Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization

Abstract

Much in vivo and in vitro evidence has shown that the α subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Gα_o on its oligomerization was explored extensively. Gα_o protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Gα_o) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Gα_o existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPγS stimulation. Palmitoylated Gα_o, on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Gα_o was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Gα_o protein. Autoradiography indicated that [¹⁴C]-palmitoylated Gα_o would in no case disaggregate into monomers after treatment with GTPγS. [³⁵S]-GTPγS binding activity assay showed that palmitoylated Gα_o was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Gα_o. Fluorescent quenching studies using BODIPY FL-GTPγS as a probe showed that the conformation of GTP-binding domain of Gα_o tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Gα_o by influencing the oligomerization state of Gα_o and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.

• Gαo
• palmitoylation
• oligomerization
• disaggregation
• GTP-binding activity
• conformation

Introduction

Heterotrimeric G proteins consisting of α, β and γ subunits on the cell membrane perform a pivotal role in signal transmembrane transduction [1], [2]. Stimulation of G proteins with activated receptors result in regulation of various enzymes and ion channels [3]. The structure alteration corresponding to the function state of G proteins [4–6] are described in two valuable G protein-coupled signal transduction models. Gilman's ‘subunit dissociation’ model [1] is based on the studies of purified G-coupled signal components reconstituted onto lipid vesicles and shows that activated heterotrimeric G protein could dissociate into α and βγ subunits that could transduce signals separately to the downstream effectors. Since G proteins cannot diffuse freely on the membrane, this model does not explain satisfactorily how an activated receptor could activate many G proteins and the signals are propagated rapidly in a reversible fashion. Rodbell and colleagues carried out a series of studies using n-octyl glucoside or digitonin to extract Gs, Gi, Go and Gq from rat brain synaptoneurosomes [6], [7] or from rat liver membrane [3] and found that the G proteins on cell membrane displayed multimeric structures larger than heterotrimeric G proteins and disaggregated by ligand-induced activation. These results suggest that heterotrimeric G proteins on the cell membrane could link together as multimers or oligomers, and the activated G-protein-coupled receptor (GPCR) could trigger the disaggregation of them into ‘monomeric’ G protein serving as messengers (coupling agents) with the effectors. This is the ‘disaggregation-coupling’ model [7]. Studies with purified Gα_o protein further showed that Gα_o itself could form oligomeric structures and disaggregate into monomeric Gα_o under the stimulation by GTPγS [8]. Thus, it seemed necessary to explore further the oligomerization mechanism of Gα subunit and its relative modulatory factors in this process.

Palmitoylation of the Gα_o subunit is a readily reversible covalent modification that usually occurs at the 3rd cystine residue from N-terminus of the protein. Palmitoylation-depalmitoylation cycle is reported to be prompted with the activation of G proteins [9], [10]. Therefore, palmitoylation is speculated to be an important means of regulation for G protein-coupled signal transduction. Correlative studies have shown that palmitoylation modification could enhance the membrane binding of Gα [11], [12] or accelerate the location of G protein on specialized membrane domains [13], [14], while there is still contradictive evidence that the palmitoylation-disabled mutant Gα_z can still properly locate in the membrane through its myristoylation modification and the assistance of Gβ/γ subunits [15–17]. On the other hand, palmitoylation shows the effects on the interaction of Gα and other proteins such as Gβ/γ subunits [18] and RGS proteins (regulator of G protein signals) [19]. Recently, Cao's work indicates that the affinity of Gα_o for GTPγS decreased obviously after being palmitoylated [20], suggesting that palmitoylation might participate directly in the G protein activity regulation as well as its membrane binding and location. While the detailed mechanism of how the dynamic change of the palmitoylation state of Gα regulates its function, the activity remains unclear. Since oligomerization has also been noticed to be related with the activation state of Gα in vivo and in vitro as mentioned above, in this paper we have used various approaches to study further the effect of palmitoylation on Gα_o oligomerization and its functional activity, so as to explore the regulatory mechanism of Gα palmitoylation on G protein-coupled signal transduction process.

Materials and methods

Materials

Guanosine 5′-3-O-(thio)triphosphate (GTPγS) was purchased from Roche Molecular Biochemicals (Basel, Switzerland); Phenyl Sepharose High Performance, DEAE Sephacel and Q Sepharose High Performance, Superdex™ 200 HR 10/30 gel filtration column were from Amersham Pharmacia (Piscataway, NJ, USA); palmitoyl-Coenzyme A and acrylamide were from Sigma (St. Louis, MO, USA); [¹⁴C]-palmitoyl-Coenzyme A and [³⁵S]-GTPγS were from Perkin-Elmer (Waltham, MA, USA); BODIPY FL-GTPγS was from Molecular Probes (Eugene, OR, USA); Rabbit anti-mouse polyclonal antibody specific to Gα_o and goat anti-rabbit IgG conjugated with alkaline phosphatase were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the expression plasmid pQE60-Gα_o was a gift from Prof. Susanne Mumby (University of Texas, Southwestern Medical Center, USA); the N-myristoyltransferase expression plasmid pBB131-NMT was a gift from Prof. Gordon (University of Washington, USA); Hypocrellin B (HB) was generously provided by Prof. Jiachang Yue (Institute of Biophysics, Chinese Academy of Science, Beijing, PR China).

