating hormone-dependent transient association with the plasma membrane. B, 90o rotation of PKCgamma -RFP images within the box in A over the whole time course such that time is now the y axis. C, images of alpha q-GFP in the same cells and time points as in A demonstrating that stimulation with hormone does not result in detectable relocalization. D, 90° rotation of alpha q-GFP images within the boxed region in C over the whole time course. The images in A and C are 31 × 42 µm. Video is available (Video 1). In the video as in the figure, the images on the left are of PKCgamma -RFP and those on the right are of alpha q-GFP.

There was no obvious translocation of the alpha q-GFP signal following stimulation, but it is possible that the movement of alpha q-GFP is more rapid and/or less pronounced than the movement of PKCgamma -RFP. To explore the spatial limits within which a change in the localization of alpha q-GFP could have been detected, we quantified the membrane staining pattern of alpha q-GFP within the boxed region shown in Fig. 10C for the first 400 s of the experiment. For each frame, the signal across the membrane was fit to a Gaussian distribution (Fig. 11A) and the S.D. of the distribution was used to estimate the width of the signal at the membrane. Fig. 11B shows these standard deviations plotted versus time in the stimulated cell from Fig. 10 (left) and in an unstimulated control cell. In the stimulated cell, the standard deviations varied between 0.04 and 0.08 µm with no clear increase preceding the PKC response. For comparison, the membrane signal of a control cell measured in the same manner showed a variability in thickness that exceeded any measured in the responding cell. If alpha q-GFP translocates from the membrane in response to stimulation, it must do so more quickly than the frame rate (1 image/2 s) can capture and/or move only short distances that would not change the membrane profile by more than 0.04 µm.



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Fig. 11.   Width of the alpha q-GFP membrane signal over time in stimulated and unstimulated cells. To quantify the alpha q-GFP signal at the membrane, the signal across the membrane was fit to a Gaussian distribution and the S.D. of the fit was used to estimate the width of the signal. A, fit of the alpha q-GFP signal across the membrane from the boxed region of frame 100 in Fig. 10C. B, plot of the standard deviations of the Gaussian fits of the alpha q-GFP signals in the boxed region of the first 200 frames in Fig. 10C (left) versus time and from a similar region of alpha q-GFP membrane signal in a cell stimulated with vehicle (right). In the stimulated cell, the width of the membrane signal did not change during the interval between the stimulus and the PKC response. Variability in membrane thickness was greater in the control cell than in the stimulated cell.

A Change in the Cellular Localization of alpha q-GFP Is Not Observed Upon Stimulation with Aluminum Fluoride-- One potential reason why release of alpha q-GFP from the plasma membrane was not observed in response to receptor stimulation is that the activation/deactivation cycle of alpha q is too fast to be measured on the time scale over which we collected images. It has been proposed that rapid GTP hydrolysis may allow receptors and G proteins to remain bound throughout the GTPase cycle (45). In the presence of PLC or RGS4, Gq can hydrolyze GTP with a deactivation half-time of 25-75 ms at 30 °C (46).

Therefore, to prevent rapid deactivation of alpha q-GFP by GTP hydrolysis, we stimulated with AlF4-. AlF4- activates alpha  subunits by binding to the GDP-bound form and mimicking the gamma -phosphate of GTP in the transition state intermediate of the GTPase reaction (47, 48). Stimulation with AlF4- thus provided a means to test whether prolonged activation of alpha q would result in its release from the plasma membrane.

AlF4- stimulation of HEK-293 cells transiently co-transfected with alpha q-GFP and PKCgamma -RFP produced oscillating PKC translocation responses. Fig. 12A shows a PKCgamma -RFP image before the first translocation response. Fig. 12B shows a PKCgamma -RFP image in the same field during one of the membrane translocation responses. Fig. 12C, a projection of the PKCgamma -RFP image stack in the boxed region in Fig. 12B rotated such that time is the Y axis, shows the oscillating PKCgamma -RFP responses. Although AlF4- activates all G proteins, its effect on PKC localization involves activation of alpha q, because the number and magnitude of translocation responses was greater in cells that were co-transfected with alpha q-GFP than in cells transfected only with PKCgamma -RFP. Of 82 cells co-transfected with alpha q-GFP and PKCgamma -RFP (from two experiments), 46 exhibited at least one response, with a total of 216 oscillations. In contrast, of 61 cells transfected with PKCgamma -RFP alone (from two experiments), only 18 responded, with a total of 63 oscillations. Therefore, although HEK-293 cells express alpha q endogeneously, the level of expression appears to be limiting with respect to mediating PKC responses to AlF4-.



