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J. Biol. Chem., Vol. 281, Issue 33, 23999-24014, August 18, 2006

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Stable Association between G_q and Phospholipase C₁ in Living Cells^*

From the Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661

Received for publication, November 16, 2005 , and in revised form, June 2, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Signal transduction through G_q involves stimulation of phospholipase C (PLC) that results in increased intracellular Ca²⁺ and activation of protein kinase C. We have measured complex formation between G_q and PLC₁ in vitro and in living PC12 and HEK293 cells by fluorescence resonance energy transfer. In vitro measurements show that PLC₁ will bind to G_q(guanosine 5\m-'\-3-O-(thio)triphosphate) and also to G_q(GDP), and the latter association has a different protein-protein orientation. In cells, image analysis of fluorescent-tagged proteins shows that G_q is localized almost entirely to the plasma membrane, whereas PLC₁ has a significant cytosolic population. By using fluorescence resonance energy transfer, we found that these proteins are pre-associated in the unstimulated state in PC12 and HEK293 cells. By determining the cellular levels of the two proteins in transfected versus nontransfected cells, we found that under our conditions overexpression should not significantly promote complex formation. G_q-PLC₁ complexes are observed in both single cell measurements and measurements of a large (i.e. 10⁶) cell suspension. The high level (40% maximum) of FRET is surprising considering that G_q is more highly expressed than PLC₁ and that not all PLC₁ is plasma membrane-localized. Our measurements suggest a model in which G proteins and effectors can exist in stable complexes prior to activation and that activation is achieved through changes in intermolecular interactions rather than diffusion and association. These pre-formed complexes in turn give rise to rapid, localized signals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The G_q family of G proteins transduces signals connected to agents such as angiotensin II, catecholamines, endothelin 1, and prostaglandin F₂. In its activated GTP-bound state, G_q will stimulate the catalytic activity of its main effector phospholipase C (PLC).² PLC enzymes catalyze the hydrolysis of the signaling lipid, phosphatidylinositol 4,5-bisphosphate, to generate two second messengers that result in an increase in intracellular Ca²⁺ and a host of proliferative and mitogenic changes in the cell (for review see Refs. 1 and 2). There are four forms of PLC (PLC_1-4) that differ in their tissue distribution and their regulation by G protein subunits. Here we will focus on PLC₁, which is strongly activated by G_q subunits. PLC₁ is widely distributed and is most highly expressed in neuronal tissue where it may participate in rapid intracellular Ca²⁺ signaling (2).

Activation of PLC₁ by G_q(GTP) is thought to occur through direct contact between the enzyme and the activated G protein subunit. This idea stems from the close correlation between the lateral association of PLC_1-3 and G subunits and the concentration dependence of activation (3). However, this mechanism may differ from G subunits that undergo significant conformational changes upon activation unlike G subunits (4).

The rate of activation of PLC₁ by G_q(GTP) will depend on the rate of association and the rate of the conformational changes that lead to effector activation. If the two proteins are complexed prior to G_q activation, the signal will no longer depend on the diffusion rates of the two proteins, and the on rate of PLC₁ activation would be greatly accelerated. There is now accumulating evidence that higher order complexes of signaling proteins exist in cells. Ross and co-workers (5) found that the rate of G_q-PLC₁ signaling was so rapid that dissociation of G_q from the receptor was not probable, and indeed, association between seven transmembrane receptors and G_q has been observed in cells (6). In Drosophila, a signaling complex involving receptor, a PLC homolog, a protein kinase C homolog, and a scaffold protein has been identified (7). More evidence for signaling complexes comes from RGS4-dependent Ca²⁺ oscillations in cells using a PLC- agonist (8), suggesting that the G protein-coupled receptor, the G protein heterotrimer, and PLC could be localized in a signaling complex. Although these studies are suggestive, to date the physical association of a G protein subunit and its corresponding effector have not yet been reported in living cells.

Although preformed G protein-effector complexes would not only give rise to rapid signals as mentioned above, the localization of the signal would no longer depend on the localization of the two proteins but rather on the diffusion of the products generated. Most importantly, these signals would only be generated by a specific receptor type. For PLC₁-G_q, the second messengers generated are expected to have very rapid diffusion as compared with the proteins, thus further enhancing signal speed.

The ability of proteins to form complexes depends on their local concentration as well as their affinities. We have previously used fluorescence methods to quantify the affinities of PLC enzymes to G_q, G, and other components in this pathway on model membranes using purified proteins (9, 10). As expected, the binding of PLC₁ to G_q(GTPS) is extremely strong as compared with binding to deactivated G_q. Although this reduction in affinity is significant, it is important to note that if the local cellular concentrations of PLC₁ and G_q are above this dissociation constant, then they would remain together in the activated as well as the deactivated states.

