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1 Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA
2 Departments of Neurosciences and Biology, University of California, La Jolla, CA, USA
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Abstract |
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o subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABAB R1 and R2 subunits (
12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABAB R1 or R2 subunits (13% FRET), and between Go and GABAB R1 or R2 subunits (10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABAB R1 or R2 subunits (10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Go. These data are discussed in terms of a model in which GABAB receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.
(Received 19 October 2006;
accepted after revision 14 December 2006;
first published online 21 December 2006)
Corresponding author P. A. Slesinger: The Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA. Email: slesinger{at}salk.edu
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Introduction |
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subunit which, in turn, leads to activation of the G subunit and the G
dimer. G dimers bind to and activate Kir3 channels (Reuveny et al. 1994; Wickman et al. 1994; Huang et al. 1995). G subunits are required for terminating Kir3 activation. The intrinsic GTPase activity of the G subunit hydrolyses GTP, leading to inactivation of the G dimer. Regulator of G protein signalling (RGS) proteins accelerate the GTPase activity of G subunits (GAP), leading to faster activation and deactivation of Kir3 channels (Doupnik et al. 1997).
Several studies suggest that the receptors, G proteins and channels may exist in a signalling complex (Lavine et al. 2002; Peleg et al. 2002; Clancy et al. 2005). We have recently found that the pertussis toxin (Ptx)-sensitive G heterotrimer (i.e. Gi/o family) associates directly with Kir3 but not with G protein-insensitive inward rectifiers (Clancy et al. 2005). Mutations in the C-terminal domain of Kir3 that disrupt this interaction also impair channel activation by GPCRs (Clancy et al. 2005). These experiments indicate that a close association between the G protein and channel is required for efficient activation, suggesting that Kir3 channels exist in a specific signalling complex. One prediction of this hypothesis is that diffusion of proteins within the complex will be restricted. Experiments examining the lateral mobility of G proteins indicate that G proteins in the plasma membrane are constrained in their movement (Kwon et al. 1994). The protein–protein interaction of both the G and G subunits with Kir3 channels and GPCRs suggests that the G subunit may not dissociate from the G dimer upon G protein activation (Clancy et al. 2005). Although there is biochemical evidence for Gq and Gs dissociation from G, the evidence demonstrating dissociation for Gi/o G proteins is less clear (Rebois et al. 1997). Klein et al. (2000) demonstrated that dissociation is not a prerequisite for signalling. Directly fusing G to G, thereby preventing dissociation, does not alter the signalling properties in yeast. Consistent with this finding, Bunemann et al. 2003) used fluorescence resonance energy transfer (FRET) to demonstrate that Gi and G undergo a conformational rearrangement, rather than dissociation, during activation. Recent FRET studies have also suggested that some GPCRs are precoupled with the G protein (Gales et al. 2005; Nobles et al. 2005; but see Hein et al. 2005). These studies suggest that a signalling complex may exist that contains the receptor, G protein and channel.
In support of this model, Lachance et al. (1999) demonstrated that G remains associated with 2-adrenergic receptors following activation. Furthermore, Lavine et al. (2002) discovered that D2/D4 dopamine receptors form stable, G-dependent complexes with Kir3 channels. The lipid environment may further segregate specific signalling molecules. For example, Kir3 channels, GABAB receptors and Gi/o G proteins associate with lipid rafts, whereas metabotropic glutamate receptors and Gq G proteins associate with caveolin (Becher et al. 2001; Oh & Schnitzer, 2001; Koyrakh et al. 2005). Finally, recent studies have shown that RGS proteins may stably interact with G G proteins (Benians et al. 2005) and µ opioid receptors (Georgoussi et al. 2006). Taken together, these studies suggest that Kir3 channels, G proteins, GPCRs, other signalling proteins (e.g. RGS proteins and receptor kinases), lipids and cytoskeletal anchoring proteins (Bloch et al. 2001) coexist in a macromolecular signalling complex.
