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Original

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

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Pages 58-71 | Received 05 Dec 2006, Published online: 09 Jul 2009

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 Gαo on its oligomerization was explored extensively. Gαo protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Gαo) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Gαo 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 Gαo, on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Gαo 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 Gαo protein. Autoradiography indicated that [14C]-palmitoylated Gαo would in no case disaggregate into monomers after treatment with GTPγS. [35S]-GTPγS binding activity assay showed that palmitoylated Gαo was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Gαo. Fluorescent quenching studies using BODIPY FL-GTPγS as a probe showed that the conformation of GTP-binding domain of Gαo tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Gαo by influencing the oligomerization state of Gαo and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.

Introduction

Heterotrimeric G proteins consisting of α, β and γ subunits on the cell membrane perform a pivotal role in signal transmembrane transduction Citation[1], Citation[2]. Stimulation of G proteins with activated receptors result in regulation of various enzymes and ion channels Citation[3]. The structure alteration corresponding to the function state of G proteins Citation[4–6] are described in two valuable G protein-coupled signal transduction models. Gilman's ‘subunit dissociation’ model Citation[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 Citation[6], Citation[7] or from rat liver membrane Citation[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 Citation[7]. Studies with purified Gαo protein further showed that Gαo itself could form oligomeric structures and disaggregate into monomeric Gαo under the stimulation by GTPγS Citation[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 Gαo 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 Citation[9], Citation[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 Citation[11], Citation[12] or accelerate the location of G protein on specialized membrane domains Citation[13], Citation[14], while there is still contradictive evidence that the palmitoylation-disabled mutant Gαz can still properly locate in the membrane through its myristoylation modification and the assistance of Gβ/γ subunits Citation[15–17]. On the other hand, palmitoylation shows the effects on the interaction of Gα and other proteins such as Gβ/γ subunits Citation[18] and RGS proteins (regulator of G protein signals) Citation[19]. Recently, Cao's work indicates that the affinity of Gαo for GTPγS decreased obviously after being palmitoylated Citation[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 Gαo 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 Gαo and goat anti-rabbit IgG conjugated with alkaline phosphatase were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the expression plasmid pQE60-Gαo 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 Gαo

The Escherichia coli strain JM109 cotransformed with pQE60-Gαo 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 Gαo fractions were pooled and stored at −70°C. The purity of Gαo 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 Gαo

o was palmitoylated in vitro according to Duncan and Gilman Citation[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 Gαo and 200 µM palmitoyl-Coenzyme A. The reaction mixture was incubated at 22°C for 1 h to obtain palmitoylated Gαo. Non-palmitoylated Gαo was treated likewise without palmitoyl-Coenzyme A. Hence palmitoylated and non-palmitoylated Gαo were both in the hydrophobic system with 0.02% Lubrol PX. The efficiency of palmitoylation was measured according to Yang's method Citation[22] by using [14C]-palmitoyl-Coenzyme A instead of palmitoyl-Coenzyme. That is: palmitoylation efficiency=[[14C]-palmitate]bound/[Gαo]total. [Gαo]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 Gαo

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

Preparation of Gαo samples in different states

Palmitoylated Gαo (pGαo) was prepared as above. As 25 µM GDP (guanosine diphosphate) was added to the purification system of Gαo, the purified Gαo was in GDP bound state and designated as Gαo·GDP (before palmitoylation) and pGαo·GDP (after palmitoylation). Gαo·GTPγS and pGαo·GTPγS were prepared as follows: Gαo·GDP and pGαo·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 MgCl2, 0.02% (w/v) Lubrol PX and incubated at 30°C for 30 min. Likewise, Gαo·GDP and pGαo·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 Citation[23]. Gαo·GDP, Gαo·GTPγS, pGαo·GDP and pGαo·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 Gαo were performed in succession. In addition, Gαo·GDP and Gαo·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 Gαo

pGαo·GDP and pGαo·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

o·GDP, Gαo·GTPγS, pGαo·GDP and pGαo·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 Gαo 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, Gαo.GDP, Gαo·GTPγS and pGαo.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 Citation[24] from http://www.analyticalultracentrifugation.com/download.htm.

Atomic force microscopy

A drop of Gαo 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’ Citation[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 Citation[26]. Non-palmitoylated or palmitoylated Gαo 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 Gαo 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 Citation[27]: F0/F = 1 + Ksv×Q.

Results

Expression and purification of Gαo with high activity

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

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

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

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

In vitro palmitoylation of Gαo

Purified myristoylated Gαo 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 GαoCitation[22]. [14C]-Palmitoyl-CoA binding assay showed that, when the [Gαo]total in system was set as 200 µM, [[14C]-palmitate]bound was 165±12 µM (n=3), namely the Gαo palmitoylation efficiency was above 80%. The [35S]-GTPγS binding assay showed that the binding activity of the palmitoylated Gαo was about 7.8 nmol/mg (Table I), which meant that only 30% of the binding activity of Gαo 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 shows, in non-Lubrol PX system (L1 and L2) and in 0.1% (L3 and L4) Lubrol PX system, the NDE patterns of Gαo·GDP (L2 and L4) or Gαo·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 Gαo protein examined by NDE. M, protein molecular size standards; L1, L2, without Lubrol PX; L3, L4, with 0.1% Lubrol PX. ‘ + ’, Gαo activated by GTPγS before electrophoresis; ‘-’, Gαo not activated by GTPγS before electrophoresis.