In this study, we have determined the ability of PLC₁ and G_q to form complexes in the basal and stimulated states in vitro and in two cell lines using fluorescence resonance energy transfer (FRET) between fluorescent-tagged proteins. FRET measurements are based on the probability of transfer of excited energy from a donor fluorophore to an acceptor. This probability depends on the electronic properties of the donor and acceptor as well as their intermolecular distance (11, 12). Because the amount of FRET depends on the 6th power of this distance, it is a sensitive measure of protein-protein associations. For the eCFP-eYFP donor-acceptor FRET pair, the distance at which 50% of the donor fluorescence is lost to transfer is 30 Å, making this pair useful to monitor protein association (13). By using this method, we find that PLC₁ and G_q are complexed even when G_q is in the deactivated state, although the nature of the association differs. In cells, we find that these proteins are strongly complexed in the unstimulated state despite their low affinity. These pre-formed PLC₁-G_q(GTPS) complexes allow for rapid signaling through changes in protein orientation during G protein turnover and delocalization of the signal through diffusion of the second messengers produced by activation. Based on the cellular concentration of the proteins and their affinities, our results suggest that co-localization must occur through unidentified factors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents—eCFP-G_q was derived from G_q-GFP, as described previously (14), and was a generous gift from Dr. Catherine Berlot (Geisinger Clinic, Danville, PA) as were the constitutively active eCFP-G_q (R183C) (eCFP-G_qRC) (15) and eCFP-G₁ (16). The eCFP construct was originally obtained from Clontech. eYFP-PLC₁ was a generous gift from Loren Runnels (Department of Cell Biology, Rutgers University). This construct shows wild-type basal activity and activation by G_q.

In Vitro FRET Studies—In vitro affinities between PLC₁ and G_q(GTPS) or G_q(GDP) were determined as described previously (10). Briefly, the proteins were expressed in Sf9 cells through a baculovirus system and purified. GDP-bound G_q was then labeled with an amine reactive probe, coumarin SE (Molecular Probes, Inc.), reconstituted on large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) (1:1:1), and the center of spectra mass and intensity was monitored as purified, and unlabeled PLC₁ was incrementally added. G_q was activated using the procedure of Ref. 17. This procedure results in at least 80% nucleotide exchange.

Trypsin Digestion—Samples of either PLC₁ alone or in a 1:1 mixture with G_q(GTPS) or G_q(GDP) were preincubated for 10 min on ice before the addition of trypsin. Proteolysis was allowed to proceed at 37 °C for either 5 or 20 min before addition of 10% SDS. Samples were then boiled for 3 min and subjected to SDS-PAGE. Bands were visualized using silver stain.

Cell Culture and Transfection—Rat pheochromocytoma cells (PC12), derived from the adrenal gland (ATCC, CRL-1721), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% equine serum, 5% fetal bovine serum, and 100 mM sodium pyruvate and were incubated at 37 °C with 5% CO₂. Nerve growth factor (NGF, Sigma) was added to a final concentration of 100 ng/ml to induce differentiation. Prior to transfection, cells were grown in T-25 flasks to 80-90% confluency.

Plasmids were introduced into cells by electroporation using a protocol adapted from Maniatis and co-workers (18). Briefly, the DMEM was aspirated, and the PC12 cells were harvested by adding 5 ml of fresh media and pipetting multiple times over the bottom of the flask. Cells were spun down for 5 min at 1500 x g and resuspended in 5 ml of phosphate-buffered saline (PBS). One ml of cells was removed and counted, and the cell suspension was diluted to 5 x 10⁶ cells/ml. Cells (500 µl) were pipetted into 0.4-cm Bio-Rad cuvettes and incubated on ice for 10 min. Then 10-30 µg of plasmid DNA was added to each cuvette and gently mixed. The electroporator (Bio-Rad Gene Pulser Xcell) was set to 0.25 kV with a capacitance of 500 microfarads. Cuvettes were placed in the shocking chamber and pulsed once. After the pulse, the cells were allowed to rest for 1-2 min, and 1 ml of DMEM was then added to each cuvette. Cells were placed in 15-ml conical tubes containing 3 ml of DMEM. Tubes were spun down at 1500 x g for 5 min, and cells were brought up in 1.5 ml of DMEM and plated onto LabTek chambers coated with 50 µg/ml fibronectin (Sigma). Three to four hours post-transfection, the wells were washed with 1 ml of PBS, and 1.5 ml of DMEM containing 100 ng/ml NGF was added. After 3 days of incubation with NGF, transfected cells were differentiated and used for imaging.

HEK cells were cultured in DMEM plus 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO₂. Plasmids were introduced to HEK cells as described except they were not preincubated on ice before the pulse, and the electroporator was set to 0.2 kV with a 960-microfarad capacitance.

Calcium Release—PC12 cells, in T-25 flasks, were washed two times with PBS. Cells were collected in Hanks' buffered salt solution (HBSS, 15 mM Hepes (pH 7.67), 118 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, and 1 mg/ml BSA) and spun down at 1500 x g. Cells were counted and adjusted to a concentration of 1 x 10⁷ cells/ml. Fura-2 AM (5 mM) (Sigma) was added to the cells, and cells were incubated in the dark at 37 °C for 40 min with rotation. After labeling, cells were spun down at 1500 x g and resuspended in HBSS. To make measurements, cells were diluted to 1.0 x 10⁶ cells/ml in HBSS. One-ml cell suspensions were put into a cuvette with a stir bar and placed in the fluorometer. The samples were excited at 340 and 380 nm, and the emission was measured at 510 nm, and the ratios of the two excitations were recorded over time.

