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Table of Contents
Introduction
Results
Growth hormone receptor 1b (GHSR1b) and orexin type 1 receptor (OX1R) have many similar characteristics in terms of their functions and body distribution and are involved in various physiological functions. In this study, we explored the possibility of GHSR1b and OX1R dimerization. Bioluminescence (BRET), fluorescence resonance energy transfer (FRET) and coimmunoprecipitation (Co-IP) were used to analyse the formation of GHSR1b and OX1R heteromers in cells. We also explored their signal transduction pathway mechanism. The results showed that ghrelin could stimulate GHSR1b/OX1R heterodimer cellsto increase Gαs protein activation and induce downstream signalling pathway activity. GHSR1b/OX1R heteromers triggered ghrelin-induced Gαs/protein kinase A signalling pathway activity. Thus, GHSR1b can form a heterodimer with OX1R, leading to increased protein kinase A activity. At the same time, stimulation with orexinA did not alter G protein-coupled receptor (GPCR) interactions with Gα protein subunits. Moreover, ghrelin induced a significant increase in cell proliferation. These results suggest that heterodimers of ghrelin and GHSR1b/OX1R promote the upregulation of a Gαs-cAMP-cAMP-responsive element signalling pathway. The nature of this signalling pathway may have significant implications in regulating physiological functions. Keywords: GHSR1b;OX1R; heterodimerization; ghrelin
Introduction
The growth hormone secretagogue receptor (GHSR) is a growth hormone secretagogue (GHS) receptor. The GHSR gene is located in the 26.31 area of the long arm of human chromosome 3(Carriba P et al.,2008). Growth hormone receptor 1b (GHSR1b) is a member of the orphan G protein-coupled receptor (GPCR) family; under agonist action, GHSR1b promotes growth hormone secretion in elderly individuals. GHSR content is highest in the central nervous system and anterior pituitary gland. GHSR is also expressed in the hypothalamus (including the anterior inferior hypothalamus, hypothalamic ventral nucleus, anterior ventral nucleus, suprachiasmatic nucleus, nucleus, arcuate nucleus, paraventricular nucleus and nodular papillary nucleus), heart, pancreas, intestine, kidney, adipose tissue, and male and female reproductive systems, as well as in the main feeding centre (pituitary gland,ventral nucleus and hypothalamus)(He SQ et al.,2011). Even in the absence of growth factors and growth hormone secretion, GHSR plays an important role in humans and animals (Sakurai T et al., 2014) due to its constitutive activity. Furthermore, data have shown that GHSR1b can dimerize with other receptors, including MC3R, 5-HT2C, andCB1(Carriba P et al.,2008; Sakurai T et al.,1998). Upon dimerization, these receptor dimmers may induce different physiological functions;thus, the dimerized GHS system appears to play an important and complex role(Sakurai T et al., 2014). A better understanding of this dimerization may improve the treatment of physiological disorders, such as Parkinson's disease, schizophrenia, addiction, obesity, and diabetes. Orexin, a hypothalamic neuropeptide(Howard AD et al.,1996; McKee KK et al.,1997), was discovered simultaneously by two research groups in 1998. The orexin system includes two neuropeptides, orexinA and orexinB, whose receptors are orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R), which are GPCRs. OX1R may bind orexinA or orexinB but has higher affinity for orexinA. In the rat brain, orexin neurons are largely present in the hypothalamus dorsal and medial regions and lateral to the fornix region, andorexin nerve endings project into the central nervous system(McKee KK et al.,1997;Schellekens H et al.,2015). Sakurai found that neuropeptides secreted by the hypothalamus exerted modest effects on feeding activity; these neuropeptides were designated orexins (Sakurai T et al.,1998). An in-depth study of the orexin system revealed the role of orexin in regulating various behaviours and emotional reactions. The widespread distribution of orexin receptors in the hypothalamus and central nervous system supports the physiological functions of orexin, such as its regulation of drinking water intake, energy metabolism, reproduction, sleep, and waking. We hypothesized that the expression of two types of receptors in the same neurons affects signalling pathway interactions to regulate the appetite element system, resulting indifferential behavioural responses. Paradoxically, although GHSR1b is widely distributed in the brain, endogenous ghrelin is nearly undetectable. However, the results of our study indicated that GHSR1b function occurred in the absence of ligand binding and that GHSR1b could form heterologous receptor dimers with OX1R. According to the traditional view, GPCR dimers form in vitro only and do not affect physiological function. However, our findings confirm the in vivo formation of two different heterologous receptor dimers due to agonist-induced signal transduction. GHSR1b and OX1R are involved in feeding, reward and stress behaviour. The dimerization of GHSR1b and OX1R may change their original functions, which alters their reaction to orexin/ghrelin and thus plays an important role in the orexin/ghrelin system. In this research, our aim is to detect the possibility of these effects and survey the mechanism of heterodimerization-mediated signal transduction. Given the significant amounts of money that have been invested in research onthe effects of GHSR1b receptor antagonists on weight reduction, our findings have important potential applications(Masuda Y et al.,2000; Shuto Y et al.,2002).