We hypothesized that the G heterotrimer interacts with the GPCR, RGS and Kir3 proteins under resting conditions. Upon receptor activation, the G subunit undergoes a conformational rearrangement that reveals a surface of G that binds to and activates Kir3 channels (Bunemann et al. 2003; Clancy et al. 2005). Here, we asked whether these protein–protein interactions could be studied using advanced microscopic techniques. We used FRET to study these potential protein–protein interactions in living cells. The magnitude of FRET is inversely proportional to the sixth power of distance, making FRET a highly sensitive tool for detecting distances of less than 100 Å between fluorophores (Jares-Erijman & Jovin, 2003). We used total internal reflection fluorescence (TIRF) microscopy to selectively measure the FRET in the plasma membrane, avoiding contamination from cytoplasmic signals (Axelrod et al. 1983). The spectral properties of Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) are well matched for measuring FRET and these fluorophores can be genetically engineered into any protein (Heim & Tsien, 1996). We constructed a series of CFP and YFP fusion proteins (including Kir3 channels, Go subunits, GABAB receptors and RGS4 proteins) for FRET analysis of possible associations within this signalling pathway. Proteins that bring the CFP/YFP fluorophores within 100 Å of each other would be expected to generate a FRET signal.
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Methods |
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CFP and YFP (we use the terms ‘CFP’ and ‘YFP’ for enhanced CFP (eCFP) and enhanced YFP (eYFP), respectively) fusion proteins were constructed as follows. For all C-terminal fusions proteins, the stop codon was eliminated by PCR mutagenesis. For Kir3.2a–YFP and Kir3.2a–CFP, Kir3.2a (Lesage et al. 1994) cDNA was subcloned by PCR into Xho1/HindIII sites of pEYFP-N1 or pECFP-N1 (BD Biosciences Clontech, Mountain View, CA, USA). CFP–Kir3.4 and Kir3.1–YFP cDNAs were provided by Riven et al. (2003). For Kir2.1*–YFP, the GYG sequence in the pore of Kir2.1 (Kubo et al. 1993) was mutated to AAA (denoted by *) using overlapping PCR and subcloned into the Xho1 and BamH1 of pEYFP-N1. For GABAB R1/R2–YFP and R1/R2–CFP, GABAB R1 and R2 cDNAs were subcloned by PCR into EcoR1/Age1 sites of pEYFP-N1/pECFP-N1. For RGS4–CFP, RGS4 was amplified using RT-PCR with rat brain RNA and subcloned into XhoI/AgeI site of pECFP-N1. For m1–YFP muscarinic receptor, human m1 receptor was subcloned by PCR into XhoI/HindIII of pEYFP-N1. For rat Go fusions, AgeI site was created after E94, M114 and I261 in Go–pcDNA3.1+. The eCFP was amplified from pECFP-N1 by PCR with an amino acid linker flanking the eCFP (TGSGGGGSTGGGGS–CFP–GGGGSQGGGGSAG) and spliced into the AgeI site. C351 was mutated to G to make Go resistant to Ptx inactivation (Go*) (Mutneja et al. 2005). We did not observe a significant difference in FRET measurements between the wild-type and Ptx-insensitive versions of CFP-tagged Go and therefore pooled these data. For Rho-pYC, the C-terminal prenylation site of Rho (RQKKRRGCLLL) was added to the C-terminal domain of a YFP–CFP fusion (Mayr et al. 2001). In RGS4 experiments, Go, G1 and G2 cDNA were coexpressed with RGS4–CFP. All constructs were confirmed by DNA sequencing.