To measure calcium release upon stimulation, cells were stimulated with 1 µM acetylcholine or carbachol, and the ratio was measured again. To break open the cells, 10% Triton X-100 was added followed by calcium chelation with 2 mM EDTA to obtain the maximal and minimal amounts. To calculate internal calcium concentration, see Equation 1 (19),

(Eq. 1)

where R is the measured ratio (fluorescence emitted at 340 and 380 nm); R_min and R_max are the ratios with EDTA and detergent; F_min is the fluorescence in the presence of EDTA (i.e. minimum calcium), and F_max is the fluorescence in the presence of detergent (i.e. maximum calcium). The K_d of Fura-2 AM is 225 nM (19).

Cell Fractionation and Western Blot Analysis—PC12 cells were transfected with protein expression vectors as described above. Five identical transfections were combined into a T-25 flask and differentiated for 3 days with NGF. Then PC12 cells were harvested from T-25 flasks and washed two times with PBS. After the second wash, the cells were brought up in PBS containing 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin, placed on ice, and homogenized. Nuclei were removed by a low speed centrifugation at 750 x g for 5 min at 4 °C. The supernatant was removed and spun at 28,000 rpm for 35 min at 4 °C. The supernatant or cytosolic fraction was removed, and the resulting pellet or membrane fraction was brought up in PBS containing 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin. The protein concentration for the cytosolic and membrane fractions was assayed, and 4-15 µg was loaded into each well of an SDS-polyacrylamide gel. Proteins were then transferred to nitrocellulose; the membrane was blocked overnight and then probed for G_q and PLC₁ using a 1:200 dilution for primary antibody (Santa Cruz Biotechnology) and 1:2000 dilution for secondary antibody (Sigma). Western blots were developed using alkaline phosphatase reaction, and the amount of overexpressed protein per µg of protein loaded was calculated using a standard curve generated from purified G_q and PLC₁.

Immunofluorescence—For secondary immunofluorescence for endogenous expression of G_q and PLC₁, PC12 cells were plated and differentiated as described above. The cells were washed twice with PBS and fixed with 1.5 ml of 3% paraformaldehyde at room temperature for 10 min. The fixing solution was removed, and the cells were washed three times for 10 min each with MSM-PIPES Buffer (modified Shierdls media; 18 mM MgSO₄, 5 mM CaCl₂, 40 mM KCl, 24 mM NaCl, 5 mM PIPES (pH 6.8), 0.5% Triton X-100, 0.5% Nonidet P-40). After washing, the cells were blocked in PBS containing 5% goat serum, 1% BSA, and 50 mM glycine for 15 min. Cells were incubated with the primary antibody (1:200 dilution) in PBS containing 0.5% BSA at 37 °C for 1 h and then washed three times with PBS for 10 min each. Cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody (1:2000 dilution) in PBS containing 0.5% BSA at 37 °C for 1 h, and then washed three times with PBS for 10 min each. PBS was then added to wells, and the cells were imaged.

Preparation of Membrane Fractions—HEK293 cells (12.5 x 10⁶ per 150-mm dish) were transfected using DEAE-dextran (21) or using 62.5 µl of Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. 48 h after transfection, membranes were prepared as described (22). The amounts of plasmids used in the transfections are given in the legend to Fig. 7.

Instrumentation—Confocal images were taken at the University Microscopy Center on a Bio-Rad apparatus. Pixel analysis of confocal images was done using ImageJ (National Institutes of Health). All other images, time lapses, and z-stacks were taken on a Zeiss Axiovert 200M with an AxioCam MRm camera using Axiovision software. Fluorescence spectra were taken on a photon-counting spectrofluorometer, ISS-PC (ISS, Urbana, IL).

Single Cell FRET Measurements—FRET measurements were determined using the procedure of Devreotes and co-workers (24). Bleed through values were obtained by transfecting PC12 cells with 10 µg of free eCFP or free eYFP plasmid vectors and imaging under the appropriate filter sets (Chroma, Inc.). Cells expressing only CFP or YFP were then imaged under the CFP (Chroma 31044v2) or YFP (Chroma 41029) and FRET (Chroma 31052) filter sets to determine the FRET/CFP or FRET/YFP ratio. Averaging over 12 cells on our system, the bleed through values for CFP and YFP are 39 and 28%, respectively, using the background-corrected intensities calculated using ImageJ software from the National Institutes of Health. We generated a net FRET image by accounting for bleed through emission, see Equation 2,

(Eq. 2)

where a and b equal the percentage of bleed through of YFP and CFP under the FRET filter set. However, to compare FRET values among cells with varying protein expression levels, the net FRET (nF) value can be normalized (N_FRET). From the entire intensity value of the image, one can calculate N_FRET. Normalized FRET (N_FRET) is given as shown in Equation 3,

(Eq. 3)

where a and b equal the percentage of bleed through of CFP and YFP under the FRET. N_FRET was determined as described (23).

