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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

Dynamics of apelin receptor/G protein coupling in living cells Bo Baia, Yunlu Jianga, Xin Caia, Jing Chena,b,n a

Neurobiology Institute, Jining Medical University, Jining 272067, PR China Division of Metabolic and Vascular Health, Warwick Medical School, University of Warwick, Coventry, UK

b

article information

abstract

Article Chronology:

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we

Received 20 April 2014

demonstrated that apelin-13 stimulation can lead to the activation of Gαi2 or Gαi3 through

Received in revised form

undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ.

29 July 2014

Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical

Accepted 24 August 2014

dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi1 subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demon-

Keywords: G protein-coupled receptor Apelin receptor

strated that stimulation of APJ with apelin-13 causes activation of Gαi2, Gαi3, Gαo, Gαq; among which Gαi2, Gαi3 were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.

G protein subunit

& 2014 Published by Elsevier Inc.

Resonance energy transfer

Introduction Apelin receptor (putative receptor protein related to the angiotensin receptor AT1, APJ) belongs to the G-protein-coupled receptors (GPCRs) [1,2], whose endogenous ligand apelins are emerging as a key hormone in cardiovascular homoeostasis [3,4]. Amongst all apelins, apelin-13 binds with high affinity to and associates with APJ more efficiently than apelin-36 and rapidly dissociates from APJ in Chinese hamster ovary (CHO) cells engineered to express the cloned human APJ [5]. Apelins mediate a variety of physiological processes including neuroprotection, pain, fluid homoeostasis, endocrine and metabolic functions. One of the key emerging features of the apelin-APJ system is its interaction with the renin–angiotensin system [3,6]. New research

has found that APJ has a dual role in cardiac hypertrophy through different signalling pathways [7]. Apelin activates APJ signals via the G protein pathway and elicits a cardiac protective response, whereas sustained overload activates APJ to induce cardiac hypertrophy via a G protein-independent fashion. Hence, the apelin-APJ system is expected to be a therapeutic target for treatment of heart failure, hypertension, and obesity-related diseases. In the last decade, there has been a substantial reevaluation of the classic assumption that GPCRs undergo conformational alterations after agonist binding and initiate a GDP/GTP exchange at the Gα subunit in order to activate Gα and Gβγ, which interact with downstream effectors, such as phosphoinositide 3-kinases (PI3K), adenylyl cyclase (AC), phospholipases (PLC) and ion channels

Abbreviations: GPCR, G protein-coupled receptor; APJ, apelin receptor; HEK, human embryonic kidney; BRET, bioluminescence resonance energy transfer; FRET, Förster resonance energy transfer; Rluc, Renilla reniformis luciferase; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; ELISA, Enzyme-Linked Immunosorbent Assay n

Corresponding author. E-mail address: [email protected] (J. Chen).

http://dx.doi.org/10.1016/j.yexcr.2014.08.035 0014-4827/& 2014 Published by Elsevier Inc.

Please cite this article as: B. Bai, et al., Dynamics of apelin receptor/G protein coupling in living cells, Exp Cell Res (2014), http://dx.doi. org/10.1016/j.yexcr.2014.08.035

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[8,9]. For example, using Förster resonance energy transfer (FRET), Gαi subunits were discovered to undergo rearrangement instead of dissociation during adrenergic receptor activation by noradrenaline, which illustrates the complexity of G-protein signalling [10]. Understanding the profile of G-proteins coupled to APJ could provide more detailed information about the molecular link between activation of APJ and its biological effects. Previous research has discovered that forskolin-stimulated cAMP production is suppressed by apelin-13, indicating APJ was hypothesized to couple to Gαi/o [11]. Moreover, the activation of ERK1/2 by apelin is mediated via PKC in HEK293 cells expressing mouse APJ, indicative of coupling to either Gαo or Gαq [12]. Here, we use BRET and FRET to analyze the dynamics of G-protein coupling to human APJ in real time, which not only confirms the binding of Gαi2, Gαo and Gαq, but also demonstrates the activation of Gαi3 after APJ stimulated with apelin-13. We also found that during APJ activation, Gαo and Gαq subunits will dissociate with Gβ1γ2 via the classic model. Interestingly, using both BRET and FRET we found Gαi2, Gαi3 and Gβ1γ2 subunits undergo a re-arrangement without subunit dissociation upon apelin-13 stimulation, which provide new evidence to support this novel theory about this specific activation pattern of Gαi. Taken together, our findings provide evidence to highlight the diversity of G-protein activation in the apelin-APJ system, which could lead towards a better understanding of the function mediated by the apelin-APJ system in cardiovascular control and metabolic homoeostasis.