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

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

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

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

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

Oligomers of palmitoylated Gαo detected by autoradiography

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

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

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

Oligomerization of Gαo estimated by gel filtration chromatography

shows the elution profiles of Gαo·GDP, Gαo·GTPγS, pGαo·GDP and pGαo·GTPγS on gel filtration chromatography (GFC). The eluates were subjected to SDS-PAGE to identify the Gαo protein peaks (). 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 B (Gαo·GTPγS) and D (pGαo·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 C (pGαo·GDP) could be palmitate that did not bind with Gαo.

Figure 5.  Gel filtration chromatography of Gαo 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 5.  Gel filtration chromatography of Gαo 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, Gαo·GDP without gel filtration as a control; L1 and L2 corresponding to 11.1 ml and 11.91 ml elution peaks in A, respectively; L3 and L4 corresponding to 11.9 ml and 15.6 ml elution peaks in B; L5, L6, L7 and L8 corresponding to 10.5 ml, 11.9 ml, 15.3 ml and 16.0 ml elution peaks in C; L9, L10 and L11 corresponding to 10.5 ml, 11.06 ml and 15.6 ml peaks in D.

Figure 6.  SDS-PAGE of the fractions eluted from the gel filtration chromatography column. Lanes (L): L0, Gαo·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.

shows that Gαo·GDP displayed a tiny Gαo peak at 11.1 ml and a main Gαo peak at 11.91 ml (A); Gαo·GTPγS displayed a single Gαo peak at 11.9 ml (B). 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 Gαo, the eluate of the 11.1 ml peak in A was dimeric Gαo (78 kD). These results are basically consistent with , namely Gαo·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 Gαo·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 (A 11.1 ml, ≤20%) than in NDE (, ∼90%). Detailed analysis of the possible reasons will be addressed in the Discussion.

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

Oligomerization of Gαo 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 Citation[28], Citation[29], so the oligomerization of Gαo 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 Gαo in different states. shows the c(s) distribution of Gαo·GDP, Gαo·GTPγS and pGαo·GDP. The c(M) distribution transformed from the c(s) distribution indicated molecular mass of every species displayed in . As can be seen, after palmitoylation, the quantity of Gαo 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 Gαo to form oligomers, either in quantity or in oligomeric degree, was remarkably improved after Gαo was palmitoylated.

Figure 7.  Sedimentation coefficient distributions derived from sedimentation velocity profiles. (A) Gαo·GDP. (B) Gαo·GTPγS. (C) pGαo·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.

Figure 7.  Sedimentation coefficient distributions derived from sedimentation velocity profiles. (A) Gαo·GDP. (B) Gαo·GTPγS. (C) pGαo·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 Gαo in different states determined by analytical ultracentrifugation.

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 Gαo 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. A shows the scan patterns of Gαo in different states on mica substrate appearing basically as globular particles with distinct sizes. B indicated the diameter distribution of Gαo 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 pGαo·GDP was obviously bigger than that of Gαo·GDP without palmitate; also similar changes could be found on pGαo·GTPγS compared with Gαo·GTPγS. It should be mentioned that, due to the sensitivity limitation mainly from the ‘Tip broaden effect’ Citation[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 Gαo samples in different states. (A) The scan pattern of Gαo as visualized by AFM in tapping mode. Image size 1×1 µm, z-range 0–5 nm. (B) Diameter distributions of Gαo 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.

Figure 8.  AFM analysis of Gαo samples in different states. (A) The scan pattern of Gαo as visualized by AFM in tapping mode. Image size 1×1 µm, z-range 0–5 nm. (B) Diameter distributions of Gαo 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 Gαo, but not any protein aggregation.

Fluorescence quenching of BODIPY FL-GTPγS bound to Gαo

BODIPY FL-GTPγS contains a BODIPY fluorophore and a GTP analog group, GTPγS. 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 (A) or palmitoylated (B) Gαo. The increase in fluorescence resulted from the specific association of GTPγS with the GTP-binding domain of Gαo which eliminate the quenching of the guanine base on BODIPY Citation[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 Gαo. The arrow indicates the time of addition of non-palmitoylated Gαo (A) or palmitoylated Gαo (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 9.  Fluorescence changes of BODIPY FL-GTPγS incubated with non-palmitoylated and palmitoylated Gαo. The arrow indicates the time of addition of non-palmitoylated Gαo (A) or palmitoylated Gαo (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.