Preparation of myristoylated Gα_o

The Escherichia coli strain JM109 cotransformed with pQE60-Gα_o and pBB131-NMT was grown in T₇ enriched medium supplemented with 50 µg/ml kanamycin and 50 µg/ml ampicillin. When the OD₆₀₀ reached to ∼0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 60 µM and the cells were then grown at 30°C overnight. The cells were harvested and lysed by freezing-thawing with liquid nitrogen and digested with 0.2 mg/ml lysozyme before the lysate was centrifuged at 30,000 g for 1 h. The supernatant was adjusted to 1.2M (NH₄)₂SO₄ and 25 µM GDP, and then applied to Phenyl Sepharose and Q Sepharose columns for further purification. The highly purified Gα_o fractions were pooled and stored at −70°C. The purity of Gα_o was evaluated by SDS-PAGE stained with Coomassie blue R-250 and the protein concentration was determined using the BCA™ Protein Assay Kit (Pierce, Chicago, IL, USA).

In vitro palmitoylation of Gα_o

Gα_o was palmitoylated in vitro according to Duncan and Gilman [21] with some modifications. The palmitoylation reaction was conducted in buffer A (20 mM Hepes, pH8.0, 1 mM EDTA, 2 mM MgCl₂, 0.02% Lubrol PX) with 20 µM Gα_o and 200 µM palmitoyl-Coenzyme A. The reaction mixture was incubated at 22°C for 1 h to obtain palmitoylated Gα_o. Non-palmitoylated Gα_o was treated likewise without palmitoyl-Coenzyme A. Hence palmitoylated and non-palmitoylated Gα_o were both in the hydrophobic system with 0.02% Lubrol PX. The efficiency of palmitoylation was measured according to Yang's method [22] by using [¹⁴C]-palmitoyl-Coenzyme A instead of palmitoyl-Coenzyme. That is: palmitoylation efficiency=[[¹⁴C]-palmitate]_bound/[Gα_o]_total. [Gα_o]_total in assay system is known, and [[¹⁴C]-palmitoyl]_bound could be determined by Liquid scintillation counter.

The GTPγS-binding activity assay of the purified Gα_o

The [³⁵S]-GTPγS binding assay of Gα_o and palmitoylated Gα_o were performed according to Northup et al. [4]. Liquid scintillation counts were measured on a 1450 MicroBeta Liquid Scintillation & Luminescence Counter (PerkinElmer™ Life Sciences).

Preparation of Gα_o samples in different states

Palmitoylated Gα_o (pGα_o) was prepared as above. As 25 µM GDP (guanosine diphosphate) was added to the purification system of Gα_o, the purified Gα_o was in GDP bound state and designated as Gα_o·GDP (before palmitoylation) and pGα_o·GDP (after palmitoylation). Gα_o·GTPγS and pGα_o·GTPγS were prepared as follows: Gα_o·GDP and pGα_o·GDP were diluted in buffer B containing 500 µM GTPγS, 20 mM Hepes, pH 8.0, 1 mM EDTA, 1 mM DTT, 7.5 mM MgCl_2, 0.02% (w/v) Lubrol PX and incubated at 30°C for 30 min. Likewise, Gα_o·GDP and pGα_o·GDP samples were diluted and incubated in buffer B without GTPγS for 30 min.

3–20% continuous non-denaturing gel electrophoresis and western blot

3–20% continuous non-denaturing gel electrophoresis (NDE) and western blot (WB) were performed according to Protein Methods [23]. Gα_o·GDP, Gα_o·GTPγS, pGα_o·GDP and pGα_o·GTPγS samples (20 µl of 16 µM) were mixed with 5 µl 5× sample buffer (300 mM Tris-HCl, pH 8.0, 50% [v/v] glycerol, 0.05% [w/v] bromophenol blue) and subjected to 3–20% NDE with constant voltage of 200 V for 5 h. Western blot analysis of the above samples using polyclonal antibody specific to Gα_o were performed in succession. In addition, Gα_o·GDP and Gα_o·GTPγS without Lubrol PX or with 0.1% (w/v) Lubrol PX were subjected to the same 3–20% NDE as control. All the gels were strained with Gelcode Blue Stain Reagent (Pierce, Chicago, IL, USA).

Autoradiography of palmitoylated Gα_o

pGα_o·GDP and pGα_o·GTPγS samples (20 µl of 16 µM) were resolved on 3–20% NDE. The gel was dried at 80°C for 2 h on a Model 583 Gel Dryer (BioRad, Hercules, CA, USA) and then subjected to exposure cassette involving phosphorus screen for 48 h. The phosphorus screen was scanned by Typhoon TRIO+ (Amersham Bioscience, Piscataway, NJ, USA) and the result was shown on Adobe Photoshop CS2.