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Fig. 12.   A change in the cellular localization of alpha q-GFP is not observed upon stimulation with AlF4-. HEK-293 cells were co-transfected with plasmids encoding alpha q-GFP and PKCgamma -RFP. Cells were stimulated with 30 µM AlCl3, 10 mM NaF at 37 °C and images were collected on the inverted microscope in sets of 10 in which 1 alpha q-GFP image was followed by 9 PKCgamma -RFP images. AlF4- was added after frame 1 (first PKCgamma -RFP image). The interval between frames was ~1 min for the first 2 sets of images. 20 sets of images were then collected with ~10-s intervals between frames. Images were collected out to 46 min after application of the stimulus. The first response in the field shown occurred in frame 13 (12.41 min after addition of AlF4-; A) PKCgamma -RFP, before the first response (frame 9, 8.27 min after application of the stimulus). This is used as the "before" image, because cell movement in the initial frames made it impossible to make direct comparisons with later frames. B, PKCgamma -RFP during oscillation response (frame 69, 26.86 min after application of the stimulus). C, 90o rotation of PKCgamma -RFP images within the box in B over the whole time course such that time is now the y axis. Increases in signal in the plasma membrane (right) correspond to decreases in signal in the cytoplasm (left) and vice versa. In this experiment, 8 transient translocations of PKCgamma -RFP occurred. D, alpha q-GFP before the first PKCgamma -RFP response (frame 10, 10.33 min after application of the stimulus); E, alpha q-GFP during PKCgamma -RFP oscillation response (frame 70, 27.08 min after application of the stimulus). F, 90o rotation of alpha q-GFP images within the boxed region in E over the whole time course, indicating that stimulation with AlF4- does not cause detectable relocalization. Bar = 20 µm. Video is available (Video 2). In the video, as in the figure, the images on top are of PKCgamma -RFP and those on the bottom are of alpha q-GFP. Since 1 alpha q-GFP image was collected for every 9 PKCgamma -RFP images, the single alpha q-GFP image that corresponds to each set of 9 PKCgamma -RFP images is shown below the PKCgamma -RFP images.

As with hormonal stimulation, treatment of cells with AlF4- did not produce a detectable change in the localization of alpha q-GFP in cells that clearly gave PKCgamma -RFP translocation responses. Images of the alpha q-GFP signal before (Fig. 12D) and during (Fig. 12E) the PKCgamma -RFP responses showed the same distribution pattern. The consistency of the alpha q-GFP signal after stimulation with AlF4- is further demonstrated in Fig. 12F, which shows a projection of the alpha q-GFP image stack in the boxed region in Fig. 12E, rotated as in Fig. 12C. Even when activation of alpha q-GFP is irreversible, there is no movement out of the membrane within the time frame of many cellular responses. Video 2 is a movie showing the entire sequence of PKCgamma -RFP and alpha q-GFP images in this experiment.

A major difference between the mutations (RC and C9S/C10S) and stimulation with hormone or AlF4- is that the mutations affect alpha q-GFP from the moment that it is synthesized, while stimulation occurs subsequent to the initial targeting of alpha q-GFP to its cellular locations. The mechanistic implications of the different effects of the mutations versus the stimulations are discussed below.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This is the first report of a functional G protein alpha  subunit-GFP fusion protein. alpha q-GFP exhibits normal interactions with a G protein-coupled receptor, with beta gamma , and with PLC, and has made it possible to track the behavior of alpha q in living cells without the potential artifacts that can arise from antibody staining, and from studying fixed or fractionated cells. We have tested the roles of activation state and palmitoylation in targeting of newly synthesized alpha q to the plasma membrane. We have also investigated the effects of activation by receptors and by AlF4- on the localization of alpha q. These studies reveal that although activation and lack of palmitoylation can disrupt targeting of alpha q to the plasma membrane, membrane-associated alpha q does not relocate upon activation.

Our studies with alpha q-GFP provide new insights into the cellular location and behavior of alpha q in resting cells. The observed inhomogeneity of the alpha q-GFP membrane population may indicate compartmentalization with other signaling proteins, which could contribute to the efficiency and specificity of signaling. For instance, previously reported clusters of alpha 2A-adrenergic receptors (49) resemble the alpha q-GFP patches seen here. Such compartmentalization has been observed for the beta -adrenergic receptor-Gs-adenylyl cyclase system, which has been localized to low density plasma membrane fractions that can be separated from the bulk of the plasma membrane (50). In addition, although alpha  subunits have generally been thought to be primarily associated with the plasma membrane, our results with alpha q-GFP indicate that a significant amount of alpha q is also in the cytoplasm. The functional significance of this cytoplasmic alpha q population remains to be determined.