Image Analysis—Images were processed using ImageJ. Before any analysis, images were corrected for background as follows. First, the background was calculated from a 50 x 50 pixel box at the top left corner of the raw image, and this intensity was subtracted from the entire image. Then uneven illumination was removed from the image by using three iterations of the Background Correction Plugin. This image was binary thresholded and then inverted to make the cell white (255) and the background black (0). Next, the image was divided by 255 to make the pixel values for white 1. Finally, the background subtracted image was multiplied by the thresholded image. These manipulations remove the background by making the background 0. Using the method of Xia and Yuechueng (23), nF images were created by multiplying the background corrected donor image by the donor correction factor, the background corrected acceptor image by the acceptor correction factor, and then subtracting each of these from the background corrected FRET image. N_FRET images were created by multiplying the background corrected donor and acceptor images and then taking the square root creating a resultant image. The nF image was then divided by the resultant image. A global and normalized FRET value was calculated by averaging the normalized FRET value for each individual pixel. This program also calculates the corresponding normalized FRET ranges (0 - 10%, 10 - 20%, etc.) for the image.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. A, binding of purified PLC1 to either 2 nM CM-Gq(GDP) or CM-Gq(GTPS) reconstituted on 300 µM large, unilamellar vesicles composed of POPC/POPS/POPE (1:1:1), where n = 3. Binding was followed by the shift in the emission energy of the coumarin probe upon binding. Control studies substituted buffer for enzyme. The binding curves were fit to a bimolecular association constant using SigmaPlot, and the S.E. is shown. B, SDS-PAGE showing the extent of trypsin digestion at either 5 or 20 min of incubation of PLC1 when bound to Gq(GDP) or Gq(GTPS).

FRET Measurements of Membrane Preparations—FRET measurements of membranes that were co-transfected with eCFP and eYFP tags were determined by the increase in eYFP fluorescence in the absence and presence of eCFP by using the exciting wavelength of the donor. The FRET value is thus a ratio of the emission maxima of the two peaks centered 490 and 527 nM at the exciting wavelength of the donor (440 nm) (24).

Membranes, prepared as described above, were thawed and diluted with PBS buffer so that the absorption at 433 nm for CFP samples or at 514 nm YFP samples was below 0.1 absorbance unit. Fluorescence measurements were made at room temperature. A 475 nm cut-on filter was placed before the emission monochromator. After taking the verifying emission spectra of the samples, the intensity of each sample was monitored at 1-s intervals for 3 min while stirring. We note that the intensities were stable over time, and identical values were obtained taking the intensities of the samples under nitrogen.

Because these FRET measurements are done on a fluorometer where the emission is better isolated as compared with a microscope, the percent FRET values were calculated either by comparing the acceptor emission when only acceptor or donor is excited, i.e. using eCFP and eYFP excitation (i.e. = 440 and 475 nm) and monitoring the emission at 525 nm to obtain R, where R = (I(490)/I(475)). FRET was then determined by comparing the R values for eCFP/eYFP-transfected samples to samples individually transfected with one of the fluorophores. Thus, Equation 4 is given,

(Eq. 4)

where R(s) is the intensity ratio of the sample. Real time changes in FRET were also monitored by the change in the intensity ratio of the donor and acceptor (490/527 nm) when the donor is excited (440 nm).

FRAP—Fluorescence recovery after photobleaching (FRAP) measurements were carried out using an N₂ laser (Spectra Physics) of 100-milliwatt power to photobleach a circular region of 2 µmin diameter on specific regions of the cells. The intensity of the photobleached portion was allowed to recover over time, and the recoveries were fit to 1 or 2 exponential curves.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Vitro Binding of Purified PLC₁ to G_q in the Activated and Deactivated States—We have determined previously the affinity of PLC₁ to activated G_q laterally associating on model membrane surfaces (9) using fluorescence methods. Here, we have repeated this measurement and determined the decrease in affinity between the proteins when G_q is in the deactivated state. These measurements were carried out by covalently attaching the fluorescent probe coumarin to G_q, reconstituting it on large unilamellar vesicles and monitoring the change in coumarin fluorescence intensity as PLC₁ is added. We note that these studies were done at lipid concentrations exceeding the membrane binding constant of the proteins, and so only lateral associations between the membrane-bound proteins are viewed (25, 26). The binding curves were fit assuming a bimolecular association, although we cannot discount the possibility that different conformational states of G_q contribute to the curve. The data in Fig. 1A show that PLC₁ binds 300-fold (K_app = 640 ± 68 nM versus 2.8 ± 0.7 nM) more weakly to deactivated as opposed to activated G_q(GTPS). Thus, for complexes to form in the basal state, the proteins would have to be co-localized at high concentrations (i.e. above 500 nM).