Results
Identification of stable cells
The GHSR1b-specific primer samplified the target GHSR1b fragment in both the GHSR1b single transfection group and the cotransfection group but not in the OX1R single transfection group. These results indicated that GHSR1b was expressed in the GHSR1b single transfection and cotransfected cells but not in the OX1R single transfection cells. The use of the OX1R-specific primer samplified the target OX1R fragment in both the OX1R single transfection group and the cotransfection group but not in the GHSR1b single transfection group, indicating that GHSR1b stable cells did not express OX1R. However, OX1R was expressed in OX1R stable cells and co-transfected GHSR1b/OX1R stable cells. These results indicated that only OX1R was expressed in the HEK293-OX1R stable cell line, only GHSR1b was expressed in the HEK293-GHSR1b stable cell line, and both OX1R and GHSR1b were expressed in the HEK293-OX1R/GHSR1b stable cell line (Fig.1A). Thus, HEK293-GHSR1b, HEK293-OX1R and HEK293-GHSR1b/OX1R stable cell lines were successfully constructed and screened. Western blotting was performed to further confirm GHSR1b and OX1R expression levels in HEK293 cells (Fig. 1B). Colocalization of GHSR1b and OX1R In this experiment, pcDNA3.1-GHSR1b and OX1R-EYFP cotransfection was analysed by performing confocal microscopy. OX1R was detected with a red-labelled antibody, while GHSR1b was labelled with a green fluorescent protein that was detectable after expression. As shown in Fig. 1C, GHSR1b (green) and OX1R (red) were expressed in the same areas in the cell membrane, indicating potential co-localization. (Figure 1)
BRET detected heterodimerization
A BRET test based on luminescence and fluorescence detection was performed. In this experiment, the energy donor and energy receptor transfection ratio was 1:3 (Rluc fusion protein :EYFP fusion protein). As shown in Fig. 2A, the mBRET ratio of Rluc-OX1R to EYFP-GHSR1b (188.23 ±1.95) was lower than that of the positive control group (mBRET ratio 194 ±11.18), but there was no significant difference between the two. The mBRET ratio was significantly higher than that of the negative control group (mBRET ratio: 37.53 ±1.90), and there was a significant difference between the two, indicating that GHSR1b and OX1R form heterologous heterodimers. Based on the preliminary detection of OX1R and GHSR1b by BRET, we tested these at saturation to determine whether the heterodimers formed by OX1R and GHSR1b were constitutive dimers. As shown in Fig. 2A(a), the OX1R/GHSR1b dimer is a constitutive dimer, but ghrelin or orexin A enhances the interaction between OX1R and GHSR1b such that their addition causes additional dimer formation (Fig. 2A(b)). GHSR1b and OX1R heterodimerization determined by Co-IP Co-IP analysis was used to analyse the interaction between OX1R and GHSR1b in the formation of a heterodimer. After HA-OX1R/Myc-GHSR1b coexpression in HEK293 cells, anti-Myc antibodies were used for immunoprecipitation. The Co-IP results showed that GHSR1b and OX1R could form dimers when expressed in the same cell simultaneously. When HA-OX1R or Myc-GHSR1b was expressed alone, the Co-IP results were similar to those of the negative control(Fig. 2B). Heterodimerization of GHSR1b and OX1R determined by FRET assays FRET was performed to analyse the interaction between GHSR1band OX1R. In these experiments, (a) OX1R-eCFP and GHSR1b-eYFP, (b) GHSR1b-eYFP, (c) OX1R-eCFP, or (d) CRH1R-eCFP and GHSR1b-eYFP plasmids were transfected into HEK293 cells. In the acquired FRET images, the sites of interaction are marked in yellow or red, with a more intense colour indicating a stronger interaction (Fig. 2C). Lower FRET signals were observed in all singly transfected cells and control cells containing GHSR1b-eYFP and CRH1R-eCFP. However, a FRET signal was detected in cells doubly transfected with GHSR1b-eYFP and OX1R-eCFP, further indicating that GHSR1b and OX1R can form heterodimers (Fig. 2D). TM1, TM5, and TM7 provide dimer interfaces TMs are important for the formation of head-to-head interfaces in class A GPCR dimers[P. Frank M et al., 2005; Pediani J.D et al., 2016]. GHSR1b and OX1R were tested with the HIV TAT-fused TM peptides TM1, TM5 and TM7. Incubation with HIV TAT-fused TM7, but not TM1 or TM5, resulted in significantly higher BRET activity than