Tissue culture and transfections
For most experiments, HEK293T [obtained from America Type Culture Collection (ATCC)] cells were used with the objective of obtaining high expression of signalling proteins. HEK293T is a highly transfectable derivative of the HEK293 that contains the temperature sensitive gene for SV40 T-antigen. We also examined expression of cDNA in HEK293 cells and observed qualitatively similar results. Data was therefore pooled between HEK293T and HEK293 cells, which we collectively refer to as ‘HEK293’ throughout the paper. HEK293 cells were cultured under sterile conditions in DMEM supplemented with fetal bovine serum (10%), glutamine (2 m) and penicillin (50 units/ml), streptomycin (50 µg/ml; GIBCO, Invitrogen Corp) in a humidified 37°C incubator with 95% air and 5% CO2. For FRET experiments, cells were seeded into six-well plates (Corning, Inc.) 3 days prior to experiment. Cells (50% confluent) were transiently transfected 2 days prior to experiment using the calcium phosphate technique. Briefly, cDNA (0.4–0.8 µg well–1) was mixed in sterile deionized water with 0.25 M CaCl2, then combined 1: 1 with Hepes-buffered saline containing (mM): NaCl 280, KCl 10, Na2HPO4 1.5, glucose 12 and Hepes 50 (pH adjusted to 6.9 with 1 N NaOH), to yield a final volume of 10% of the total well volume. The mixture was added to cells in six-well plates and incubated for 24 h at 37°C. The day before experiments, cells were reseeded onto 35 mm glass-bottomed cell culture dishes (containing a collagen-coated, #1 thickness, 14 mm glass coverslip; MatTeck Corp. Ashland, MA, USA). For imaging, cells were bathed in Hanks' balanced salt solution (Gibco Invitrogen Corp., Carlsbad, CA, USA; 14065–056) in the absence or presence of baclofen (Sigma-Aldrich, Inc., St. Louis, MO, USA) or CPG5546 (30 µM; Tocris Bioscience, Bristol, UK) for 3–25 min. Cells were approximately 80% confluent on the day of experiment. For Ptx treatment, Ptx (250 ng ml–1) was added to each dish 4–24 h prior to experiment.
For electrophysiological recordings, HEK293 or HEK293T cells were plated onto 12 mm glass coverslips (Warner Instruments) coated with poly-D-lysine (20 µg ml–1; Sigma-Aldrich, Inc) and collagen (100 µg ml–1; BD Biosciences) in 24-well plates. Cells were transiently transfected using the calcium phosphate method as above except that the DNA mixture (0.05–0.1 µg well–1) was added to cells in 24-well plates and incubated for 16–32 h at 37°C, and cells were not reseeded.
Electrophysiology
Whole-cell, patch-clamp technique (Hamill et al. 1981) was used to record macroscopic currents from HEK293 cells. Borosilicate glass (Warner; P6165T) electrodes had resistances of 1–3 M
and were coated with Sylgard to reduce capacitance. Membrane currents were recorded with an Axopatch 200B (Axon Instruments-Molecular Devices Corp., Sunnyvale, USA) amplifier, adjusted electronically for cell capacitance and series resistance (80–100%), filtered at 2 kHz with an 8 pole Bessel filter, digitized at 5 kHz with a Digidata 1320 interface (Axon Instruments-Molecular Devices Corp., Sunnyvale, USA) and stored on a laboratory computer. The intracellular pipette solution contained (mM): KCl 130, NaCl 20, EGTA 5, K2ATP 2.56, MgCl2 5.46 and Hepes 10; pH was adjusted to 7.2 with 14 mM KOH. There was 140 mM K+, 1.5 mM free Mg2+ and 2 mM MgATP in the intracellular solution. Li3-GTP (300 µM; Sigma) was added fresh to the intracellular pipette solution. The external bath solution (20K) contained (mM): NaCl 140, KCl 20, CaCl2 0.5, MgCl2 2 and Hepes 10 (pH 7.2); osmolarity 310–330 mosmol l–1. For measurement of leakage current, 20 mM KCl was replaced by 20 mM NaCl to give 160 mM extracellular Na+ (160Na). Currents were elicited with one of two protocols: a 200 ms voltage ramp from –100 to +50 mV delivered at 0.33 Hz, or continuous current recording at –80 mV. Agonist-independent current (basal) was determined by subtracting the current in 160Na from that in 20K. For ethanol activation, 100% ethanol was added directly to the 20K solution to give 200 mM ethanol (density, 0.7893 g ml–1). GABAB receptors were activated with 100–300 µM (±)-baclofen (Sigma-Aldrich, Inc). Current–voltage relations were not corrected for the junction potential of 4 mV, estimated using the Junction Potential Calculator (Axon Instruments - Molecular Devices Corp.). Activation and deactivation time constants were measured by fitting the current traces with a single exponential.