Materials and Methods Cells and reagents HEK 293T cells were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). All restriction enzymes were from NEB (New England BioLabs Inc, Ipswich, MA, USA). Human apelin13 (EC50 ¼0.37 nM [13]) and Nociceptin/Orphanin FQ (Human) (EC50 ¼ 12 nM [14]) were obtained from Phoenix Pharmaceuticals (Phoenix Pharmaceuticals, Europe GMBH, and Germany).

Cell culture and transfection HEK293T cells were cultured in MEM (Hyclone, China) with 10% FBS in a humidified 37 1C incubator with 5% CO2. For transient expression, cells were transfected with Lipofectamine 2000 (Invitrogen, Paisley, UK) according to the manufacturer's protocol.

Plasmid construction Plasmids expressing Gαi1-YFP, Gαi2-YFP, Gαi3-YFP, and Gαo-YFP were kindly provided by Professor Moritz Bünemann, University of Marburg, Marburg, Germany, which contains cDNA encoding the enhanced YFP F46L [10] inserted between position 91 and 92 of PTX-insensitive C351I mutants of Gαi subunits and Gαo. The inserted coding region of YFP all bears the glycine rich-linker sequences coding for Glu-Phe-Met-Val in the 50 end and Leu-TyrSer-Ser in the 30 end. pCFP-Gγ2 was kindly provided by S. Ikeda (Guthrie Research Institute, Sayre, PA), which express CFP attached to C-terminus of

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Gγ2-subunit [15]. For construction of pGγ2-CFP (expressing CFP attached to N-terminus of Gγ2-subunit), the human Gγ2 gene was amplified by PCR using the plasmid pGγ2-CFP as template with sense primer: 50 -CCG GAA TTC TGA TGG CCA GCA ACA ACA CCG CC -30 (EcoR I restriction site), and antisense primer: 50 -CGC GGA TCC CGA AGG ATG GCA CAG AA A AAC TTC-30 (BamH I restriction site) followed by cloning into pECFP-N1 vector (Clontech Laboratories, Inc. USA). For construction of pGαq-YFP and pGαq-Rluc, site-directed mutagenesis was first performed on the cDNA of the human Gαq to insert a BamH I site between position 97 and 98. Then, the intermediate construct (Gαq-BamH I) was used to insert in phase at the BamH I site the coding region of enhanced YFP F46L or the coding region of Rluc, both without stop codon and bearing the glycine rich-linker sequences coding for Gly-Asn-Ser-Gly-Gly in the 50 end and Gly-Gly-Gly-Asn-Ser in the 30 end, respectively. For expressing Gαi1-Rluc, Gαi2-Rluc, Gαi3-Rluc, and Gαo-Rluc fusion protein, similar strategies were adopted. First, site-directed mutagenesis was performed on the cDNA of the human Gαi1, Gαi2, Gαi3, and Gαo to insert an EcoR I site between position 91 and 92, Then, the intermediate construct (Gα subunits EcoR I) was used to insert at the EcoR I site the coding region of Rluc without stop codon and bearing the glycine rich-linker sequences coding for Gly-Asn-Ser-Gly-Gly in the 50 end and Gly-Gly-Gly-Asn-Ser in the 30 end, respectively. pRluc-Gγ2: the human Gγ2 gene was amplified by PCR using the plasmid CFP-Gγ2 as template with sense primer: 50 -CCG GAA TTC TAT GGC CAG CAA CAA CAC CGC C-30 (EcoR I restriction site) and antisense primer: 50 -CGC GGA TCC CAA GGA TAG CAC AGA AAA ACT TC-30 (BamH I restriction site) followed by cloning into the pRluc-C1 (BioSignal Packard, Canada). Similarly, the human Gγ2 gene was amplified by PCR and inserted into EcoR I and BamH I site of pRluc-N1 plasmid to construct pGγ2-Rluc All constructs were verified by direct DNA sequencing. pcDNA3.1-3  HA-APJ (AGTL10TN00), pcDNA3.1-Gβ1 (GNB0100000) and pcDNA3.1 3  HA ORL (OPRL10TN00) were obtained from UMR cDNA Resource Centre (University of Missouri-Rolla).