treatment with ghrelin or orexinA alone (Fig. 3). Finally, incubation with TM1 and TM5 decreased the induction of BRET activity upon orexin A treatment but increased BRET activity in response to ghrelin treatment (Fig. 3). Thus, TM1 and TM5 impaired GHSR1b and OX1R heterodimerization. TM7 is less important for GHSR1b and OX1R heterologous dimerization and had little effect on BRET signals(Fig. 3). (Figure 3) NFAT, CRE and SRE contents were measured using the dualluciferase method The expression of NFAT, CRE and SRE in HEK293-GHSR1b, HEK293-OX1R and HEK293-GHSR1b/OX1R cells reflects the effects of GHSR1b and OX1R on the intracellular signal transduction pathway[15,18]. In the HEK293-OX1R cell line, NFAT-luc activity increased significantly after treatment with orexin A (P< 0.01) (Fig. 4A). The intracellular CRE-luc activity of GHSR1b/OX1R cells treated with ghrelin or dual agonists (orexinA/ghrelin) was increased significantly (P< 0.01), and treatment of HEK293 GHSR1b/OX1R cells with orexinA had no effect on CRE-luc activity (Fig. 4B).The intracellular CRE-luc activity of GHSR1b/OX1R cells treated with ghrelin or dual agonists (orexinA/ghrelin) was increased significantly (P< 0.01), and treatment of HEK293 GHSR1b/OX1R cells with orexinA had no effect on CRE-luc activity. Gαi-coupled receptors inhibited adenylyl cyclase (AC) and activated the ERK pathway through the βγ subunits via SRE-luc in HEK293 GHSR1b/OX1R cells treated with ghrelin and/or orexinA; at the same time, the activity of SRE-luc did not change obviously (Fig. 4C). All the results showed that heterodimerization of GHSR1b and OX1R increased CRE activity dependent on protein kinaseA (PKA) activity when ghrelin was added to HEK293-GHSR1b/OX1R cells, which further confirmed that the GHSR1b/OX1R heterodimer promotes the Gαs signalling pathway. Detection of intracellular cAMP and calcium content The detection of intracellular cAMP content permits further characterization of the effects of GHSR1b/OX1R heterologous dimerization on the intracellular signal transduction pathway. The detection results are shown in Fig.5A(a); the interaction of orexin A and ghrelin in HEK293-GHSR1b/OX1R cells resulted in a significant increase in cAMP content in a dose-dependent manner compared with HEK293-GHSR1b cells or HEK293-OX1R cells. Calcium is a downstream second messenger of the Gαq subtype. The detection of intracellular calcium facilitates the elucidation of the effects of the GHSR1b/OX1R dimer on the intracellular signal transduction pathway[37]. The calcium content of HEK293-OX1R cells was significantly increased compared with that in the basal grouptreated with orexinA (Fig.5A(c)). The calcium concentration in HEK293-GHSR1b cells was significantly different from that in the control group. OrexinA, ghrelin or both affected HEK293-OX1R cells or HEK293-GHSR1b cells, but calcium levels were significantly lower in HEK293-GHSR1b/OX1R cells (ghrelin: 1.793±0.1308, orexinA: 1.958 ± 0.0906, ghrelin/orexinA: 1.6723 ± 0.1126). The calcium levels in HEK293-GHSR1b/OX1R dimer cells were not different from those in HEK293-OX1R cells or HEK293-GHSR1b cells. Based on the results, the GHSR1b/OX1R dimer is not associated with the G protein binding of the Gαq subtype, and the GHSR1b/OX1R dimer alters the signal pathway of the monomer. Gα protein subunit assays GHSR1b/OX1R heterodimers can participate insignalling that differs from that of their monomers. Gα BiFC-BRET assays were performed to survey interactions between GHSR1b/OX1R heterodimers. As shown in Fig.10, changes in the BRET ratio between Rluc8-tagged Gαq (Fig. 5B(a) ), Gαs (Fig. 5B(b)), or Gαi2 (Fig. 5B(c)) and BiFC (OX1R-VC155 and GHSR1b-VN173) were observed. The expression of Rluc8-Gαi2 and BiFC (GHSR1b/OX1R) in HEK293 cells did not significantly change the BRET ratio. However, the expression of BRET Rluc8-Gαi2 and Venus-GHSR1b (GHSR1b/OX1R) increased with 80 nM ghrelin or orexinA compared with the expression levels observed for Rluc8-Gαi2 and Venus-labelled OX1R cells during the 15-min duration prior to the peak, strongly suggesting that GHSR1b activation regulates GHSR1b-OX1R heterodimerization. cAMP accumulation is a major indicator of Gs protein activation and usually significantly decreases when the Gi protein is activated(Ward RJ et al.,2017).In HEK293cells expressing Rluc8-Gs