Gel filtration chromatography

Gα_o·GDP, Gα_o·GTPγS, pGα_o·GDP and pGα_o·GTPγS samples were dialyzed against buffer C (20 mM Hepes, pH 8.0, 1 mM EDTA, 1 mM DTT, 0.02% [w/v] Lubrol PX) at 4°C separately. 100 µl of each Gα_o sample (10 µM) was applied to a Superdex™ 200 HR 10/30 gel filtration column using an AKTA purifier system (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The elution monitored at 280 nm was conducted with buffer C at 0.5 ml/min. The molecular mass standards were applied to the same column in parallel experiments.

Analytical ultracentrifugation

For analytical ultracentrifugation (AU) detection, Gα_o.GDP, Gα_o·GTPγS and pGα_o.GDP were dialyzed against the buffer C and further purified on a Superdex™ 200 HR 10/30 gel filtration column eluted with the buffer C. Sedimentation velocity experiments were performed at 50,000 rpm/min on a ProteomelLab™ XL-1 centrifuge with a An-60 Ti rotor (Beckman, Fullerton, CA, USA) at a protein concentration of 0.75 mg/ml. Scans were taken at 275 nm with a radial step size of 0.003 cm. Differential sedimentation coefficients, c(s), were calculated by least-squares boundary modeling of the sedimentation velocity data using SEDFIT software [24] from http://www.analyticalultracentrifugation.com/download.htm.

Atomic force microscopy

A drop of Gα_o solution (10 µl, 2.5 µg/ml) in 50 mM sodium acetate buffer (pH 4.6) was deposited onto a piece of freshly cleaved mica, incubated at room temperature for 5 min, and then rinsed with the same sodium acetate buffer to remove loosely bound proteins. Imaging was performed using a NanoScope IIIa Multi-mode AFM (Veeco Instruments, USA) with an E-scanner (1 µm×1 µm) operating in tapping mode. The cantilevers (100 µm, NP-S, Veeco Instruments) were oscillated at a frequency of ∼ 8 kHz, and the set point was regulated to have the minimum possible force exerted on the sample while maintaining the sharpness of the image. Height images were collected with a scan rate of 1 Hz. All samples were scanned with the same tip to unify the influence of ‘tip broadening’ [25] on the nanometer size particles.

Fluorescent quenching assay

The fluorescent assay using BODIPY FL-GTPγS as a probe was conducted according to McEwen's method with a few modifications [26]. Non-palmitoylated or palmitoylated Gα_o was diluted to 500 nM in buffer D (50 mM Hepes, pH 8.0, 1 mM EDTA, 20 mM MgCl₂, 0.02% [w/v] Lubrol PX) and BODIPY FL-GTPγS was added to a final concentration of 50 nM. After stabilization of the fluorescence change resulting from the association of Gα_o and BODIPY FL-GTPγS, the fluorescence quencher was added in different concentrations and the fluorescence changes were monitored with a Hitachi 4500 spectrofluorometer (Tokyo, Japan) at 22°C. The classical Stern-Volmer equation was used to analyze the quenching data [27]: F₀/F = 1 + Ksv×Q.

Results

Expression and purification of Gα_o with high activity

Myristoylated Gα_o expressed in E. coli strain JM109 could be separated from non-myristoylated Gα_o for its stronger affinity to Phenyl Sepharose and was applied to Q-Sepharose column for further purification. Purity of the myristoylated Gα_o was above 90% as examined by SDS-PAGE (Figure 1a), while the immune active character of the object protein band was determined by western blot analysis (Figure 1b). The [³⁵S]-GTPγS binding assay indicated that the binding activity of the purified Gα_o was about 21.8 nmol/mg (Table I; the theoretical binding activity is 24.4 nmol/mg), which would mean that more than 85% of the purified Gα_o remained high binding activity.

Figure 1.  SDS-PAGE (a) and Western blot (b) analysis of the recombinant Gα_o protein. 2 µg purified recombinant Gα_o was subjected to 12% SDS-PAGE and 20 ng purified recombinant Gα_o was applied in Western blot. Mobility of molecular size standards were indicated at left.

Table I.  [³⁵S]-GTPγS binding activity of Gα_o with or without palmitate.

	[^35S]-GTPγS binding quantity (nmol/ml)	Protein concentration (mg/ml)	GTPγS binding activity (nmol/mg)	% of active protein in all
Non-pal Gαo	16.35±0.62×10^−2	0.75×10^-2	21.8	89.3
Pal-Gαo	5.85±0.39×10^−2	0.75×10^-2	7.8	31.9

* Non-palmitoylated Gα_o and palmitoylated Gα_o were both diluted into 0.75×10⁻² mg/ml in the assay systems, [³⁵S]-GTPγS binding quantity were determined by Liquid scintillation counts. Data represent means±SEM of three independent experiments.