TIRF microscopy and FRET measurements
Through-the-objective TIRF microscopy is achieved when collimated laser light is offset to illuminate the back focal plane of the objective, which causes the laser light to arrive at the coverslip at an angle (Fig. 3A). When this angle is greater than the critical angle (
), an evanescent wave of excitation light is produced at the interface between two media having different refractive indices, the glass coverslip and the media or cell membrane (Axelrod et al. 1983). The intensity of this evanescent wave falls off exponentially with distance above the interface, allowing selective imaging within 100 nm of the glass–medium interface (i.e. plasma membrane and submembrane regions). For TIRF microscopy, we used a Nikon TE2000 microscope, a 60x oil-immersion TIRF objective (Nikon; 1.45 NA), and either a tunable krypton–argon laser tuned to 514 nm (Melles Griot; model, 643-AP-A01) or a solid state DPSS 442 nm laser (Melles Griot; model, 85 BTL 010). The light from a Polychrome IV monochromator (Till Photonics) was also combined with the laser into a single condenser at 50% intensity for both laser and monochromator. The Nikon filter cube contained a polychroic mirror with reflection bands at 440 and 510 nm and band-passes at 475/30 and 560/60 nm (z442/514rpc; Chroma Technology Corp., Bockingham, VT, USA). No excitation filters were used. CFP and YFP emission filters (470/30 for CFPEm and 535/50 nm for YFPEm, respectively) were placed in a filter wheel (Sutter Instruments) and controlled by a Lambda 10–2 controller (Sutter Instruments). Images 16 bit were acquired with a Till 12.5 MHz Imago CCD camera (Till Photonics). The camera, laser shutters and filter wheel were electronically controlled by TILLvisION 4.0 software. Images were analysed using TILLvisION 4.0 software and NIH ImageJ software (FRETcalcv1 plugin).
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Images (16 bit) were acquired for CFP fluorescence (400 ms exposure, 2 x 2 binning – 442 nm laser, CFPEm filter), FRET fluorescence (400 ms exposure, 2 x 2 binning – 442 nm laser, YFPEm filter) and YFP fluorescence (100 ms exposure, 2 x 2 binning – 514 nm laser, YFPEm filter) before and after photobleaching (60–90 s) with the 514 nm laser and monochromator tuned to 514 ± 8 nm. The combination of laser and monochromator consistently produced 20% more bleaching of YFP (92% total bleaching), increasing the FRET efficiency (see online Supplemental Material Fig. S1). Furthermore, because proteins are in dynamic equilibrium between plasma membrane and cytoplasm, it was advantageous to photobleach cytoplasmic, as well as membrane localized acceptor fluorophores. Measuring CFP only at the membrane ensures that the FRET signal originates only from membrane fluorophores. %FRET was calculated as the percentage increase in CFP emission after photobleaching YFP (eqn (1)):
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cAMP assay
HEK293 cells were seeded into 48-well plates and transiently transfected 24 h later (using the calcium phosphate method) with cDNA encoding GABAB R1 and R2 subunits (0.25 µg well–1), together with either empty plasmid (pcDNA3), Go, Go* or Go*-94–CFP (0.05 µg well–1). Cells were treated with Ptx (250 ng ml–1) the day after transfection, and the cAMP assay performed in triplicate 2 days after transfection. For the cAMP assay, cells were washed in fresh medium, and treated with 3-isobutyl-1-methylxanthine (IBMX) (0.1 µM; 15 min) to block phosphodiesterases, and then treated with forskolin (5 µM; 10 min) to stimulate adenylyl cyclase, in the absence or presence of baclofen (100 µM; 10 min). To rapidly stop the reaction, the entire plate was inverted to remove the liquid and 95% ethanol–0.1 N HCl (200 µl well–1) was added rapidly to each well. Plates were placed in a –20°C freezer for 30 min. The contents of each well was transferred to an Eppendorf tube, and placed in a vacuum concentrator (Speedvac, Savant) to dry completely. cAMP concentrations were measured using a radioimmunoassay kit, according to the manufacturer's instructions (Biomedical Technologies, BT-300). The assay is based on competitive binding of cAMP in the sample with iodinated cAMP (cAMP- 125I) for a highly specific cAMP antibody. Samples were counted in a gamma counter (Apex Automatic; Micromedic Systems) and normalized to forskolin-stimulated data.