and BiFC (GHSR1b/OX1R), the BRET ratio did not change significantly. When treated with 80 nM ghrelin or orexinA, the BRET ratio in cells expressing Rluc8-Gs and Venus-GHSR1b (GHSR1b/OX1R) abruptly increased (P<0.05) compared with that in cells expressing Rluc8-Gs and Venus-tagged OX1R, strongly suggesting the ability of activated GHSR1b to modulate GHSR1b-OX1R heterodimerization. In cells expressing GHSR1b-VN173 and OX1R-VC155 tagged with VN173 and VC155, an increase in BRET was observed only for Gαi2 and Gαs. No changes in BRET were observed forthe negative controls (Fig. 5B). (Figure 5) β-arrestin assay β-arrestin1 and 2 are important metabolites that mitigate receptor desensitization and play important roles in the regulation of G protein-mediated signal transduction for most receptors. β-arrestin1 and 2 are closely associated with GPCR desensitization, internalization, complex formation, cell proliferation and gene transcription (Ward RJ et al.,2017; Parenty G et al.,2008). When HEK293cells expressing Rluc8-tagged β-arrestins, GHSR1b-VN173, and OX1R-VC155 were stimulated with 80 nM orexinA, the results indicated an increase in ligand-induced BRET signals, suggesting that the recruitment of β-arrestin1 and β-arrestin2 to GHSR1b depends on OX1R expression and orexinA stimulation (Fig.5C). The negative controls (GHSR1b-VN173 and β-arrestin1-Rluc8, or OX1R-VC155 and β-arrestin1-Rluc8) had no changes in BRET (Fig. 5C(a)). When stable cells coexpressing GHSR1b-VN173 and OX1R-VC155 were stimulated with ghrelin or/and orexin A, BRET signals increased, indicating that GHSR1b and OX1R heteromeric dimerization recruited β-arrestin1 (Fig. 5C(a)). Similar to β-arrestin1, β-arrestin2 was recruited to GHSR1b and OX1R heteromeric dimers depending on the ligand (ghrelin or/and orexin A) (Fig. 5C(b) ). The results showed that the GHSR1b/OX1R heterodimer does not alter the recruitment of β-arrestin1 and/or β-arrestin2. Effects of growth hormone on the proliferation of transfected cell lines After incubation for 24 h in 96-well plates, 80 nM growth hormone was added to transfected cells, followed by starvation overnight in serum-free medium. Cell proliferation was then measured; transfected HEK293 cells were used as controls. The effects of different doses of growth hormone on HEK293 cell proliferation are shown according to thedata from four independent experiments. Each experiment was repeated 4 times; **P<0.01, *P<0.05. (Figure 6)
DISCUSSION
Growth factors are associated with energy balance, diet, depression and addiction. Following their initial discovery, growth factors were also found to be related to obesity in modern society. One of the most difficult questions concerning growth factors is the primary role of growth hormone receptors in the brain, given that GHSR1b dimerization complexes (5-HT2c, MC3R, D1R, D2R, and CB1) act as receptors in most areas of the brain(Ward RJ et al.,2017; Parenty G et al.,2008). These systems are associated with rewards, eating and memory; therefore, understanding the dimerization of these factors, including their signalling initiation and modification and the different pathways and systems that affect these factors, is an area of interest. Recent studies have shown that GHSR is highly correlated with food intake and body weight regulation(Jeffery PL et al.,2003; Wang J et al.,2015; Tian C et al.,2010). Animal experiments have shown that injecting GHS into the central nervous system causes a food intake increase, fat oxidation decrease, and animal obesity(Howick K et al.,2018) . The expression of GHSR mRNA in the main feeding centre (pituitary gland, middle nucleus and hypothalamus) is also increased; furthermore, expression is significantly lower, and body weight is significantly lower in GHSR gene knockout rats than incontrols(Xu Y et al.,2013; Drake MT et al.,2007). The most important role of GHSR occurs through interactions with growth factors, thus affecting growth hormone secretion, food intake, fat accumulation, human growth, development and obesity(Millar RP et al.,2010) . We sought to observe the interactions between GHSR1b and OX1R at the molecular, cellular and physiological levels both in vivo and in vitro. In addition to native tissues, GPCRs exist as monomeric entities that can form dimers and/or oligos when expressed in a cell system. In