In vitro palmitoylation of Gα_o

Purified myristoylated Gα_o was further palmitoylated in vitro. The palmitoylation site has been shown by our laboratory to be specially located at the N-terminus 3rd cystine residue of Gα_o[22]. [¹⁴C]-Palmitoyl-CoA binding assay showed that, when the [Gα_o]_total in system was set as 200 µM, [[¹⁴C]-palmitate]_bound was 165±12 µM (n=3), namely the Gα_o palmitoylation efficiency was above 80%. The [³⁵S]-GTPγS binding assay showed that the binding activity of the palmitoylated Gα_o was about 7.8 nmol/mg (Table I), which meant that only 30% of the binding activity of Gα_o remained after palmitoylation.

Non-denaturing gel electrophoresis and western blot analysis

Non-denaturing gel electrophoresis (NDE) is one of the most reliable methods to detect oligomerization or disaggregation of proteins and protein subunits. Lubrol PX was used in the assay system to mimic hydrophobic environment of membrane. As Figure 2 shows, in non-Lubrol PX system (L1 and L2) and in 0.1% (L3 and L4) Lubrol PX system, the NDE patterns of Gα_o·GDP (L2 and L4) or Gα_o·GTPγS (L1 and L3) were similar respectively, which indicated that Lubrol PX used in the system would not interfere with the oligomerization assays. Thus, 0.02% Lubrol PX was added in all the following assays of this study.

Figure 2.  The effect of detergent Lubrol PX on oligomerization and disaggregation of Gα_o protein examined by NDE. M, protein molecular size standards; L1, L2, without Lubrol PX; L3, L4, with 0.1% Lubrol PX. ‘ + ’, Gα_o activated by GTPγS before electrophoresis; ‘-’, Gα_o not activated by GTPγS before electrophoresis.

The action of palmitoylation on oligomerization and disaggregation of Gα_o was then performed on the same NDE as above. It could be found that Gα_o·GDP in the electrophoresis gel exhibited several bands above 66 kD (Figure 3a, L1). As the Gα_o monomer is ∼39 kD, it is readily determined that Gα_o·GDP exist as dimers, trimers, tetramers and pentamers according to molecular mass standards. Indeed, there was still a very dim band below 66 kD which could be Gα_o·GDP monomers. Preactivation with GTPγS induced obvious structure changes of Gα_o as the oligomers entirely disaggregated into monomers (Figure 3a, L2). The interest in palmitoylated Gα_o, was that pGα_o·GDP existed entirely as oligomers including dimers, trimers, tetramers and pentamers without any detectable monomer (Figure 3a, L3); being strikingly different from, pGα_o·GTPγS pretreated with GTPγS existed mainly as oligomers (Figure 3a, L4; ∼80%, Table II) and a small amount of monomers (∼20%, Table II). Subsequent immunoblot analysis (Figure 3b) further validated the oligomerization of Gα_o (Figure 3b, L1) and pGα_o (Figure 3b, L3), and the complete disaggregation of Gα_o (Figure 3b, L2) but not of pGα_o (Figure 3b, L4) induced by GTPγS. These results indicated that the oligomers of palmitoylated Gα_o might differ from that of non-palmitoylated Gα_o as they were less prone to disaggregate by the addition of GTPγS. Table II shows the percentage of Gα_o oligomers and monomers obtained from density scanning of the stained electrophoresis gels from several parallel experiments.

Figure 3.  Oligomerization of non-palmitoylated and palmitoylated Gα_o evaluated by NDE (a) and WB (b). Electrophoresis of the Gα_o samples were conducted with 0.02% Lubrol PX in the system. ‘ + ’, Gα_o, palmitoylated or activated by GTPγS before the electrophoresis. ‘-’, Gα_o, non-palmitoylated or not activated by GTPγS before the electrophoresis, respectively.

Table II.  Proportion of Gα_o in monomer and oligomer states determined by scans of stained non-denaturing polyacrylamide gels.

Protein	Monomer%	Oligomer%
Gαo·GDP	10±3.8	90±4.6
Gαo·GTPγS	100	0
pGαo·GDP	0	100
pGαo·GTPγS	15±3.6	80±4.1

* Data represent means±SEM of three independent experiments

Oligomers of palmitoylated Gα_o detected by autoradiography

In order to further confirm the oligomerization state of palmitoylated Gα_o, [¹⁴C]-palmitoylated Gα_o was first subjected to NDE gel and then to autoradiography developing. Figure 4 illustrates that, for pGα_o·GDP, the autoradiographic bands (Figure 4b, L3) coincided with the stained bands in the NDE gel (Figure 4a, L2); whereas for pGα_o·GTPγS, the autoradiographic bands of Gα_o involved only oligomers (Figure 4b, L4) but no monomers, as shown in the stained gel (Figure 4a, L1). These results demonstrated that oligomers of the palmitoylated Gα_o could in no case disaggregate into monomers after treatment with GTPγS, the small amount of disaggregated monomer pGα_o·GTPγS shown in the NDE gel (Figure 4a, L1 or Figure 3a, L4) was, in fact, non-palmitoylated (Figure 4, L4).