FRET imaging FRET was carried out in order to study the dynamics of APJ and G protein coupling in living cells [16]. FRET was calculated using the equation FRET¼raw FRET (coefficient A  YFP) (coefficient B  CFP), coefficient A and coefficient B describe bleed through and are used to correct the raw FRET image using the sensitized emission algorithm. Coefficient A¼Average threshold intensity from FRET filter set/Average threshold intensity from Acceptor filter set (samples contain acceptor only), coefficient B¼ Average threshold intensity from FRET filter set/Average threshold intensity from donor filter set (samples contain donor only) [10,17,18]. To obtain these coefficients, HEK293T cells were grown on poly-Llysine (PLL) coated 6-well plate and transiently transfected with a receptor plasmid pGαi (1,2,3)-YFP, pGαo-YFP or pGαq-YFP, a donor plasmid pCFP-Gγ2 or pGγ2-CFP, and a expressing plasmid pcDNA3.1-Gβ1 individually. After obtaining these coefficients, FRET experiment was performed using an OLYMPUS 1  71 fluorescence inverted microscope. Donor plasmid CFP-Gγ2, receptor plasmid Gαi (1,2,3)-YFP, Gαo-YFP or Gαq-YFP and plasmid expressing APJ, Gβ1 were co-transfected into HEK293T cells. In addition, cells expressing

Please cite this article as: B. Bai, et al., Dynamics of apelin receptor/G protein coupling in living cells, Exp Cell Res (2014), http://dx.doi. org/10.1016/j.yexcr.2014.08.035

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only donor fluorescent protein or receptor fluorescent protein with APJ, or expressing fluorescent proteins without G-protein subunits with APJ were also co-transfected into HEK293T cells as a control. After 12–24 h, FRET signal was detected upon apelin-13 stimulation (100 nM). In order to decrease loss of time-dependent FRET, the illumination time was 300 ms at a frequency of 10 Hz (CFP excitation 436710 nm and emission 480720 nm, YFP excitation 480720 nm and emission 535715 nm, FRET excitation 436710 nm and emission 535715 nm). Pseudocolor FRET images were generated by MetaFluor 7.0 Software, with the lowest FRET intensity in black and the highest FRET intensity in red.

BRET measurements To study the dynamics of APJ and G protein coupling in living cells, Kinetic BRET assays were carried out using eBRET as described previously [19]. eBRET measurements were carried out as follows. A. HEK293T cells were trypsinized when grown to  90% confluence and seeded into 24-well plates [20]. After HEK293T cells were grown in 24-well plates at 37 1C in a humidified atmosphere of 5% CO2, cells were transiently transfected with indicated plasmids to express APJ, Gα-YFP, Gβ1 and Gγ2-Rluc. 6 h after transfection, the medium was replaced with fresh DMEM containing 10% FBS. 12 h post-transfection, the cells were distributed in 96-well microplates

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at a density of 1–3  104 cells with 100 ml fresh medium and incubated for 12–24 h before use [20,21]. B: BRET detection, aspirates of the 40 ml medium from each well and incubated cells with the substrate of EnduRen™ (60 μM final concentration) were used. 96-well microplates with aluminium foil were stored 2 h at 37 1C, 5% CO2 [22]. eBRET measurement was observed by sequential integration of the signals detected in the 460720 nm (luciferase) and 535720 nm (EYFP) windows. The BRET ratio was calculated as the ratio of light emitted by acceptor (EYFP) over the light emitted by donor (Rluc). The BRET ratio observed between interacting proteins was normalized by subtracting the background BRET ratio (Donoronly controls). The sample of cells expressing APJ, Gβ1 and Gγ2-Rluc represents the background ratio. This signal is described as the ‘ligand-induced BRET ratio’. For BRET kinetic assays, the final pre-treatment reading is presented at the zero time point (time of PBS or apelin-13 addition). eBRET ratio was detected and showed by mBRET (mBRET¼BRET ratio  1000) [20,22].