vivo, GPCR dimerization has been demonstrated using resonance energy transfer Co-IP(Borroto-Escuela D et al.,2013; Gomes I et al.,2016). In many brain regions, GHSR1b and OX1R are coexpressed. We speculated that coexpression of GHSR1b and OX1R in the same neurons can lead to interactions via heterodimerization. In this research, FRET and traditional Co-IP provide solid evidence for the formation of GHSR1b/OX1R heterodimers. We selected the HEK293 cell line to stably express GHSR1b and OX1R proteins to evaluate whether their dimerization affects cell viability. HEK293 cell proliferation was investigated after we selected the cell lines stably expressing GHSR1b and OX1R. Proliferation was significantly higher in HEK293 GHSR1b and OX1R cells than in HEK293 GHSR1b or HEK293 OX1R cells alone after treatment with ghrelin. Furthermore, ghrelin induced the proliferation of human HEK293 GHSR1b and OX1R cells in a dose-dependent manner. These results show that ghrelin is important in the proliferation of HEK293 GHSR1b and OX1R cells. GPCR heterologous fusion is an important process in regulating receptor function. Receptor-receptor interactions may result in the formation of a stable structure and alter downstream signal coupling, leading to dimer-specific signal transduction(Rozenfeld R et al., 2007). In this study, we found that even in the absence of ghrelin, OX1R signalling was still present, suggesting that GHSR1b acts as an allosteric regulator of OX1R signalling. This finding confirms that GHSR1b still functions in the absence of ghrelin in the brain. Coexpression of GHSR1b and OX1R was confirmed by observing the binding of YFP and OX1R via immunofluorescence histochemistry. The colocalization of the two receptors on most hypothalamic neurons is consistent with the results of previous in situ hybridization and RT-PCR studies(Panetta R et al.,2008; Thompson M.D et al.,2008). We questioned whether coexpression of these GHSR1b receptors and OX1R receptors in these neurons affects the signalling pathway induced by orexin. Experimental evidence obtained from HEK293 cells and primary cultured hypothalamic neurons confirmed that coexpression of the two receptors altered the traditional signalling pathway of OX1R and induced orexin-induced Ca 2+ release. In natural tissues, the expression of GHSR1b is very low(Costantini V.J et al.,2011; Wren A.M et al.,2001). In a heterodimer structure, GHSR1b exhibits significantly higher basal activity than that observed innatural tissue, indicating that the basal activity of GHSR1b fulfils a physiological function (R.J. Ward et al.,2011; Pediani J.D et al., 2016), potentially explaining the signalling effects of GHSR1b on OX1R regulation. Receptor interaction may also regulate the signalling pathways of receptors(J. Bonaventura et al.,2015). Our results indicate that receptor alterations occurred due to the formation of dimers by GHSR1b and OX1R. GPCRs assume homologous or heterodimeric forms in vitro, which alters receptor signalling and transport. However, due to a lack of experimental evidence, the presence of receptor dimers in natural tissue remains controversial. FRET is an ideal method for detecting homologous or heterogeneous GPCR enrichment with high sensitivity; thus, we applied this method to observe the expression of physiological levels of GPCRs. To evaluate whether GHSR1b and OX1R dimerization affects cellular functions, we selected HEK293 cells stably expressing GHSR1b and OX1R. The results showed that proliferation was significantly higher in GHSR1b/OX1R cells than in OX1R or GHSR1b cells alone. At the same time, ghrelin induced the proliferation of GHSR1b/OX1R cells in a dose-dependent manner. This indicates that ghrelin plays an important role in the proliferation of GHSR1b/OX1R cells. Because ghrelin stimulates food intake, anorexia may result if a GHSR1b antagonist is administered or if GHSR1b is eliminated to block the action of endogenous ghrelin signalling. Our experiments provide important evidence that neurons coexpressing GHSR1b and OX1R perform a regulatory role, leading to a differential GHSR1b response to endogenous orexin. More importantly, coexpression of GHSR1b and OX1R in neurons administered a GHSR1b antagonist leads to orexin signal transduction blockade, while neurons expressing OX1R alone are not affected.