Figure 4.  Autoradiography of palmitoylated Gα_o performed after the non-denaturing gel electrophoresis. (a) Stained NDE gel. (b) Autoradiography of the electrophoresis gel of (a).

Oligomerization of Gα_o estimated by gel filtration chromatography

Figure 5 shows the elution profiles of Gα_o·GDP, Gα_o·GTPγS, pGα_o·GDP and pGα_o·GTPγS on gel filtration chromatography (GFC). The eluates were subjected to SDS-PAGE to identify the Gα_o protein peaks (Figure 6). As can be seen, the elution peaks corresponding to L4, L7, L8 and L11 were not protein peaks. The large elution peaks of 15.6 ml (L4, L11) in both Figure 5B (Gα_o·GTPγS) and Figure 5D (pGα_o·GTPγS) could be the GTPγS molecule with a strong absorbance at 280 nm, while the15.3 ml (L7) and 16.0 ml (L8) elution peaks in Figure 5C (pGα_o·GDP) could be palmitate that did not bind with Gα_o.

Figure 5.  Gel filtration chromatography of Gα_o in different states on a Superdex™ 200 HR 10/30 column. The elution positions of the standard proteins were indicated as: (a) trimer of bovine serum albumin, BSA (210 kD, 8.9 ml); (b) dimer of BSA (140 kD, 9.8 ml); (c) monomer of BSA (68 kD, 11.2 ml).

Figure 6.  SDS-PAGE of the fractions eluted from the gel filtration chromatography column. Lanes (L): L0, Gα_o·GDP without gel filtration as a control; L1 and L2 corresponding to 11.1 ml and 11.91 ml elution peaks in Figure 5A, respectively; L3 and L4 corresponding to 11.9 ml and 15.6 ml elution peaks in Figure 5B; L5, L6, L7 and L8 corresponding to 10.5 ml, 11.9 ml, 15.3 ml and 16.0 ml elution peaks in Figure 5C; L9, L10 and L11 corresponding to 10.5 ml, 11.06 ml and 15.6 ml peaks in Figure 5D.

Figure 5 shows that Gα_o·GDP displayed a tiny Gα_o peak at 11.1 ml and a main Gα_o peak at 11.91 ml (Figure 5A); Gα_o·GTPγS displayed a single Gα_o peak at 11.9 ml (Figure 5B). From elution positions of the standard proteins, it can be deduced that the eluate of the 11.91 ml (or 11.9 ml) peak was monomeric Gα_o, the eluate of the 11.1 ml peak in Figure 5A was dimeric Gα_o (78 kD). These results are basically consistent with Figure 3, namely Gα_o·GDP in itself can form oligomers and disaggregate entirely to monomers in the presence of GTPγS. It should be mentioned that there are two differences for Gα_o·GDP between the GFC and NDE: (i) The oligomers observed in GFC consist only of dimers without any trimer, tetramer and pentamer that were evidently displayed in NDE; and (ii) the amount of oligomers was lower in GFC (Figure 5A 11.1 ml, ≤20%) than in NDE (Table II, ∼90%). Detailed analysis of the possible reasons will be addressed in the Discussion.

After palmitoylation, pGα_o·GDP display a distinctly different elution profile (Figure 5C) compared with Gα_o·GDP (Figure 5A). There was a larger Gα_o protein peak at 10.5 ml (including 70–80% pGα_o) and a smaller Gα_o protein peak at 11.9 ml (including 20–30% pGα_o). From the standard protein elution positions, the former should be dimeric and/or trimeric Gα_o, while the latter should be monomeric Gα_o. On the other hand, pGα_o·GTPγS eluted as 10.5 ml and 11.06 ml Gα_o protein peaks which should correspond to trimeric and dimeric Gα_o, respectively (Figure 5D). GFC analysis clearly showed that, after palmitoylation, the oligomers amount of Gα_o increased considerably and the oligomers remained more stable even under the GTPγS stimulation.

Oligomerization of Gα_o identified by potent biophysical methods

In some cases, restrictions of apparatus or methods would confuse the identification of protein oligomers based on molecular mass determination [28], [29], so the oligomerization of Gα_o protein was further confirmed by two additional biophysical approaches.

Analytical ultracentrifugation analysis

Sedimentation velocity experiments were performed by using analytical ultracentrifugation (AU) approach to determine the oligomerization of Gα_o in different states. Figure 7 shows the c(s) distribution of Gα_o·GDP, Gα_o·GTPγS and pGα_o·GDP. The c(M) distribution transformed from the c(s) distribution indicated molecular mass of every species displayed in Table III. As can be seen, after palmitoylation, the quantity of Gα_o oligomers changes from 18% to 90% and the species of oligomers changes from single dimers to a mixture of dimers and trimers. These results confirmed that the ability of Gα_o to form oligomers, either in quantity or in oligomeric degree, was remarkably improved after Gα_o was palmitoylated.