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

Figure 10.  Fluorescence quenching of Gαo bound BODIPY FL-GTPγS probe with acrylamide. The assay system contained 50 nM BODIPY FL-GTPγS and 500 nM either non-palmitoylated Gαo(▪) or palmitoylated Gαo(•). The acrylamide quencher (0∼0.11 M in 0.01 M increments) was added after the fluorescence level of Gαo 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 10.  Fluorescence quenching of Gαo bound BODIPY FL-GTPγS probe with acrylamide. The assay system contained 50 nM BODIPY FL-GTPγS and 500 nM either non-palmitoylated Gαo(▪) or palmitoylated Gαo(•). The acrylamide quencher (0∼0.11 M in 0.01 M increments) was added after the fluorescence level of Gαo 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 Gαo bound BODIPY-FL GTPγS probe with Hypocrellin B (HB). The assay system was the same as in , 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.

Figure 11.  Fluorescence quenching of Gαo 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 Gαo 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 () and HB ().

Discussion

Nearly all the α subunits of the four G protein families (Gs, Gi, Gq, and G12) could be palmitoylated at their cysteine residues near the N-terminus Citation[30–33]. Depalmitoylation of Gα subunit occurs extensively in the G protein-coupled signal transmembrane transduction process triggered by activated receptors Citation[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 Gαo organization structure changes accompanied with its palmitoylation. The experimental results of NDE showed that oligomers of Gαo·GDP disaggregated into Gαo monomers when treated with GTPγS, but oligomers of palmitoylated Gαo could hardly be disaggregated by the stimulation of GTPγS ( and ). Further investigations by GFC (C, 5D), AU (C and ) and AFM () analysis also indicated that palmitoylation may enhance the ‘potential’ or ‘propensity’ of the Gαo to form oligomers, reflected by the increase of the quantity and/or oligomerization degree of Gαo, and by the phenomena that pGαo·GDP oligomers were difficult to disaggregate into monomers by GTPγS stimulation as Gαo·GDP oligomers were. All these results consistently confirmed that palmitoylation modification may enhance and maintain the oligomerization state of Gαo. One point should be mentioned: Gαo·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 Gαo·GDP oligomers themselves. As 1 mM DTT was added to all Gαo samples for maintaining the optimal conformation and activity of Gαo, the force that induces Gαo oligomerization should not be the disulfide bond, but non-covalent bond such as hydrogen bond and hydrophobic interactions between the Gαo 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 Gαo oligomers compared with GFC. In other words, Gαo·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 Gαo·GDP was caused by the ease at which it was disaggregated by GTPγS. On the contrary, pGαo·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 pGαo·GDP stronger than Gαo·GDP could be further confirmed.

Based on the results described above, an interesting problem about the relationship between palmitoylation modification and Gαo activity as well as its accompanying conformational changes was further explored. The result showed that [35S]-GTPγS binding activity of the Gαo dramatically decreased after Gαo was palmitoylated; correspondingly, the palmitoylation modification would promote Gαo 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 Gαo, namely, palmitoylation might regulate the GDP/GTP exchange of Gαo by enhancing its oligomerization. Therefore, by understanding the effect of palmitoylation on Gαo conformation should be necessary to clarify this functional link. As shown in and , the quenching on BODIPY fluorophore with either hydrophobic (HB) or hydrophilic (acrylamide) quenchers consistently decreased upon palmitoylation of Gαo, which implied that the GTP-binding domain of Gαo became more compact from the space block after Gαo 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 Gαo. So it could be deduced that the enhancement of Gαo oligomerization is relative to the decrease of GTPγS binding activity of Gαo, and its structure basis might be the increase of space block around the GTP binding domain after Gαo 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 Citation[20], Citation[34]. From the results of NDE, non-palmitoylated Gαo displayed similar oligomerization-disaggregation characters in the systems with or without detergent (), implying that the presence of detergent might have little influence on Gαo 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 Gαo and micelles is fairly weak without the existence of Gβγ Citation[35], and (ii) the system containing phospholipids might bring complexities to the study of Gαo palmitoylation and oligomerization. The effects of phospholipids on the structure/conformation and function of membrane proteins have been reported extensively Citation[36–38]. Recently, preliminary results from our group showed that phosphaticic acid (PA) influenced the binding activity and oligomerization of Gαo (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 Gαo protein. As previously reported, depalmitoylation when accompanied with the receptor activation, would occur on Gα subunit Citation[9–11]. In this article, we have presented powerful evidence that palmitoylation could enhance and maintain the Gαo 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 Gαo. In other words, depalmitoylation could depress the Gαo oligomerization, and consequently facilitate the GDP/GTP exchange of Gαo. These actions would suggest that palmitoylation modification of Gαo might be involved in the G protein-coupled signal transduction process by influencing Gα subunit oligomerization. Accordingly, some amendments to the ‘disaggregation-coupling’ model (A) by Rodbell Citation[7], Citation[39] are proposed, involving potential steps in which palmitoylation might be involved (using the symbol ‘’ in B). 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 Citation[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.

Figure 12.  Proposed amendments to the disaggregation-coupling model. (A) The ‘disaggregation-coupling model’. Reproduced with permission from Reference Citation[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.

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