Enzyme-Linked Immunosorbent Assay (ELISA) Transient transfection was performed by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. After a further 24 h, cells were fixed using 4% paraformaldehyde in PBS (30 min at room temperature). After fixation, the cells were washed three times with PBS, and the nonspecific

Fig. 1 – Gαi2 and Gβ1γ2 undergo a subunit rearrangement upon APJ activation. (A and B) Analysis of Gαi2 activation upon apelin stimulation using FRET in HEK293T cells co-transfected with pcDNA3.1-3  HA-APJ, Gαi2-YFP, pcDNA3.1-Gβ1, pCFP-Gγ2 (A) or pcDNA3.1-3  HA-APJ, Gαi2-YFP, pcDNA3.1-Gβ1, pGγ2-CFP (B). Upon stimulation with 100 nM apelin-13, there is significant change in FRET, assessed as the ratio of YFP over CFP emission. (C and D) Analysis of Gαi2 activation upon apelin-13 stimulation using Realtime BRET. Real-time BRET was measured in HEK293T cells co-transfected pcDNA3.1-3  HA-APJ, Gαi2-YFP, pcDNA3.1-Gβ1, pRluc-Gγ2 (C) or pcDNA3.1-3  HA-APJ, Gαi2-YFP, pcDNA3.1-Gβ1, pGγ2-Rluc (D). The BRET ratios are calculated as previously described. All results were expressed as the mean7SEM of six experiments. Please cite this article as: B. Bai, et al., Dynamics of apelin receptor/G protein coupling in living cells, Exp Cell Res (2014), http://dx.doi. org/10.1016/j.yexcr.2014.08.035

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binding sites were blocked with blocking buffer (3% dry milk). Cells were incubated with the primary rabbit polyclonal anti-HA antibody (Cell Signalling, USA) overnight at 4 1C, subsequently washed with PBS three times and then further incubated with a peroxidase conjugated goat/rabbit secondary antibody in DMEM for 1 h at 37 1C. Primary antibodies were used as 1:500, secondary antibodies as 1:2000 dilutions in blocking buffer. The final substrate (200 ml) 3,30 ,5,50 -tetramethylbenzidine (Sigma-Aldrich) was added and incubated for 30 min at 37 1C and the enzymatic reaction stopped with 50 ml of 2 N H2SO4 solution. An orange colour reaction was produced when the substrate reacted with the peroxidase enzyme conjugated on the second antibody and the colour density of each well measured Colorimetric reaction was measured using an imarkTM Microplate reader (Bio-Rad, USA) at 450 nm wavelength.

Statistical analysis To evaluate recordings we used Excel 2009 (Microsoft Corporation) and GraphPad Prism 5. Results are expressed as mean standard error of the mean7SEM. Student's paired t-test was used to assess the statistical significance of differences between two groups. In the case of multiple group comparisons, ANOVA

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was adopted. P-valued o0.05 were used for consideration of significance of the findings.

Results Apelin receptor activation causes Gαi2 and Gαi3 subunits activation through a unique rearrangement process FRET and BRET were carried out to explore the Gαi2 and Gαi3 subunits activation after apelin-13 stimulation in living cells. For FRET assay, HEK293 cells were co-transfected with pcDNA3.13  HA-APJ, pGαi2-YFP or pGαi3-YFP, pcDNA3.1-Gβ1 and pGγ2-CFP plasmids. Upon apelin-13 stimulation, FRET signal was detected (coefficients A and B are 0.02 and 0.58, 0.04 and 0.54 respectively) and exhibited a substantial increase followed by a decrease after wash out (Figs.1 and 2). In BRET assay, similarly, upon activation of APJ by apelin-13, there was also an agonist-induced increase in BRET ratio of Gαi2 or Gαi3-YFP to Rluc-Gγ2. These results indicate a decrease in the distance between the αAB-loop of Gαi subunit and N-terminus of Gγ2-subunit, which was in accordance to previous reports that Gαi activation in intact cells involves subunit rearrangement rather than dissociation. Therefore, it suggests that