Figure 7.  Sedimentation coefficient distributions derived from sedimentation velocity profiles. (A) Gα_o·GDP. (B) Gα_o·GTPγS. (C) pGα_o·GDP. Differential sedimentation coefficients, c(s), in the lower panel of (A), (B) and (C) were calculated from the sedimentation velocity data using SEDFIT software. After loading the complete data sets into SEDFIT, the bottom, meniscus, and the outer and inner fitting limits should be selected on the data plot (not shown). The residuals plot in the top panel of (A), (B) and (C) shows the residuals distribution between the fitting limits, and the absence of very large residuals at the extreme radius values indicated that the fitting limits selection was appropriate.

Table III.  Parameters of the Gα_o in different states determined by analytical ultracentrifugation.

		Sedimentation velocity
Protein samples	Mcalculated (kD)	S^a (S)	S20^w (S)	Msv^a (kD)	Proportion (% (%)*	Association state
Gαo·GDP	39.443	2.09	2.38	46.3	68	Monomer
		3.68	3.89	91	18	Dimer
Gαo·GTPγS	39.456	2.12	2.33	40.9	72	Monomer
		3.51	3.86	83	13	Dimer
pGαo·GDP	39.457	3.62		82.5	70	Dimer
		4.20		125	20	Trimer

*Proportion is obtained by SEDFIT software. The data set shown is a representative result from three parallel experiments.

Atomic Force Microscopy imaging

Atomic Force Microscopy (AFM) is a powerful tool to study the shape and size of protein molecules. It is generally assumed that sizes of molecules may reflect interaction between the molecules such as oligomerization. To get more evidence of Gα_o oligomerization influenced by palmitoylation and GTPγS activation from various biophysical approaches, the AFM technique, as a supplement of morphological examination, was also used in the present studies. Figure 8A shows the scan patterns of Gα_o in different states on mica substrate appearing basically as globular particles with distinct sizes. Figure 8B indicated the diameter distribution of Gα_o particles evaluated by the method of section analysis using Nanoscope version 6.12. As can be seen from the scan pattern and diameter distribution, the size of pGα_o·GDP was obviously bigger than that of Gα_o·GDP without palmitate; also similar changes could be found on pGα_o·GTPγS compared with Gα_o·GTPγS. It should be mentioned that, due to the sensitivity limitation mainly from the ‘Tip broaden effect’ [25] of the AFM used in this study, the types of oligomers and their disassociation could not be exactly detected at the moment.

Figure 8.  AFM analysis of Gα_o samples in different states. (A) The scan pattern of Gα_o as visualized by AFM in tapping mode. Image size 1×1 µm, z-range 0–5 nm. (B) Diameter distributions of Gα_o particles in pattern A. The particles maximum height was determined by vert distance program, particles diameter were determined by particle analysis program (Nanoscope 111a 6.12 rl software offered by Veeco Instruments). For any given particle, the value of particle maximum height was positive correlative with its diameter, so the diameter was further taken as a measurement for particle size in this study.

Since the AFM assay could provide a stable liquid circumstance for the detected molecules, and there are no components in our analysis system that could induce protein denaturing, the size changes of the molecular granules shown on AFM pattern should reflect the oligomerization of Gα_o, but not any protein aggregation.

Fluorescence quenching of BODIPY FL-GTPγS bound to Gα_o

BODIPY FL-GTPγS contains a BODIPY fluorophore and a GTP analog group, GTPγS. Figure 9 shows that the fluorescence increased to a maximum 3- to 4-fold higher than the baseline when BODIPY FL-GTPγS was incubated with either non-palmitoylated (Figure 9A) or palmitoylated (Figure 9B) Gα_o. The increase in fluorescence resulted from the specific association of GTPγS with the GTP-binding domain of Gα_o which eliminate the quenching of the guanine base on BODIPY [26]. In a subsequent quenching experiment, the maximum fluorescence remained stable for about 10 min.

Figure 9.  Fluorescence changes of BODIPY FL-GTPγS incubated with non-palmitoylated and palmitoylated Gα_o. The arrow indicates the time of addition of non-palmitoylated Gα_o (A) or palmitoylated Gα_o (B) to the same final concentration of 500 nM. The fluorescence was monitored on a Hitachi 4500 spectrofluorometer at 22°C with λex at 470 nm and λem at 510 nm. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 10 shows that the fluorescence of BODIPY FL-GTPγS bound to non-palmitoylated Gα_o was significantly quenched by acrylamide (Figure 10A), while the fluorescence for the palmitoylated Gα_o remained unchanged during the corresponding experiment (Figure 10B). When using the hydrophobic quencher, Hypocrellin B (HB), a similar difference in the fluorescence quenching was observed (Figure 11). Although the fluorescence of BODIPY FL-GTPγS was quenched in the presence of either non-palmitoylated (Figure 11A) or palmitoylated (Figure 11B) Gα_o, analysis of HB quenching data indicate that the K_SV value of non-palmitoylated Gα_o (K_SV=41.92±2.71) was higher than that of palmitoylated Gα_o (K_SV=32.38±1.25).