Materials and methods
Plasmids, reagents, antibodies and cells pcDNA3.1-OX1R, which contains human OX1R cDNA, and pcDNA3.1-GHSR1b, which contains GHSR1b, plasmids were obtained from the UMR cDNA Resource Centre (University of Missouri-Rolla). pEYFP-N1,containing enhanced yellow fluorescent protein (eYFP), and pRluc-N1, containing the Rluc plasmid, were purchased from Addgene (Cambridge, MA, USA). SRE-luc, CRE-luc and NFAT-RE-luc were obtained from Promega (Wisconsin, USA).Human orexinA and ghrelin were purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). Lipofectamine 2000 was obtained from Invitrogen (Grand Island, USA). Anti-HA and anti-Myc antibodies were purchased from Cell Signaling Technology (Danvers, USA). Anti-GHSR1b and anti-OX1R antibodies were obtained from Novus Biologicals (Abingdon, UK). Human embryonic kidney 293 (HEK293) cells were obtained from the Peking Union Medical College Cell Centre (Beijing, China). Construction of expression vectors eYFP-GHSR1b and eCFP-OX1R,which encode an eYFP-tagged and an enhanced cyan fluorescent protein (eCFP)-tagged protein, respectively, were constructed as previously described[11,12]. eCFP and eYFP were attached to the C-termini of OX1R and GHSR1b by inserting the open reading frames (ORFs) of OX1R and GHSR1b into the eCFP-N1 and eYFP-N1 vectors, respectively, to obtaineCFP-OX1R and eYFP-GHSR1b. Finally, the recombinant vectors were transfected into HEK293 or HEK293T cells using the liposome method. Recombinant vector expression was observed with a laser scanning confocal microscope. Plasmid expression was observed via fluorescence microscopy. The total transfected DNA was constant in all experiments via the use of empty vectors. Transfected cell lines stably expressing GHSR1b orOX1Ror coexpressing GHSR1b and OX1R for 10 weeks were selected with G418 (0.6 mg/mL). Identification of stable cells The endogenous expression of GHSR1 and OX1R was detected by performing RT-PCR. Total RNA was extracted and purified from human HEK293 cells. According to the instructions of the reverse transcription kit (Fermentas), cDNA was obtained, and PCR was then performed to identify HEK293-GHSR1b, HEK293-OX1R and HEK293-GHSR1b/OX1R stable cell lines. Immunostaining and confocal microscopy HEK293T cells were cultured in 12-well plates. When the cell density reached 60%, GHSR1b-eYFP and pcDNA3.1-OX1R-HA (1 : 1) were cotransfected into cultured cells and maintained for 24 h. Then, the cells were transferred to glass coverslips in 6-well plates. After three 5-min PBS washes, the cells were incubated in 1% bovine serum albumin (BSA) (0.1% Triton X-100) at room temperature for 1 h. The cells were washed 3 times for 5 min each and then incubated overnight with an anti-HA antibody (3% BSA, diluted 1:500) at 4°C. After 3 additional 5-min PBS washes, the cells were incubated with Alexa-Fluor®594 (red)-conjugated secondary antibodies (goat anti-rabbit IgG-Cy3, 1 :400) (Molecular Probes, Invitrogen) for 1 h at room temperature. The cells were washed 3 times in PBS for 5 min each, and 4′,6-diamidino-2-phenylindole (DAPI, purple) was thenappliedfor 20 min to stain the nuclei. Confocal microscopy (Leica, Milton Keynes, UK) was used to detect immunofluorescence. Biological resonance energy transfer (BRET) assays The experimental assay method was carried out as previously described(Lohse M.J et al.,2012). mVenus-GHSR1b and OX1R-Rluc plasmids encoding the eYFP and Rluc fusion proteins, respectively, were cotransfected into HEK293 cells in a 24-well plate. After 24 h, the cells were distributed into 96-well microplates at a density of 5×10 4 cells/well. Cotransfected cells were cultured with high-glucose DMEM without phenol red. Then, coelenterazine H substrate was added. Finally, the data were analysed using Graph Pad Prism v6.0software. Fluorescence resonance energy transfer (FRET) assays FRET assays were performed as described previously (Howick K et al.,2018). Briefly, the donor plasmid OX1R-eCFP and the receptor plasmid GHSR1b-eYFP were cotransfected into HEK293 cells. The donor and acceptor channels were used to eliminate crosstalk in the FRET channel. To determine the calibration coefficients to correct for excitation and emission crosstalk, OX1R-eCFP or GHSR1b-eYFP was transfected into HEK293 cells as donor-only and acceptor-only constructs, respectively. After 18 h, FRET signals were detected with a FRET Kit for a Leica AM TIRF MC system. The efficiency of FRET (𝐸𝐴(𝑖)) was calculated using the following equation. A, B, and C correspond to the intensities of the three signals (donor, FRET, and acceptor, respectively), and α, β, γ, and δ are the calibration factors generated by the acceptor-only and donor-only references. 𝐸𝐴(𝑖) = B − A ∗ β − C ∗ (γ − α ∗ β) C ∗ (1 − β ∗ δ)
Coimmunoprecipitation(Co-IP)
The Myc-GHSR1b and HA-OX1R plasmids were cotransfected into HEK293 cells. After 24 h, the cells were collected in a 1.5-mL tube containing 200 µL of weak lysis buffer for lysis at 4°C. After centrifugation at 16,000g for 30 min, the supernatant was transferred to a 1.5-mL centrifuge tube. Then, 200 µL of supernatant and 40 µL of anti-HA were mixed for 6 h at 4°C. Next, the mixture was centrifuged at 18,000 g for 15 seconds, and the precipitate was washed 5 times with PBS. Then, the proteins were separated via 10% SDS-PAGE and analysed by Western blotting.