View larger version (51K): [in this window] [in a new window]   FIGURE 2. A, localization of GFP-Gq in a PC12 cell. The Gq image shows a cell of 10 x 20 µm and is a reconstruction from 82 slices. A rotated side view is presented at the bottom of the figure. In the inset we show a red dot corresponding to a point in the x-y plane (9 pixels) where we collected the intensity through the z axis of the cell (red line). B, histograph of the intensity distribution of eGFP-Gq along the red line running through the cell.

Quantifying the Endogenous and Overexpressed G_a and PLC₁ in Cells—To visualize complexes between PLC₁ and G_q in cells, we have overexpressed fluorescent-tagged G_q and PLC₁ and monitored association formation by fluorescence spectroscopy. Because overexpression could promote complex formation between the proteins, we quantified the amount of overexpressed eCFP-G_q and eYFP-PLC₁ in differentiated PC12 cells and compared these to endogenous levels found in cells transfected with empty vector by Western blot analysis (see "Materials and Methods"). This was accomplished by placing four different amounts of purified G_q or PLC₁ (7) to generate a calibration curve. The growth and morphology of the transfected cells were identical to untransfected cells, suggesting that at our levels of expression neither protein is disruptive to cell function. This observation correlates well with studies showing that PC12 clones that are stably expressing PLC₁ have the same sensitivity to NGF stimulation, proliferation, and survival upon serum deprivation as compared with wild-type cells (27). We note that transfection of HEK293 cells with G_q and PLC₁ did not affect the growth or morphology of these cells.

Differentiated PC12 cells (see "Materials and Methods") were harvested and ruptured, and the membrane and cytosolic fractions were isolated. We find that eCFP-G_q levels in the membrane fractions were 0.33 ± 0.06 µg/mg total protein compared with 0.17 ± 0.02 µg/mg protein of endogenous protein. Thus overexpressed protein is 2-fold higher than endogenous protein under our transfection conditions.

For eYFP-PLC₁, the ratio of transfected to endogenous enzyme is roughly 3-fold (0.006 versus 0.002 µg/mg protein). Similarly, the cellular PLC activity is 10% higher in the transfected cells consistent with increased expression of functional PLC₁ and also noting that other active PLC enzymes (e.g. PLC and PLC) are expressed in PC12 cells (2). This study shows that the overexpressed amounts of eCFP-G_q and eYFP-PLC₁ are proportional and not high enough to elicit abnormal cellular responses.

View larger version (84K): [in this window] [in a new window]   FIGURE 3. A, localization of YFP-PLC1 microscopy in a PC12 cell distinct from the one shown in Fig. 2. In the eYFP-PLC1 image, a cell of 12 x 16 µm is a reconstruction from 45 slices. In the insets we show a red dot corresponding to a point in the x-y plane (9 pixels) where we collected the intensity through the z axis of the cell. B, histograph of the intensity distribution of YFP-PLC1 running through the cell.

We assessed the amount of GFP-G_q in the cytoplasm by pooling the intensities from 5.0 to 14.6 µm, which should correspond to the cytosol, and dividing by the pooled intensities from the bottom to top distance of 0 - 4.8 µm, which should correspond to the plasma membrane. This calculation gives an intensity ratio of the cytosolic/plasma membrane of 0.06.

The localization of eYFP-PLC₁ was also viewed. Unlike GFP-G_q, a larger percent of the protein was seen in the cytosol (Fig. 3, A and B). To ensure that the cytosolic population of eYFP-PLC₁ was not caused by overexpression, we collected z-stack images of endogenous PLC₁ and fixed PC12 cells stained with a PLC₁ monoclonal antibody (Fig. 4, A and B). The distribution of endogenous PLC₁ matched that of overexpressed eYFP-PLC₁. We verified that PC12 cells contain a significant population of cytosolic PLC₁ by Western blot analysis of the cytosolic and membrane fractions of nontransfected and transfected cells (data not shown). Comparing the pooled ratios of the histograph intensities from the cytosolic (2.4-6.0 µm) divided by plasma membrane populations (0-2.2 µm), we obtain a value of 0.48. This value is far higher than the ratio obtained for GFP-G_q of 0.06. Similar results were obtained over a wide range of cells allowing us to conclude that differentiated PC12 cells have two populations of PLC₁ in the resting state, one localized on the plasma membrane and another in the cytosol. In contrast, G_q is almost entirely localized on the plasma membrane.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. A, immunofluorescence of endogenous expression of PLC1 in differentiated PC12 cells. Each image shown is a reconstruction from 41 slices. B, distribution analysis in cell sections averaged over six cells.

All cells expressing eCFP-G_q/eYFP-PLC₁ displayed a significant level of FRET with the mean value close to 40%. The transfection efficiency for eCFP-G_q and eYFP-PLC₁ was 60-80% for both proteins, and we note that significantly decreasing the level of expression of either one of the proteins reduced the FRET to non-specific values (i.e. less than 20% FRET, see below), correlating well with the properties of FRET (11).