Analysis
All values are reported as mean ± S.E.M. Statistical significance was assessed using one-way ANOVA followed by post hoc test (Bonferroni), using a significance level of P < 0.05 (SigmaStat 3.0).
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Results |
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We showed previously that Go interacted directly with Kir3.2a, suggesting that Kir3 channels exist in a signalling complex that contains the GPCR, G protein and Kir3 channel (Clancy et al. 2005). To investigate potential protein–protein interactions in living cells, we measured FRET in transiently transfected HEK293 cells using TIRF microscopy. TIRF microscopy enables fluorescence excitation only near the interface between the glass coverslip and the aqueous solution bathing the cells, thereby allowing selective visualization of plasma membrane fluorophores without contamination from fluorophores in the cytoplasm (Axelrod et al. 1983). CFP or YFP were fused to the C-terminal domain of GABAB receptor R1 or R2 subunits, to the N- and C-terminal domains of Kir3.1, Kir3.2a and Kir3.4 channels, and to the C-terminal domain of RGS4. The Kir3.1–YFP and CFP–Kir3.4 constructs were tested previously and shown to undergo FRET (Riven et al. 2003). Introduction of CFP into G was complicated because the N-terminal domain of G is required for anchoring to the plasma membrane and the C-terminal domain is essential for coupling to the GPCR (Wall et al. 1995). We therefore inserted CFP into three different loops of Go (Fig. 1A). Two were located in the helical domain; CFP was inserted after E94 in the loop connecting A and B helices, similar to yeast G (Janetopoulos et al. 2001), and after M114 (loop connecting B–C). A third CFP was inserted after I261, just following switch III (between 3 and 5) (Wall et al. 1995).
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o by examining the functional coupling of GABAB receptors to Kir3.2a channels expressed in HEK293 cells. To study the function of ectopically expressed Go in the absence of signalling through endogenous G, a C-terminal cysteine was mutated thereby rendering the Go protein insensitive to Ptx (Avigan et al. 1992). Figure 1 shows an example of whole-cell current responses in HEK293 cells pretreated with Ptx (250 ng ml–1; 4 h) to uncouple endogenous G proteins from the GABAB receptor. Ptx-treated HEK293 cells transfected with wild-type Go showed no baclofen-activated Kir3 current, but retained an ethanol-activated Kir3 current, which is G protein independent (Kobayashi et al. 1999; Zhou et al. 2001). By contrast, HEK293 cells transfected with Go*-94–CFP showed robust rescue of the baclofen responses. The basal, ethanol-activated and baclofen-activated Kir3 currents were measured in cells transfected with the different Go constructs. Go*, Go*-94–CFP and Go*-114–CFP each rescued baclofen-activated currents to a similar extent in Ptx-treated cells (Fig. 1D), indicating that the CFP did not interfere with G protein coupling to Kir3 channel. In Ptx-treated cells, stimulation of Go*-94–CFP inhibited forskolin-stimulated cAMP accumulation to a similar extent as Go*. The normalized values were 1.15 ± 0.05 (n
= 3) for pcDNA3.1 vector, 0.99 ± 0.12 (n
= 3) for wild-type Go, 0.22 ± 0.04 (n
= 3) for Go*, and 0.25 ± 0.07 (n
= 3) for Go*-94–CFP (see Methods for details). Go-261–CFP, on the other hand, did not appear to couple to Kir3 channels, suggesting the placement of the CFP in this construct interfered with its function (data not shown). We then examined the G protein coupling between YFP-tagged receptors and CFP-tagged channels (Fig. 2A and B). Coexpression of GABAB R1–YFP or GABAB R2–YFP with CFP-tagged channels (Kir3.2a–CFP or CFP–Kir3.4/Kir3.1) resulted in baclofen-induced currents indistinguishable from control (Fig. 2A and B), suggesting the fluorophores do not demonstrably interfere with the signalling of the proteins. We also studied the ability of RGS4–CFP to modulate Kir3 channel activation. RGS4 accelerates the GTPase activity of Go/i G proteins, leading to faster activation and deactivation rates (Doupnik et al. 1997). Co-expression of RGS4–CFP with the m2 muscarinic receptor and Kir3.2a channels resulted in carbachol-activated currents that activate and deactivate more rapidly (Fig. 2C and D).