Design and synthesis of TM peptides
An HIV transactivator of transcription (HIV TAT)-linked peptide (YGRKKRRQRRR) was fused to the C-termini of the OX1R TM1, TM5 and TM7 peptides. All TMs were synthesized correctly. TM1, TM5 and TM7 (10 mmol/L) were added 1 h prior to the BRET assay and then measured using a BRET instrument. HEK-293 cells cotransfected with OX1R-Rluc and GHSR1b-eYFP (1:3) were incubated with the TM1, TM5, or TM7 peptides at 37°C (4 μM) and detected using a BRET instrument.
1.12. BiFCconstruction
BiFC-BRET (Howick K et al.,2018) was determined to detect GHSR1b/OX1R heterologous dimerization in living cells. pCE-BiFC-VN173 and pCE-BiFC-VC155, containing the Venus fluorescent protein, were separately fused with GHSR1b and OX1R, respectively (GHSR1b-VN173 and OX1R-VC155), to create heterologous dimers. HEK293 cells were then cotransfected with GHSR1b-VN173 and OX1R-VC155 and observed with a fluorescence microscope. The detected Venus fluorescence indicated the successful construction of BiFC, confirming the heterologous dimerization of the GHSR1b and OX1R receptors. The signalwas monitored by BRET to generate kinetic curves, and monitoring was carried out for 50 min after the addition of ghrelin and/or orexinA (100 nM). NFAT-RE, CRE and SRE luciferase reporter assays The activity of NFAT-RE, CRE, and SRE in HEK293-OX1R, HEK293-GHSR1b, and HEK293-OX1R/GHSR-1 stable expression cells was detected to study the effects of GHSR1b and OX1R heterodimers on downstream signalling. Three types of downstream signalling factors were selected to detect OX1R/GHSR-1b, OX1R and GHSR-1b binding to Gαq, Gαs and Gαi, respectively. To carry out nuclear factor of activated T-cell-response element (NFAT-RE), cAMP-response element (CRE), and serum response element (SRE) luciferase reporter assays, cells stably expressing GHSR1b, OX1R, or GHSR1b/OX1R were cotransfected with pSRE-Luc, pCRE-Luc, or pNFAT-Luc, together with pRL-Tk. Twenty-four hours after transfection, the cells were starved and stimulated with orexin A and/or ghrelin at a final concentration of 80 nM for 8 h prior to harvest(Nakazato M et al., 2001; Masuda Y et al.,2000). Detection of intracellular cAMPand calciumcontent HEK293-OX1R, HEK293-GHSR1b and HEK293-OX1R/GHSR1b stable cells were cultured in 24-well plates. cAMP levels in each group were measured using a commercial cAMP ELISA kit (Cell Biolabs, San Diego, CA, USA). The assay methods were performed as described previously (Kojima M et al., 1999; Muller C. E et al., 2012; P. Frank M et al.,2005). The YFP-Epac-RLuc plasmid was also used to measure cAMP levels. YFP-Epac-RLuc was transfected into HEK293-OX1R, HEK293-GHSR1b and HEK293-OX1R/GHSR1b cells. After 24 h, the cells were distributed and cultured in HEPES-buffered phenol red-free medium in a 96-well white microplate for another 24 h. Cells were stimulated with 500 μM IBMX (3-isobutyl-1-methylxanthine) for 20 min and agonists (ghrelin or orexin A, 10 -7 μM) for 5 min. Forskolin (FSK) at a concentration of 10μM was added, and the cells were incubated for another 5 min. The BRET ratio was measured in the presence of 5 μM coelenterazine H. BRET signals were determined by calculating the ratio of YFP emission (535 nm) to Rluc emission (460 nm). A cAMP BRET biosensor was used to determine whether heterodimerization affects cAMP accumulation. When the sensor binds to cAMP, the BRET ratio decreases. Thus, the BRET ratio permits the quantitation of changes in cAMP. We used a Fluo-4NW assay kit (Invitrogen, USA) to detect changes in intracellular calcium content. Stable cell lines were plated at 6×10 4 cells/well and incubated with lysine (Lys). After 24 h, the culture medium was removed, and 1× loading dye (2.5 mM, 100 μl/well) was added to the cells. Cells were incubated at 37°C for 20 min and then at room temperature for another 20 min. The loading dye was