To assess the significance of the maximal FRET values shown in Fig. 5, we can compare the average FRET observed for these proteins to the 55 ± 4% FRET observed for eCFP-G_q and eYFP-G₁₇ and the 50 ± 7% FRET observed for eCFP-G_q and eYFP-bradykinin receptor when overexpressed in HEK293 cells.³ The FRET from this latter complex decreases to a level below resolution (i.e. less than 20%) upon stimulation because of receptor internalization. Thus, the magnitude of the maximum FRET value correlates well with other complexes.

View larger version (61K): [in this window] [in a new window]   FIGURE 5. Examples of normalized FRET images of four PC12 cells expressing eCFP-Gq and eYFP-PLC1 after NGF treatment (see "Materials and Methods"). For each example, the 1st image corresponds to the basal FRET, and the 2nd image is 30 s after stimulation with 1 µM acetylcholine. These images were taken focusing on or close to the bottom of the cells, and the normalized FRET was analyzed using the method of Ref. 23. Also shown are surface plots depicting the distribution of the FRET signal over time for each cell, where the y axis is the percent FRET, the x axis is the percent pixels in each FRET range, and the z axis is time. One can see the percentage of pixels within each FRET range does significantly change upon stimulation, indicating that there is not a redistribution of the signal.

To determine whether a basal population of eCFP-G_q-eYFP-PLC₁ complexes could be seen in other types of cells, we carried out the same study using HEK293 cells. Similar to that seen in PC12 cells, these cells showed a cytosolic population of eYFP-PLC₁ and a plasma membrane population that is complexed with eCFP-G_q in the unstimulated state. Thus, this same cellular localization is seen in other cell lines.

The measurements described above were performed on single cells. Even though many cells were viewed, there is a possibility that eYFP-PLC₁ and eCFP-G_q FRET in the basal state only occurs in limited cases. To determine the amount of FRET in a large number of cells, we co-transfected cells with eYFP-PLC₁ and eCFP-G_q and measured the degree of FRET for 10⁶ cells in a spectrofluorometer. We found that the range of FRET for this large population of cells was identical to single cell measurements as described below (Fig. 5) verifying the single cell measurements that show that a significant fraction of eYFP-PLC₁ and eCFP-G_q are complexed in the basal state.

Cytoplasmic eYFP-PLC₁ Is Not Recruited to the Membrane by eCFP-G_q during Stimulation—As shown in Fig. 1, activation of G_q increases its affinity to PLC₁ by over 2 orders of magnitude. With this in mind, we measured changes in FRET with stimulation of PC12 cells expressing eCFP-G_q and eYFP-PLC₁ by the addition of either 1 µM carbachol or 1 µM acetylcholine. The results for four single cells are shown in Fig. 5. In all cells viewed and analyzed (n >12), we find that the amount of FRET does not significantly change upon the addition of a G_q agonist. Moreover, as mentioned above, a stable co-localization of eYFP-PLC₁/eCFP-G_q was observed for eYFP-PLC₁-eCFP-G_q complexes in HEK293 cells by both FRET and by co-immunoprecipitation, suggesting that this behavior is not specific to PC12 cells. These results suggest that even though the affinity between eYFP-PLC₁/eCFP-G_q is expected to increase significantly upon activation, the cytoplasmic population of eYFP-PLC₁ is not recruited to the plasma membrane surface by eCFP-G_q.

To confirm the single cell studies above, we measured relative changes in FRET between eYFP-PLC₁/eCFP-G_q expressed in 10⁶ PC12 cells in a fluorometer as described above. Similar to the single cell studies, these results (Fig. 6A) also show that the number of eYFP-PLC₁-eCFP-G_q complexes does not significantly change with cell stimulation.

A trivial explanation of the constant FRET between eCFP-G_q and eYFP-PLC₁ with stimulation is that activation is not efficient. To determine whether this is the case, we measured the amount of FRET between eCFP-G_q-RC and eYFP-PLC₁. This point mutant produces a constitutively active G_q (15), which is expected to be strongly associated with PLC₁ in the unstimulated state, and we expect this association to change little with the addition of agonist. We find that both the basal and stimulated FRET values between eCFP-G_qRC and eYFP-PLC₁ are similar to wild type (Fig. 6B) suggesting that association between the two proteins is not related to the stimulated state.

To ensure that the eCFP-G_q/eYFP-PLC₁ expressed in cells is part of a signaling complex coupled to a G_q receptor, specifically the m1 and m5 muscarinic receptors in PC12 cells (29), and has the ability to increase intracellular calcium upon stimulation, we measured the amount of Ca²⁺ released upon the addition of 1 µM acetylcholine in 10⁶ PC12 cells. The results, presented in Fig. 7, show that in the basal state all four cell groups have similar levels of internal Ca²⁺ as expected from its tight cellular regulation. Upon the addition of agonist, a similar robust increase in intracellular Ca²⁺ from nontransfected and singly transfected cells is observed. However, the doubly transfected cells gave a significantly higher amount of released Ca²⁺. Considering that the G_q-coupled m1 and m5 receptors constitute only 5% of the muscarinic receptors in these cells (29), this increase suggests that a large and significant population of the transfected proteins is functional and coupled to muscarinic receptors.