Fig. 2 – Gαi3 and Gβ1γ2 undergo a subunit rearrangement upon APJ activation. (A and B) Analysis of Gαi3 activation upon apelin stimulation using FRET in HEK293T cells co-transfected with pcDNA3.1-3  HA-APJ, Gαi3-YFP, pcDNA3.1-Gβ1, pCFP-Gγ2 (A) or pcDNA3.1-3  HA-APJ, Gαi3-YFP, pcDNA3.1-Gβ1, pGγ2-CFP (B).Upon stimulation with 100 nM apelin-13, there is significant change in FRET, assessed as the ratio of YFP over CFP emission. (C and D) Analysis of Gαi3 activation upon apelin-13 stimulation using Realtime BRET. Real-time BRET was measured in HEK293T cells co-transfected pcDNA3.1-3  HA-APJ, Gαi3-YFP, pcDNA3.1-Gβ1, pRluc-Gγ2 (C) or pcDNA3.1-3  HA-APJ, Gαi3-YFP, pcDNA3.1-Gβ1, pGγ2-Rluc (D). The BRET ratios are calculated as previously described. All results were expressed as the mean7SEM of six experiments. Please cite this article as: B. Bai, et al., Dynamics of apelin receptor/G protein coupling in living cells, Exp Cell Res (2014), http://dx.doi. org/10.1016/j.yexcr.2014.08.035

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during apelin-13 stimulated APJ activation, Gαi2 and Gα3 were activated through a molecular rearrangement of heterotrimeric G protein subunits. To further support and strengthen this hypothesis, we also used plasmids expressing CFP labelled Gγ2-subunit at the C-terminus instead of N-terminus to explore the FRET signals change after apelin-13 stimulation. According to the Gαi-rearrangementactivation model discovered by Moritz Bünemann, placing CFP on the C terminus of Gγ2-subunit would cause a decrease in FRET signal if the effects are caused by a rearrangement of Gβγ relative to Gαi. Here, we found FRET signal between YFP attached to Gαi2/3 subunits and CFP attached to C-terminus of Gγ2-subunit significantly decrease after apelin-13 stimulation and gradually return to basal level after wash-out. Besides, the BRET assays also demonstrated that upon apelin-13 stimulation, the BRET ratio of YFP attached to Gαi2/3 subunits to CFP attached to C-terminus of Gγ-subunits decreases, indicating an increase in the distance between the C terminus of the Gγ2-subunit and the αAB-loop of Gαi2/3 during APJ activation, which is perfectly accordance to Gαi-rearrangement-activation model proposed by Bünemann that Gαi protein subunits undergo a molecular rearrangement during activation involving a decrease in distance between αA/B loop of Gαi subunit and the N-terminus of Gβγ subunits with an increase

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in distance between αA/B loop and C-terminus of Gβγ subunits. Taken together, our results suggest interestingly that upon stimulation, APJ activates Gαi2, Gαi3. Moreover, these G proteins activations undergo a novel subunit rearrangement process instead of classical dissociation.

Apelin receptor activation leads to Gαo and Gαq disassociation with Gβγ We also investigate whether Gαo and Gαq are also activated as Gαi2/Gαi3 during apelin receptor activation. The interaction of Gαo and Gαq with Gβ1γ2 was also detected with BRET and FRET as above. Our results indicated that in contrast to Gαi2/Gαi3, both N-terminal fluorescent tagged and C-terminal fluorescent tagged Gγ2subunits exhibited an agonist-induced decrease in FRET signal and BRET ratio with fluorescent tagged Gαo and Gαq (Figs. 3 and 4), suggesting the distance between αA/B loop of Gα subunits and whole Gβγ subunits increases upon apelin-13 stimulation. These results fit to the classical model of G-protein activation that Gα subunits and Gβγ subunits undergo dissociation during activation, which indicates that APJ may also couple to Gαi and Gαo upon activation and the mechanism of these activations