discarded, and 90 μL ofassay buffer solution was added to each well. The corresponding agonist (final concentration: 100 nM) was added to each well. Fluorescence was detected at an excitation wavelength of 485 nm, and the emission wavelength was 515 nm. Gα protein subunit assays Gαs-Rluc8, Gαi2-Rluc8 and Gαq-Rluc8 were used to test for GHSR1b and/or OX1R activity. Gαs-Rluc8, Gαi2-Rluc8 and Gαq-Rluc8 were transfected into HEK293-GHSR1b, HEK293-OX1R and HEK293-GHSR1b/OX1R stable cells (cell density: 5×10 4 /well). After 24 h, the cells were transferred to poly-lysine-coated white 96-well cell culture plates. Then, the cells were cultured in HEPES-buffered phenol red-free medium (Invitrogen, Life Technologies). After 24 h, agonists were added, and cells were stimulated for 15 min. The BRET ratio was measured in the presence of 500 nM coelenterazine H. Real-time kinetics of the GHSR1b/OX1R heterodimer as a function of β-arrestin recruitment β-arrestin1-Rluc8 and β-arrestin2-Rluc8 were transfected into HEK293-GHSR1b-VN173, HEK293-OX1R-VC155 and HEK293-GHSR1b-VN173/OX1R-VC155 stable cells. After 24 h, the cells were transferred to poly-lysine-coated white 96-well cell culture plates (cell passage density: 5×10 4 /well). Then, the cells were cultured in HEPES-buffered phenol red-free medium (Invitrogen, Life Technologies). After 24 h, agonists (ghrelin and/or orexin (80 nM) were added, and cells were stimulated for 15 min. The BRET ratio was measured in the presence of 500 nM coelenterazine H.To generate kinetic curves, BiFC-BRET was monitored for 10 min. After the addition of agonists (80 nM), BiFC-BRET monitoring continued for an additional 60 min.
Cell proliferation assays
HEK293 cells were seeded in a 96-well plate at a density of 4×10 4 cells/well. After 24 h, the cells were treated with 1, 10, and 80 nM ghrelin for another 24 h. HEK293 cell proliferation was measured using a CCK-8 (Gibco, Invitrogen) viability assay. HEK293, HEK293-GHSR1b, HEK293-OX1R, and HEK293-GHSR1b/OX1R cells were seeded in plates at a density of 5×10 4 cells/well. After 18 h, all cells were stimulated with 80 nM ghrelin (and/or orexinA) for24 h. Then, the CCK-8 assay was performed. After incubation, absorbance was measured at 450 nm. Each sample was repeated four times. Wells containing media alone were included as a control.
Statistical analysis
The data from all experiments are shown as the mean± SEM. The data were analysed using SPSS11.5 software (SPSS, Inc., Chicago, IL, USA).T-tests were performed, and P≤0.05 was considered statistically significant.
Acknowledgements
The study was approved by the regional ethics committee and hospital authority. The work was funded by the Shandong Key Research and Development Plan Project (2018GSF118137), the Shandong Medical and Health Technology Development Plan Project of Shandong Province (2017WS339), the Science and Technology Projectof Shandong Province (J17KB085), the Young Teachers Research Support Fund of Jining Medical University (JY2017KJ019), the National Natural Science Foundation of Jining Medical College(2016)and the Growth Program of Young Teachers in Shandong Province (2017). Compliance with ethical standards Shulin Chen is a member of the advisory board of Endocrine. The remaining authors declare that they have no conflict of interest.
Ethicsapproval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the experiments involving mice were carried out in accordance with the ethical standards of institutional and national research committees and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. This article does not contain any studies with human participants performed by any of the authors.
ConflictofInterest
The authors declare that they have no conflicts of interest. The authors declare that they consent to the publication of this research.
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