Although eYFP-PLC₁-eCFP-G_q complexes may not dissociate upon stimulation, it is possible that their localization in the cell changes during activation. We tested this idea using the commercially available plasma membrane markers YFP-MEM and CFP-MEM (Clontech, Inc.). These markers show a transfection efficiency of 100%. To follow protein localization, we co-transfected the CFP-MEM or YFP-MEM markers at a high enough level to give a significant and reproducible amount of FRET with eCFP-G_q or eYFP-PLC₁ (i.e. 46 and 41%, respectively).

Terminally differentiated PC12 cells were either co-transfected with CFP-MEM and eYFP-PLC₁ or YFP-MEM and eCFP-G_q, and changes in FRET with stimulation were measured for 10⁶ cells in a fluorometer (see above) using the procedure described by Lohse and co-workers (30). After measuring a stable basal level of eYFP/eCFP emission for 5 min, the cells were stimulated, and the CFP and YFP emission intensity was monitored over 30 min. Both time and stimulation did not affect the intensity of the CFP and YFP emissions. These results (Fig. 8) indicate that the proteins do not move off the membrane upon stimulation with acetylcholine or carbachol.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. A, analysis of time lapse FRET images of PC12 cells expressing eCFP-Gq and eYFP-PLC1. The variation of the percentage of FRET for stimulated and unstimulated cells is shown in the insets where stimulation was carried out by the addition of 1 µM acetylcholine (ACh) after obtaining a flat base line for 30 s. Error bars indicate S.E. for each time point. B, identical study as in A, except that cells expressed the constitutively active eCFP-Gq-RC, rather than wild type.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Internal Ca2+ levels of PC12 cells in the basal, stimulated, and recovery states (200 s after stimulation) for four groups of transfected cells labeled with Fura-2AM as follows: empty vector, Gq-eCFP, PLC1-eYFP, and co-transfected Gq-eCFP and PLC1-eYFP. Asterisks indicate significant difference (p < 0.05) between groups or between different states within each individual group. The only significant difference between groups resulted from comparing the stimulated state of empty vector and the co-transfected cells. Error bars indicate S.E.

Studies of YFP-PLC₁-eCFP-G_q Complexes in Membrane Preparations—The FRET results of live cell studies performed above clearly showed eYFP-PLC₁-eCFP-G_q complexes in the quiescent state. The presence of these complexes suggests that some cellular factors, such as protein scaffolds, may stabilize eCFP-G_q/eYFP-PLC₁ in the basal state. We extended the cellular studies using membrane preparations of cells expressing these proteins and related partners to better understand whether eYFP-PLC₁-eCFP-G_q complexes are stabilized by cytoskeletal or cytosolic proteins. We expressed pairs of G_qRC, G_q, PLC₁, G₁₇, and G_s with either CFP or YFP tags in HEK293 cells by transient transfection and prepared membrane fractions (see "Materials and Methods"). We then determined the extent of eCFP/eYFP FRET in each membrane preparation by the ratio of donor and acceptor emission maximum intensities at 490:527 nm using an exciting wavelength of 440 nm as described (21). In an initial series of studies, the degree of FRET was measured as a function of the amount of cDNA used in the transfection. Similar to whole cell studies, our results show a systematic increase in the amount of FRET between eCFP-G_q and eYFP-PLC₁ from 18 to 100% with increasing cDNA used for transfection. Taken together, these results show that in the absence of cytoskeletal and cytosolic components, eCFP-G_q and eYFP-PLC₁ form stable and titratable complexes on membrane surfaces, and these complexes can be seen in the basal state even under low levels of transfection.

In Fig. 10 we show a comparison of different protein pairs in the basal and stimulated states at moderate levels of transfection. High levels of FRET are seen with the eCFP-G_qRC/eYFP-PLC₁ probe pair, but comparably high levels are also seen for eCFP-G_q/eYFP-PLC₁ pair. Stimulation of membranes containing the eCFP-G_q/eYFP-PLC₁ pair does not cause a significant increase in the level of FRET in accordance with the results seen in living cells. This lack of change occurred for 18:21 samples regardless of the initial FRET value that ranged from 68 to 92% for identical transfection conditions. Treating the samples with SDS reduced the level of FRET to close to those obtained for a negative control using eCFP-G_q/eYFP-G_s. Thus, the low FRET values for the SDS-treated samples are considered background noise because of the contribution of overlapping donor/acceptor emission when calculating FRET. The important point is that the relative FRET values between eYFP-PLC₁ and eCFP-G_q and between eYFP-PLC₁ and eCFP-G_q(RC) are high and similar to those obtained for eYFP-G_s and eCFP-G₁₇. This latter protein pair forms strong complexes in the basal state that weaken upon stimulation (i.e. isoproterenol and GTPS) (7), and the data in Fig. 10 support this idea.