Figure 10.  Fluorescence quenching of Gα_o bound BODIPY FL-GTPγS probe with acrylamide. The assay system contained 50 nM BODIPY FL-GTPγS and 500 nM either non-palmitoylated Gα_o(▪) or palmitoylated Gα_o(•). The acrylamide quencher (0∼0.11 M in 0.01 M increments) was added after the fluorescence level of Gα_o bound BODIPY FL-GTPγS stabilized. Fluorescence change was monitored on a Hitachi 4500 Spectrofluorometer. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 11.  Fluorescence quenching of Gα_o bound BODIPY-FL GTPγS probe with Hypocrellin B (HB). The assay system was the same as in Figure 10, and the concentration of HB quencher varied from 0–0.058 mM in 0.0105 mM increments (or 0.00525 mM, the first increment only). This Figure is reproduced in colour in Molecular Membrane Biology online.

The quenching data here reflect the conformation change of GTP binding domain that the BODIPY FL-GTPγS fluorescence probe bound with. In this study, both acrylamide and HB are nonionic quenchers, so the electric charge around the fluorophore could not influence the action of quencher. Furthermore, from the similar difference in the fluorescence quenching by hydrophobic and hydrophilic quenchers, it could deduced that the main factor influencing the quenching capacity might not be the change on hydrophobic property, but the increase of space block around the fluorophore after Gα_o was palmitoylated. This space block limited the access of both hydrophobic and hydrophilic quenchers to GTP binding domain, resulting in the decrease of quenching capacity by acrylamide (Figure 10) and HB (Figure 11).

Discussion

Nearly all the α subunits of the four G protein families (G_s, G_i, G_q, and G₁₂) could be palmitoylated at their cysteine residues near the N-terminus [30–33]. Depalmitoylation of Gα subunit occurs extensively in the G protein-coupled signal transmembrane transduction process triggered by activated receptors [9–11]. But, the changes of G protein organization structure in the palmitoylation-depalmitoylation dynamic cycle and involved regulatory factors are largely unknown to date. Several lines of evidence in this article demonstrated the Gα_o organization structure changes accompanied with its palmitoylation. The experimental results of NDE showed that oligomers of Gα_o·GDP disaggregated into Gα_o monomers when treated with GTPγS, but oligomers of palmitoylated Gα_o could hardly be disaggregated by the stimulation of GTPγS (Figure 3 and Table II). Further investigations by GFC (Figure 5C, 5D), AU (Figure 7C and Table III) and AFM (Figure 8) analysis also indicated that palmitoylation may enhance the ‘potential’ or ‘propensity’ of the Gα_o to form oligomers, reflected by the increase of the quantity and/or oligomerization degree of Gα_o, and by the phenomena that pGα_o·GDP oligomers were difficult to disaggregate into monomers by GTPγS stimulation as Gα_o·GDP oligomers were. All these results consistently confirmed that palmitoylation modification may enhance and maintain the oligomerization state of Gα_o. One point should be mentioned: Gα_o·GDP behaved to some extent dissimilarly in NDE (existing mainly as oligomers) compared with other analysis means in this study such as GFC (existing as both monomers and oligomers). The difference might come from different separation conditions of these two methods and the instability of Gα_o·GDP oligomers themselves. As 1 mM DTT was added to all Gα_o samples for maintaining the optimal conformation and activity of Gα_o, the force that induces Gα_o oligomerization should not be the disulfide bond, but non-covalent bond such as hydrogen bond and hydrophobic interactions between the Gα_o proteins. Oligomers linked by the weaker non-covalent bond might be unstable and change in some conditions. In our study, the buffer system and the separation media of NDE and GFC were different (see Materials and methods). It seemed that NDE provided a more advantageous condition to keep the Gα_o oligomers compared with GFC. In other words, Gα_o·GDP without palmitate might exist as ‘loose’ or ‘unstable’ oligomers and it is difficult to retain it in some analysis conditions such as GFC. The ‘instability’ of Gα_o·GDP was caused by the ease at which it was disaggregated by GTPγS. On the contrary, pGα_o·GDP was validated by all the approaches used in this paper to exist permanently as oligomers which could not disaggregate by GTPγS stimulation. Should the difference between GFC and NDE be considered, stability of pGα_o·GDP stronger than Gα_o·GDP could be further confirmed.