Fig. 3 – Gαo and Gβ1γ2 exhibit a substantial decrease in FRET and BRET upon activation. (A and B) Analysis of Gαo activation upon apelin stimulation using FRET in HEK293T cells co-transfected with pcDNA3.1-3  HA-APJ, Gαo-YFP, pcDNA3.1-Gβ1, pCFP-Gγ2 (A) or pcDNA3.1-3  HA-APJ, Gαo-YFP, pcDNA3.1-Gβ1, pGγ2-CFP (B). Upon stimulation with 100 nM apelin-13, there is significant change in FRET, assessed as the ratio of YFP over CFP emission. (C and D) Analysis of Gαo activation upon apelin-13 stimulation using Realtime BRET. Real-time BRET was measured in HEK293T cells co-transfected pcDNA3.1-3  HA-APJ, Gαo-YFP, pcDNA3.1-Gβ1, pRluc-Gγ2 (C) or pcDNA3.1-3  HA-APJ, Gαo-YFP, pcDNA3.1-Gβ1, pGγ2-Rluc (D). The BRET ratios are calculated as previously described. All results were expressed as the mean7SEM of six experiments. Please cite this article as: B. Bai, et al., Dynamics of apelin receptor/G protein coupling in living cells, Exp Cell Res (2014), http://dx.doi. org/10.1016/j.yexcr.2014.08.035

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Fig. 4 – Gαq and Gβ1γ2 exhibit a substantial decrease in FRET and BRET upon activation. (A and B) Analysis of Gαq activation upon apelin stimulation using FRET in HEK293T cells co-transfected with pcDNA3.1-3  HA-APJ, Gαq-YFP, pcDNA3.1-Gβ1, pCFP-Gγ2 (A) or pcDNA3.1-3  HA-APJ, Gαq-YFP, pcDNA3.1-Gβ1, pGγ2-CFP (B). Upon stimulation with 100 nM apelin-13, there is significant change in FRET, assessed as the ratio of YFP over CFP emission. (C and D) Analysis of Gαq activation upon apelin-13 stimulation using Realtime BRET. Real-time BRET was measured in HEK293T cells co-transfected pcDNA3.1-3  HA-APJ, Gαq-YFP, pcDNA3.1-Gβ1, pRluc-Gγ2 (C) or pcDNA3.1-3  HA-APJ, Gαq-YFP, pcDNA3.1-Gβ1, pGγ2-Rluc (D). The BRET ratios are calculated as previously described. All results were expressed as the mean7SEM of six experiments.

involve the dissociation between Gαo or Gαq subunits and Gβγ subunits.

Apelin receptor activation does not induce significant changes of BRET and FRET signals between fluorescentGαi1 and Gβγ To analyze whether apelin-13 stimulation leads to Gαi1 subunits activation in living cells, we used FRET and BRET approaches to analyze the activation of Gαi1 in HEK293T cells. We found there were no significant FRET changes between Gαi1-YFP and CFP-Gγ2 upon activation (Fig. 5A). Similarly, there were no significant BRET changes between Gαi1-YFP and Rluc-Gγ2 (Fig. 5B). To exclude the possibility that insertion of fluorescence tag at N-terminus interferes the Gαi1 activation, Gγ2-CFP was also used to detect the G-protein activation. Again, we could not detect any significant change of FRET signal after apelin stimulation. Besides, to exclude the possibility that the discrepancy between Gαi1 and Gα2/3 stem from the difference of expression level, we also detected the APJ cell surface expression level by ELISA and Gα subunits expression level by measuring YFP. The results indicate that there is no significant difference between each group (Supplementary Fig. 1). Furthermore, to demonstrate FRET and BRET are sensitive enough to detect the Gαi1 activation, we also used HEK 293T cells expressing ORL as a positive control, which has been

demonstrated as Gαi1coupled GPCR upon Orphanin FQ activation (EC50 ¼ 12 nM) [14]. As predicated, we measured a rapid increase of FRET signal and BRET ratio of fluorescent Gαi1 to Gβγ subunits after agonist stimulation (Supplementary Fig. 2). This suggests that the binding status between Gαi1 and Gβ1γ2 does not change after apelin-13 stimulation that no FRET and BRET signals change upon activation.