The effects of mutational activation and removal of the palmitoylation sites of alpha q-GFP are consistent with the "two-signal model" for plasma membrane binding (17). According to this model, the initial localization signal for alpha  subunits such as alpha q that can be palmitoylated, but not myristoylated is association with beta gamma , which is prenylated on the gamma  subunit. After localization of the heterotrimer to the plasma membrane, in which palmitoyl transferase is proposed to be located (51), palmitoylation takes place, which leads to stable association with the plasma membrane. In support of this model, mutation of alpha q residues important for beta gamma interaction decreases both palmitoylation and association with the plasma membrane (17). Mutational activation of alpha q-GFP would thus decrease plasma membrane binding by impairing association with beta gamma , which binds preferentially to the GDP-bound form of alpha  subunits. Although both mutational activation and removal of the palmitoylation sites reduce association of alpha q-GFP with the plasma membrane, the effect of removing the palmitoylation sites is stronger. The partial effect of the RC mutation is consistent with the fact that it inhibits but does not abolish GTPase activity. The fraction of alpha qRC-GFP that is in the GDP-bound form will be able to associate with the plasma membrane.

It is initially surprising that the RC mutation, which has an incomplete inhibitory effect on GTPase activity, decreases the association of alpha q-GFP with the plasma membrane, while treatment with AlF4-, which causes irreversible activation, has no such effect. However, there is a significant difference between these two manipulations of alpha q-GFP. Whereas the mutation is present from the moment that the protein is synthesized, AlF4- is applied to cells in which there is already a membrane-associated pool of alpha q-GFP. The simplest explanation for the apparent discrepancy is that the activated state can prevent targeting of newly synthesized alpha q-GFP to the plasma membrane, but after alpha q-GFP associates with the membrane, this association is resistant to subsequent activation.

There are several potential reasons why membrane-associated alpha q-GFP does not relocate to the cytoplasm upon activation. Although release from the membrane might be predicted if palmitoylation and association with beta gamma were the only signals important for membrane localization, one scenario is that upon membrane association an additional localization signal is acquired that ensures that alpha q-GFP remains there throughout the activation cycle. This hypothetical signal could be a protein-protein interaction or an additional modification that occurs once alpha q-GFP reaches the plasma membrane. For instance, a feature of the amino terminus of alpha q other than palmitoylation of Cys9 and Cys10 is proposed to contribute to hydrophobicity and membrane association (11).

An alternative explanation for why activation does not cause release of membrane-associated alpha q-GFP is that upon activation the palmitates may not be completely removed and/or alpha q-GFP may not completely dissociate from beta gamma . It is also possible that only one of these two localization signals is required to retain as opposed to initially target alpha q-GFP in the membrane. With regard to the level of palmitate that remains after activation, although receptor stimulation increases turnover of palmitate on alpha s, the stoichiometry of palmitoylation on alpha s isolated from basal and stimulated cells was found to be the same (21). In the case of alpha q, receptor stimulation causes turnover of palmitate (9, 10), but the effect on the stoichiometry of palmitoylation has not been determined.

With respect to interactions between alpha  and beta gamma during the GTPase cycle, the generally accepted model for G protein activation assumes that alpha  and beta gamma dissociate from each other upon activation. However, it is possible that alpha  and beta gamma merely shift relative to each other, rather than completely separate. The contact between the beta  subunit and switch II is likely to be severed during activation, since switch II residues are required for the interactions of most if not all alpha  subunits with their effectors (52). However, it is possible that the connection of the beta  subunit to the amino terminus of the alpha  subunit is preserved throughout the cycle. Given that alpha q and alpha q-GFP inhibit beta gamma -dependent basal activity of GIRK4 with equal effectiveness, they are likely to possess similar affinities for beta gamma .

The studies presented here provide the starting point for further investigations of how G protein alpha  subunits are organized in vivo under basal and activated conditions. Given the overall structural similarity of the alpha  subunits, the GFP insertion site that worked for alpha q is likely to be useful for other alpha  subunits as well. For instance, residues in this region of alpha s (53) and alpha i2 (33) can be replaced with homologs from other alpha  subunits without disrupting function. GFP-tagged alpha  subunits will also make it possible to follow alpha  subunits over time during development in specialized cells such as neurons and polarized epithelia in tissue culture and intact animals. Additionally, these reagents will be useful for colocalization studies with RFP-tagged beta  subunits and receptors. Fluorescence resonance energy transfer between GFP-tagged alpha  subunits, beta  subunits, and receptors may also be possible, which would enable monitoring of the interactions of these proteins during activation cycles.


    ACKNOWLEDGEMENTS

We thank Galina Grishina and Rolando Medina for producing the alpha q-GFP construct, and Thomas Hynes for the computer graphics, helpful discussions, and critical reading of the text.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants GM50369 (to C. H. B.), EY08362 (to T. E. H.), and HL54185 (to D. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Video 1 (Fig. 10) and Video 2 (Fig. 12).

Established Investigator of the American Heart Association.

** Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 203-785-3202; Fax: 203-785-4951; E-mail: catherine.berlot@yale.edu.

Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007608200


    ABBREVIATIONS

The abbreviations used are: GFP, green fluorescent protein; RFP, red fluorescent protein; PKC, protein kinase C; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PLC, phosphoinositide phospholipase C; PBS, phosphate-buffered saline.


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