Based on the results described above, an interesting problem about the relationship between palmitoylation modification and Gα_o activity as well as its accompanying conformational changes was further explored. The result showed that [³⁵S]-GTPγS binding activity of the Gα_o dramatically decreased after Gα_o was palmitoylated; correspondingly, the palmitoylation modification would promote Gα_o to form more and stable oligomers even under the GTPγS treatment. These revealed an important functional link between the GTPγS binding activity and the oligomerization-disaggregation of Gα_o, namely, palmitoylation might regulate the GDP/GTP exchange of Gα_o by enhancing its oligomerization. Therefore, by understanding the effect of palmitoylation on Gα_o conformation should be necessary to clarify this functional link. As shown in Figures 10 and 11, the quenching on BODIPY fluorophore with either hydrophobic (HB) or hydrophilic (acrylamide) quenchers consistently decreased upon palmitoylation of Gα_o, which implied that the GTP-binding domain of Gα_o became more compact from the space block after Gα_o palmitoylation, and hence was less accessible to the quenching agents. Also the increase of space block around GTP binding domain might limit the access of GTPγS to this area, resulting in the decrease of GTPγS binding activity of Gα_o. So it could be deduced that the enhancement of Gα_o oligomerization is relative to the decrease of GTPγS binding activity of Gα_o, and its structure basis might be the increase of space block around the GTP binding domain after Gα_o palmitoylation.

Because G proteins are peripheral membrane proteins and perform their functions on the membrane, a hydrophobic environment should be important for their natural conformation and function. In this study, we used 0.02% Lubrol PX to mimic hydrophobic membrane circumstance, as reported [20], [34]. From the results of NDE, non-palmitoylated Gα_o displayed similar oligomerization-disaggregation characters in the systems with or without detergent (Figure 2), implying that the presence of detergent might have little influence on Gα_o oligomerization assay and that the performance in vitro could be applied to mimic the hydrophobic membrane environment in its native state. Certainly, there are some other methods to mimic the cellular membrane circumstance by using micelles/liposomes made of soybean lipids or phospholipids. However, we gave up using micelles and phospholipids because of their obvious drawbacks in this study: (i) The binding of Gα_o and micelles is fairly weak without the existence of Gβγ [35], and (ii) the system containing phospholipids might bring complexities to the study of Gα_o palmitoylation and oligomerization. The effects of phospholipids on the structure/conformation and function of membrane proteins have been reported extensively [36–38]. Recently, preliminary results from our group showed that phosphaticic acid (PA) influenced the binding activity and oligomerization of Gα_o (unpublished results) and the relative studies are still in progress. So, the effects of membrane lipids on the oligomerization and disaggregation process might be worthy of further investigation.

This is the first report on the relationship between palmitoylation modification and oligomerization character of Gα_o protein. As previously reported, depalmitoylation when accompanied with the receptor activation, would occur on Gα subunit [9–11]. In this article, we have presented powerful evidence that palmitoylation could enhance and maintain the Gα_o oligomerization, and depress its GTPγS binding activity by inducing a more compact conformation on the GTP-binding domain which influences the GDP/GTP exchange of Gα_o. In other words, depalmitoylation could depress the Gα_o oligomerization, and consequently facilitate the GDP/GTP exchange of Gα_o. These actions would suggest that palmitoylation modification of Gα_o might be involved in the G protein-coupled signal transduction process by influencing Gα subunit oligomerization. Accordingly, some amendments to the ‘disaggregation-coupling’ model (Figure 12A) by Rodbell [7], [39] are proposed, involving potential steps in which palmitoylation might be involved (using the symbol ‘’ in Figure 12B). Further studies on the mechanism of oligomerization and GTP binding activity modulated by palmitoylation with different G proteins should shed light on the general role of palmitoylation in the G protein-coupled signal transduction pathway.

Figure 12.  Proposed amendments to the disaggregation-coupling model. (A) The ‘disaggregation-coupling model’. Reproduced with permission from Reference [7]: Copyright (1993) National Academy of Sciences, USA. (B) The schematic illustration of improved ‘disaggregation-coupling model’ for G protein-coupled signal transduction. (a) In the basal state, palmitoylated Gα proteins exist as tighter constructed oligomers. (b) An activated receptor induces the depalmitoylation of Gα either nonenzymatically or by thioesterase activity, depalmitoylated Gα tends to become looser constructed oligomers. So (c) GDP/GTP exchange occurs and GTP-bound Gα disassociates from the oligomers. (d) Disassociated Gα protein could modulate the activity of relevant effectors until the signals are terminated by on-site hydrolysis of GTP to GDP and Pi. (e) Then GDP-bound Gα, repalmitoylated by palmitoyl transferase, tend to form tighter constructed oligomers and return to basal state. However, in this model, the way by which the Gβγ subunit participates in the oligomerization-disaggregation process is not fully described. Here we take the Gα and Gβγ as a whole through the cycle because the possible disassociation or conformation change between Gα and Gβγ might occur after GDP/GTP exchange accelerated by Gα disaggregation. This Figure is reproduced in colour in Molecular Membrane Biology online.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (30370350). The authors are grateful to Dr Geir Skogerbo for his help in the preparation of the manuscript.