Discussion G-proteins convert many pharmacological and physiological stimuli to cellular responses [23]. Canonically, G-protein coupled receptors (GPCRs) can activate G-protein subunit dissociation into Gα and Gβγ [9,24], which initiate particular signalling pathways, respectively, responding to neurotransmitters and hormones. For a long time, it is generally accepted that only dissociated G-protein α subunits play a specified role in receptor signal transduction. However, in recent years this dissociation model has been challenged as being a robust cellular response mechanism to stimuli [10,16,24,25]. Recently, Robishaw and Berlot [26] have brought up a ‘clamshell’ model, predicting that each βγ dimer remains closely associated with the alpha subunit on the receptor. In the past decade, with the emergence of novel technology,

Please cite this article as: B. Bai, et al., Dynamics of apelin receptor/G protein coupling in living cells, Exp Cell Res (2014), http://dx.doi. org/10.1016/j.yexcr.2014.08.035

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Fig. 5 – Gαi1 and Gβ1γ2 exhibit no FRET and BRET changes upon apelin-13 stimulation. (A and B) Analysis of Gαi1 activation upon apelin stimulation using FRET in HEK293T cells co-transfected with pcDNA3.1-3  HA-APJ, Gαi1-YFP, pcDNA3.1-Gβ1, pCFP-Gγ2 (A) or pcDNA3.1-3  HA-APJ, Gαi1-YFP, pcDNA3.1-Gβ1, pGγ2-CFP (B).Upon stimulation with 100 nM apelin-13, there is significant change in FRET, assessed as the ratio of YFP over CFP emission. (C and D) Analysis of Gαi1 activation upon apelin-13 stimulation using Realtime BRET. Real-time BRET was measured in HEK293T cells co-transfected pcDNA3.1-3  HA-APJ, Gαi1-YFP, pcDNA3.1-Gβ1, pRluc-Gγ2 (C) or pcDNA3.1-3  HA-APJ, Gαi1-YFP, pcDNA3.1-Gβ1, pGγ2-Rluc (D). The BRET ratios are calculated as previously described. All results were expressed as the mean7SEM of six experiments.

especially FRET and BRET, real-time and dynamic observation of the interaction between two proteins in living cells became topical and achievable [17,22,27,28]. Monika Frank has revealed the specific mechanisms of Gαi activation through rearrangement instead of dissociation using FRET in noradrenaline stimulated adrenergic receptor model [24]. Here we also found that activation of APJ with apelin-13 also leads to the activation of Gαi through this unique mechanism which was also confirmed by BRET technology. It indicates that the rearrangement of Gαi during the activation is independent of the type of GPCRs, or at least widely exists. In our approach using BRET and FRET, there is a substantial increase in BRET and FRET signals between Giα2 or Giα3-YFP and CFP- Gγ2/Rluc-Gγ2 upon apelin-13 activation, indicating that Gαi2, Gαi3 and N-terminus of Gβ1γ2 subunits become closer without subunit dissociation. In contrast, both the FRET and BRET signals decrease when the fluorescent protein is tagged at C-terminal of Gγ2, clearly indicating there is a heterotrimeric composition between Gαi2, Gai3 and Gβ1γ2 during apelin-13 simulated APJ activation. However, we only observed a substantial decrease in both BRET ratio and FRET signal between fluorescent Gαo and Gαq subunit and Gβγ subunits independent the position of fluorescent tags on Gγ2, indicating Gαo and Gαq subunits dissociate with Gβγ subunits in a classical manner during APJ activation. These data

are consistent with previous findings that APJ activates intracellular effectors via Gαi2, Gαo and Gαq subunits. Meanwhile, to the best of our knowledge, this evidence is novel in showing APJ couple to Gαi3 upon apelin-13 activation. Previous findings that APJ couples to Gαq upon activation were conducted in mouse models. Our results also provide the direct evidence to support the human APJ is also coupled to Gαq after apelin-13 stimulation for the first time. The ability of coupling to variety kinds of G-protein has been suggested to be important for APJ differentially couple to downstream effectors (e.g. MAPK signalling). Recently, Kang et al. also demonstrated in human umbilical vein endothelial cells (HUVECs), APJ activates Gα13 resulting in translocation of HDAC4 and HDAC5 and activation of transcription factor MEF2, indicating the complexity of APJ signals [29]. Besides, using dominant-negative Gαi2, Bo Bai et al. also found that apelin13 induce ERK1/2 activation through coupling APJ to Gαi2 pathway in HEK293 cells [30]. Moreover, the activation of ERK1/2 by apelin is mediated via PKC in HEK293 cells expressing mouse APJ, indicative of coupling to either Go or Gq/11 [31]. Although it was reported that Gαi1 was activated during APJ activation, however, here we did not observe changes of FRET and BRET signals between fluorescent Gαi1 and Gβγ subunits. This discrepancy could be due to the difference of G-protein species used in the assays and using difference cell lines. Combined with