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TIRF microscopy enables the study of fluorescent proteins at a distance of 100 nm above the glass coverslip, which includes the plasma membrane and submembrane regions (Fig. 3A). To illustrate this, we examined the difference between epifluorescence and TIRF microscopy with cells transfected with Go-94–CFP. Figure 3A shows CCD images of the same cell collected using either epifluorescence or TIRF illumination (excited at 442 nm and collected using the CFPEm filter). Notice that the surface proteins are readily visible in the TIRF image. We also examined FRET with a construct intrinsically designed to produce basal FRET at the plasma membrane – YFP was fused directly to CFP and anchored to the lipids via a Rho-lipid binding motif (Rho-pYC). Using eqn (1), we calculated the FRET efficiency for Rho-pYC to be 28.0%
± 2.0% (n
= 13) under TIRF microscopy (see Methods for details).
We next examined possible FRET between Go-114–CFP and either GABAB R1–YFP, GABAB R2–YFP or Kir3.2a–YFP (Fig. 3B–E, see online Supplemental Material Fig. S2). For all experiments using tagged GABAB receptors, a YFP-labelled subunit was always coexpressed with the untagged partner subunit to form functional heterodimeric receptors (Kaupmann et al. 1998; White et al. 1998). For controls, CFP-tagged proteins were coexpressed with untagged protein (‘no YFP’) (Fig. 3D, Table 1). Photobleaching of YFP consistently reduced YFP intensity by 90% (Fig. 3B and C, lower panels). An increase (0–5% increase) in CFP following YFP photobleaching was observed in most negative control experiments (i.e. CFP alone). A similar small increase in CFP fluorescence was observed for non-interacting CFP/YFP-tagged protein pairs, following photobleaching YFP (see Table 1). The increase in CFP fluorescence may represent a small fraction of CFP molecules that resensitize and/or move into the evanescent wave during the bleaching protocol (see Methods). Controls were therefore included in every FRET experiment. CFPEm fluorescence clearly increased following APB for Go-114–CFP/R1–YFP pair compared with Go-114–CFP/Kir3.2a–YFP pair (Fig. 3B and C, upper panels; see online Supplemental Material Fig. S2). To illustrate this, a histogram of FRET efficiency calculated pixel-by-pixel was compiled, demonstrating a 10–15% increase for R1 and Go and –5 to +5% change for Kir3.2a and Go (Fig. 3B and C). The mean %FRET measured over several cells was significantly higher for cells coexpressing R1–YFP or R2–YFP and Go-114–CFP (Fig. 3D). We also examined possible FRET between Go-94–CFP and R1–YFP, R2–YFP or Kir3.2a–YFP. Like Go-114–CFP, Go-94–CFP showed statistically significant FRET with the GABAB receptor but not with the channel (Fig. 3E). In addition, no significant FRET was observed between the Go constructs and either Kir3.1, Kir3.2a or Kir3.4 channels tagged on the N- or C- terminal domain (data not shown). We conclude from these experiments that some Go subunits are situated near GABAB receptors to produce FRET. By contrast, Go and Kir3 channels did not show any FRET though these two proteins are presumed to be close. One possible reason for the lack of FRET could be that insertion of fluorophore disrupted the signalling of GCFP. However, CFP-tagged Go subunits can couple to GABAB receptors and Kir3 channels (Fig. 1). Alternatively, the lack of FRET could indicate that the fluorophore is located in a position unfavourable for FRET with the channel (see Discussion).