FRAP Studies of Complex Diffusion—If eYFP-PLC₁-eCFP-G_q complexes were also stably associated with other proteins in higher order complexes, their cellular diffusion would be limited. To determine whether this is the case, we used FRAP to measure the diffusion rates of eCFP-G_q and eYFP-PLC₁. This was accomplished by laser pulsing a 2-µm² spot on the bottom of the cell to bleach the fluorophores and taking images every 0.5-2 s, depending on the sample. Images were analyzed by determining the change in intensity with time of the region that was bleached versus a region of the cell far from the bleach. For simplicity, we fit the data to 1-3 exponential decay curves to determine the number of decay mechanisms and their corresponding time constants. Free eYFP diffusing in the cell body fits best to a single exponential with a time constant of 0.5 ± 0.05 s (n = 5) correlating well with previous work (31). For simplicity, we will give comparative values of these rates in PC12 cells as follows: 76.2% of eYFP-PLC₁ is mobile and diffuses with a single exponential rate that is 40-fold slower than free YFP (n = 7). 63.5% of eCFP-G_q is mobile and shows two diffusion rates that are 10- and 40-fold slower than free YFP (n = 5). These results show that the proteins have restricted diffusion; both proteins have a significant immobile population and a slower similar diffusion that is on the order of those reported for membrane-bound cellular proteins (31).

View larger version (22K): [in this window] [in a new window]   FIGURE 8. A, emission signal and ratio from cells co-transfected with eCFP-Gq and eYFP-PLC1. The spike in the emission signal indicates when the stimulus was added. B, emission signal and ratio from cells co-transfected with eCFP-Gq and MEM-YFP. C, emission signal and ratio from cells co-transfected with eYFP-PLC1 and MEM-CFP. Cells in A were stimulated at the 120-s time point, and cells in B and C were stimulated at the 300-s time point.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We measured the association of G_q and PLC₁ by overexpressing fluorescent-tagged proteins. First, we determined whether overexpression of the protein would promote complex formation by comparing the level of expressed protein to endogenous levels. We find that our level of expression is only 2-3-fold higher than that of the endogenous protein which, as detailed below, is not expected to significantly promote complex formation. Western blot analysis of the protein levels found in a known number of cells allows us to roughly estimate the endogenous cellular concentrations. We find that these approximate concentrations are low in the cell (e.g. 20 fM for G_q and 3 fM for PLC₁). Moreover, these values are far lower than the dissociation constants estimated for these proteins (Fig. 1) even taking into account that their association may be slightly promoted by the tendency of the GFP analog tags to dimerize (K_d GFP is 100 µM (32)) and considering that attachment to the membrane may promote association more than the confinement to 300 µM lipid vesicles using for the in vitro measurements (9). Given the magnitude of these affinities, increasing the levels of the proteins 2-3-fold is not expected to alter the results obtained here.

We find that the cellular concentration of G_q far exceeds PLC₁. Considering that G_q interacts with other proteins such as receptor, G subunits, and a second effector phosphatidylinositol 3-kinase (33), it is probable that this "excess" G_q is bound to other partners. It is also possible that G_q has nonoverlapping interacting sites between these partners allowing for multiple interactions.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. A, behavior of the normalized FRET for unstimulated and stimulated (i. e. 1 µM acetylcholine 30s) cells starting at the bottom of the cell (slice 1) and moving up in the z plane at 0.24-µm intervals. The data shown are a composite of nine cells. where the error bars indicate S.E. B, montage of z-stack images showing the FRET through a PC12 cell in the unstimulated and stimulated states where each frame corresponds to 0.24 µ. B, top and bottom panels were stimulated at the 300-s time point.

View larger version (18K): [in this window] [in a new window]   FIGURE 10. Percent FRET of membranes prepared from HEK293 cells (see "Materials and Methods") expressing the constructs as noted where stimulation was carried out by the addition of 200 µM carbachol for membranes expressing eCFP-Gq and 100 µM isoproterenol for membranes expressing eYFP-Gs in the presence of 100 µM GTPS.

To understand the basis of the cytosolic and plasma membrane populations of PLC₁, we note that its cellular localization should be a function of its intrinsic membrane binding constant and the presence of protein partners. We have characterized the membrane binding properties of purified PLC₁ to lipid bilayers of varying composition (25). These studies showed that PLC₁ binds strongly and fairly nonspecifically to membranes with a membrane partition coefficient of 50 µM. Based on these membrane affinities, we expect almost all PLC₁ to be bound to cellular membranes. Localization of PLC₁ because of membrane interactions rather than specific interactions with G_q would be expected to give rise to both cytosolic and plasma membrane populations. This idea also correlates well with the low cellular concentrations of the two proteins.

View larger version (66K): [in this window] [in a new window]   FIGURE 11. Model of activation of pre-associated PLC1-Gq(GDP) in which the change in interaction between G and G subunits because of agonist binding to a G protein-coupled receptor causes a change in the protein interface between G and its PLC effector.