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our findings, it suggests the apelin signalling pathway shows a great diversity and might exhibit functional selectivity or biased signalling. Meanwhile, our data also provide a real-time analysis of dynamics of G-protein coupling to apelin receptor in living cells. It has revealed that upon activation, Gαi2, Gαi3, Gαo, and Gαq undergo very rapid conformational changes (less than 5 s) demonstrated by both FRET and BRET. APJ binds numerous apelin isoforms and signals through various G-proteins to a variety of signalling pathways to culminate in different patterns of activation and desensitization that may be tissue- and cell type-specific. Therefore, our assays could provide a powerful tool to analyze crowded intracellular signalling associated with APJ. In the last decade, there has been a substantial reevaluation of the assumption that GPCRs exist primarily as monomeric polypeptides, with support increasing for a model in which GPCRs can exist as homo or heterodimers or even highorder oligomers [32–34]. Recent reports suggest that homo or heterodimers exhibit specific functional properties which are distinct from monomeric receptors, involving signalling, trafficking amongst other pathways [35–37]. We have also employed these technologies which suggest that human APJ forms a heterodimer with KOR and leads to increased PKC and decreased protein kinase A (PKA) signal transduction evoked by apelin-13 or DynA(1-13)[20]. Our work here could help us clearly understand the G-protein coupling profiling of APJ and lays a solid foundation to identify whether APJ homo or heterodimers affect G-protein coupling in future studies. In addition, it is strongly convincing that Gβγ could be a promising pharmaceutical target in the treatment of some diseases. Turning on or off all of the signalling pathways downstream through targeting specific GPCRs has been very successful in the treatment of many diseases. As described above, Gβγ undergoing dissociation or rearrangement is a critical prerequisite for some signal transduction. Therefore, measuring distinct Gβγ signalling upon apelin-13 activation can be useful to decipher salutary effects in a number of pathological models. Apelin induced Gαi activation has been demonstrated as a benefiting effect on cardiac contractility [12,38,39], and a vasodilator activity that protects against angiotensin-II-induced cardiovascular fibrosis and atheroma [40,41]. In contrast, stretch causes hypertrophy through diminishing G protein activation while augmenting β-arrestins recruitment. The APJ mediated GRK/βarrestin signals induced by stretch signals increase cardiomyocyte cell size and play as a crucial role to cause hypertrophy in a G-protein-independent fashion. Further work will be needed to determine the APJ mediated GRK/β-arrestin signals in living cells by BRET and FRET. In conclusion, these data may provide a previously unknown basis for apelin receptor/G-protein coupling and further indicate that BRET and FRET signals can be used to measure G-protein activation in real time and in living cells. The detection of different compositions during apelin induced G-protein activation will improve the understanding of how the apelin-APJ system achieves subtype-selective coupling to effectors and their unique signal pathways.

Declaration of interest The authors have nothing to declare.

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Funding This work was supported by National Natural Science Foundation Q2 of China (Grant nos. 30971081, 30870932, and 81070961) and Shandong Provincial Natural Science Foundation (Grant nos. Q3 ZR2011CM027 and ZR2009DZ004) and Taishan Scholar Construction Special Fund.

Author contributions Jiang Yunlu carried out the experiments, Cai Xin analyzed the data, Bai Bo and Jing Chen designed the experiments, drafted and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgment We acknowledge Msc Jiamiao Hu, University of Warwick and Dr Zhongbo Chen, King's College London for assisting in drafting this manuscript.

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.08.035.

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