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We next examined whether stimulation of the GABAB receptors altered the FRET signal between Kir3 channels and GABAB receptors. Cells were divided into two groups: ‘activated’ – cells were incubated in baclofen (100–300 µM) for 3–25 min, and ‘control’ – cells were incubated in GABAB receptor antagonist CPG5546 (30 µM) for 3–25min. We observed no statistical difference in the %FRET in cells treated with agonist or antagonist (Fig. 5A and B). Therefore, the association between GABAB receptors and Kir3 channels appears to exist in the absence of G protein activation, and persists during receptor activation. In HEK293 cells coexpressing GABAB receptors and Kir3.2a channels, the baclofen-induced current desensitizes by 60–70% over 2–3 min (Mutneja et al. 2005). Thus, many of the activated receptors have desensitized with the agonist stimulation for 3–25 min, suggesting that the FRET measured may reflect receptors in activated and desensitized states (see Discussion).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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o G proteins, Kir3 channels and RGS4 proteins. We found that GABAB receptors and Kir3 channels, as well as GABAB receptors and RGS4 proteins, move within the 100 Å needed to generate FRET. It is important to note that the fluorophore-tagged proteins used in our study were functional; they could mediate G protein activation and modulation of Kir3 channels via stimulation of GABAB receptors.
One advantage of using the APB method is the ability to compare the %FRET among different studies. The %FRET (8–13%) in our study compares favourably with 15% FRET for GPCR and G (Nobles et al. 2005), 20% FRET for Kir3.1–YFP and CFP–Kir3.4 channels (Riven et al. 2003), and 30% FRET for GABAB R1 and R2 subunits (Uezono et al. 2006). It is interesting that we did not detect FRET between Go and Kir3 channels or between RGS4 and Kir3 channels. There are several different scenarios that can result in little or no FRET. First, if the two fluorophores are not close enough (> 100 Å), then there will be no FRET. Second, FRET will not occur if the dipoles of CFP and YFP are perpendicular (even if the two proteins are within 100 Å). This possibility seems unlikely for Go because CFP was inserted into two different positions, probably leading to two different orientations of CFP. Third, the insertion of the CFP could interfere with the ability of the tagged protein to directly associate with the channel. However, GABAB receptor stimulation of Go–CFP led to activation of Kir3 channels and RGS4–CFP expression resulted in faster Kir3 channel kinetics. In addition, Go–CFP or RGS4–CFP were able to undergo FRET with other YFP-tagged proteins. As we cannot distinguish between these possibilities, our experiments do not provide evidence for or against a direct association between Kir3 channels and either Go or RGS4 proteins. It is interesting that Riven et al. (2006) recently used FRET measurements to provide evidence that the G heterotrimer associates with Kir3 channels in the resting state.
For one model of GABAB receptor signalling, the R1 subunit is believed to bind ligand while the R2 subunit signals to G proteins (Bettler et al. 2004). We found that FRET occurred between Go–CFP and both YFP-tagged GABAB R1 and R2 receptor subunits. These findings suggest that Go–CFP is situated near both R1 and R2 subunits in the resting state. We cannot estimate from our measurements what fraction of receptors and Go associate. The FRET signal may represent a fraction of Go–CFP that stably associates with R1 and R2, or a time-averaged signal from continuously associating/disassociating receptors and G proteins, or a combination of both. Two recent studies provide evidence that components of the G protein signalling pathway move independently within the membrane. Azpiazu & Gautam (2004) used a FRET-based approach to show that the same pool of G proteins couple consecutively with different receptors, indicating that if a stable complex exists, the G G proteins do not (or at least not all of them) form part of it. Similarly, Hein et al. (2005) found little evidence for precoupling between the 2a-adrenergic receptor and Gi G proteins, and suggested a collision coupling as a mechanism for this receptor–G protein pair. In contrast to our study, no basal FRET between receptor and G protein was detected in their study. Gales et al. (2005), on the other hand, measured FRET between Gs and the 2-adrenergic receptor in the absence of receptor stimulation, as did Benians et al. (2003) for Go with either the 2-adrenergic receptor, m4 muscarinic receptor, A1 adenosine receptor or D2S dopamine receptor. The disparities between the results of these studies underscore the need to study the detailed mechanism of G protein coupling with specific receptors and effectors expressed in their native environment.
In addition to the Go–CFP, significant FRET was measured between YFP-tagged GABAB R1 and R2 subunits and Kir3.2a–CFP or CFP–Kir3.4. The %FRET was similar to that measured between the R1 and R2 subunits of the GABAB receptor, which are known to heterodimerize. Discovering that Kir3 associates closely with GABAB receptors was unexpected. Initially, we hypothesized that G proteins are docked on Kir3 channels in the absence of receptor stimulation (Clancy et al. 2005) – the GABAB receptor would be predicted to be near the G G protein but probably too far from Kir3 to generate a FRET signal. Our current FRET data, however, suggest that Kir3 channels associate closely with GABAB receptors in the absence of receptor activation. Recent biochemical data support this model. Dopamine D2 receptors can coprecipitate with Kir3 channels (Lavine et al. 2002). Furthermore, using bioluminescence resonance energy transfer measurements to detect associations, 2-adrenergic receptors were found to be near both Kir3 channels and another effector, adenylyl cyclase (Zamah et al. 2002). Together, these studies suggest that precoupling may involve the GPCR, G protein, RGS protein and channel. Additional biochemical studies with natively expressed proteins will help confirm these associations between GABAB receptors and Kir3 channels. We speculate that a preformed signalling complex may not apply to all G protein signalling pathways. For example, receptor activation of Gs causes translocation of the G subunit from the plasma membrane to the cytoplasm (Thiyagarajan et al. 2002). The lipid composition and formation of subcellular compartments may also be important for determining the structure and stability of signalling complexes. GABAB receptors, Kir3 channels and Gi G proteins associate with lipid rafts (Becher et al. 2001; Oh & Schnitzer, 2001; Koyrakh et al. 2005). The formation of specific receptor–channel complexes could be an important requirement for signalling in neurons. For example, GABAB receptors couple efficiently to Kir3 channels in dendrites of hippocampal neurons (Lüscher et al. 1997). Consistent with the view of a Kir3 signalling complex, immunohistochemical studies have demonstrated that GABAB receptors and Kir3.2 channels are situated physically close in these neurons (Kulik et al. 2006) and FRET was detected between Kir3 channels and G protein heterotrimer (Riven et al. 2006).
The FRET measured between GABAB receptors and Kir3 channels did not change significantly upon exposure to agonist. In our experiments, the cells are continuously bathed in baclofen raising the possibility that the receptors have partially or completely desensitized. Although GABAB receptors do not undergo endocytosis during chronic stimulation, they can exhibit desensitization through a G protein-dependent mechanism (Mutneja et al. 2005). Thus, receptors may remain associated (< 100 Å) with Kir3 channels during receptor activation and desensitization. Consistent with this, Lavine et al. (2002) reported no change in FRET between 2-adrenergic receptors and Kir3 channels upon agonist stimulation and could coprecipitate D2 receptors with Kir3 channels under basal and activated conditions. Together, these data suggest that these receptor–effector interactions persist during signalling; however, more studies are needed to examine what fraction of receptors stably associate with Kir3 channels in complexes.
Finally, we observed significant FRET between the GABAB receptor and RGS4 protein. This suggests that RGS protein, acting as a GAP for G, is located near to the GABAB receptor. Consistent with this, Georgoussi et al. (2006) demonstrated biochemically that RGS4 can interact directly with both the 
- and µ-opioid receptors. It will be interesting to determine whether RGS4 binds directly to the GABAB receptor or associates with the receptor via the G protein heterotrimer. Benians et al. (2005) did not detect FRET between RGS8 and D2 dopamine receptors, suggesting the interaction could be dependent on the type of RGS and/or receptor subtype. If there is a difference in association of different RGS isoforms with the GABAB receptor, then this may correlate with their relative GAP activity in signalling pathways involving different receptors (Benians et al. 2005).
In summary, we have detected FRET between several proteins within the Kir3 signalling complex. These data argue for a close association (within 100 Å) between GABAB receptors, Go G proteins, RGS4 proteins and Kir3 channels. Although we were unable to detect FRET between G and channel, the proximity of receptor and G protein, and receptor and channel, favour a close association between G and Kir3 channels. The close association of these proteins is probably important for the rapid and specific activation of Kir3 channels in neuronal, endocrine and cardiac cells.
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