Cloning and Characterization of Spliced Variants of the Porcine G Protein Coupled Receptor 120

The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120) has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform) was detected in spleen, while the transcript 2 (coding 310-aa isoform) was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.


Introduction
Free fatty acids (FFAs) are well known in serving as important nutrients to provide energy for the body and play important roles in various physiological regulation as signaling molecules [1][2][3]. Recently, a group of G-protein coupled receptors (GPCRs) have been identified as FFA receptors which mediate FFA-induced signaling in various tissues [2][3][4][5][6][7][8][9]. In particular, GPR120, which is described as an orphan receptor originally, functions as a polyunsaturated fatty acids (PUFAs) receptor and plays critical roles in systemic nutrient homeostasis regulation [8,10].
In human, mouse, and rat, GPR120 is expressed abundantly in intestinal tract where GPR120 mediates FFAinduced the secretion of the gut hormones (glucagon-like peptide-1 and cholecystokinin), thereby regulating appetite [5,11]. Additionally, the expression of GPR120 is also significantly abundant in adipose tissue where it participates in lipid and glucose metabolism, as well as adipogenesis regulation [12][13][14]. Ligands of GPR120, including n-3 PUFAs and synthetic GW9508, reduce inflammatory and increase insulin sensitivityin a GPR120-dependent manner in mice [8]. Moreover, GPR120 is found to mediate the taste of fatty acids and FFAs-induced antiapoptosis effects [15,16].
G q/11 is well known as a coupled G protein which can improve the cytoplasmic calcium released from endoplasmic reticulum and result in activation of pathways dependent on the calcium [5,8]. In addition, several studies have showed that GPR120 activates G q/11 signaling in various cell types [5,8,17]. The ligands of GPR120 activate the mitogenactivated protein kinase (MAPKA) pathway, leading to phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) [5]. Hence, both [Ca 2+ ]i and ERK1/2 activation are used as indexes to reflect the function of GPR120 [5,13,18].
Human GPR120 has two isoforms, a long (L) form and a short (S) form [19]. However, in mouse and rat, only 2 BioMed Research International one isoform of GPR120 is identified [11,20]. Most recent lines of evidence show that the hGPR120-L and hGPR120-S have different tissue distributions and pharmacological characteristics [16,21]. Compared with human and rodents, much less is known about the porcine GPR120 (pGPR120).
In database of mRNA sequence, pigs have 3 splice variants (NM 001204766, JQ437360, and JQ437361). However, there are no relative reports to characterize pGPR120. Recently, it has been reported that feeding n-3 PUFA-enriched diets could improve the intramuscular fat content and inhibit the expression of inflammation related genes, leading to increasing the mass of skeletal muscle of pigs [22,23]. However the detailed mechanism is largely unknown. In the current study, we presented the first evidence for tissue expression of splice variants of the pGPR120 and pharmacology of different pGPR120 isoforms. These results may provide a way to help understand the nutrichemical role of n-3 PUFA in pigs.

Materials and Methods
2.1. Cloning of pGPR120s into pcDNA3.1. The coding sequence (CDS) of pGPR120s was obtained by PCR amplification using mixed porcine complimentary DNA (cDNA) extracted from the spleen tissue of 3 Landrace barrows as templates.
The CDS of pGPR120 were inserted between restriction sites EcoRI (5 ) and XbaI (3 ) in pcDNA3.1 (+) (Clontech, Pato Alto, CA, USA) [21]. The primer sequences and optimal PCR annealing temperatures are listed in Table 1. The identities of all constructs were confirmed by sequencing.

Phylogenetic and Comparative
Analysis. The amino acids sequences of GPR120s from various species were retrieved from GenBank. Then the retrieved amino acids sequences and the deduced amino acid sequences from deposited porcine CDS sequences of pGPR120 were subjected to multiple sequence alignment using ClustalW program (http:// www.ebi.ac.uk/Tools/clustalw2/) using default settings. The MEGA 5.0 program was used to reconstruct the phylogenetic trees by the Neighbor-Joining (NJ) method (http://www .megasoftware.net/).

RNA Isolation, Semiquantitative RT-PCR, and Quantitative PCR (qPCR).
Total RNA was extracted from adipose, skeletal muscle, ileum, jejunum, duodenum, kidney, lung, spleen, liver, and heart of 6 Landrace barrows with Trizol (Invitrogen) following the manufacturer's instructions. Total RNA from 3D4/2, PK15, IPECJ2, ADSC, and DFAT, as well as differentiated ADSC and DFAT cells, was also isolated and extracted using Trizol reagent. The 2.5 g of RNA was reverse transcribed using First-Strand cDNA Synthesis Kit (TOYOBO, Japan) and cDNAs were stored at −20 ∘ C.
Total GPR120 expression in various tissues was determined by semiquantitative RT-PCR. The cross intron-exon boundaries primers (GPR120-407) were designed according to the common sequence of the pGPR120 transcripts as shown in Figure 3(a). The standard PCR program for amplification consisted of an initial denaturation step at 94 ∘ C for 4 min, followed by 35 cycles of 30 sec at 94 ∘ C, 30 sec at the indicated annealing temperature, min at 72 ∘ C ( = size in kilobases of amplified product), and a final elongation at 72 ∘ C for 5 min.
The PCR products were separated by 2% agarose gel electrophoresis (Sigma, St. Louis, MO, USA). After staining, net intensity of each band was scanned using GeneSnap system (Syngene, Cambridge, UK). The expression of pGPR120s was quantized after normalization of samples using intensity ofactin. To further check the tissue expression of total pGPR120 expression, we also determine the expression of total GPR120 in culture cells, which corresponded to pGPR120 highly expressed tissues by qPCR using a Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, Richmond, CA, USA). The PK15 and IPEC-J2 are cell lines isolated from the porcine kidney and small intestine, respectively. The 3D4/2 cell is the porcine macrophage, which is one of the common cell types in spleen. ADSC and DFAT cells are preadipocytes, whereas differentiated ADSC and DFAT cells represented mature adipocytes. Gene expression levels were calculated after normalization to the standard housekeeping gene -actin using the ΔΔCT method. In brief, the mean of the triplicate cycle thresholds (CT) of the pGPR120 was normalized to the mean of triplicate CT of the referenceactin using the calculation formula "2 CT -actin −CT pGPR120 ", which indicated a relative value as a fraction of pGPR120.
The relative expression levels of the 3 spliced variants in pGPR120 highly expressed tissues were also detected by semiquantitative RT-PCR. The primer pair (GPR120-Variants) designed for amplification of 3 spliced variants is shown in Figure 4(a). By using this primer pair, amplification will produce 3 different fragments from 3 spliced variants. The PCR products were separated by 2% agarose gel electrophoresis or 10% polyacrylamide gel electrophoresis and detected by using ethidium bromide (Sigma) to measure the levels of pGPR120s mRNA from various tissues and cell lines. All the primers sequences were listed in Table 1.

Luciferase Reporter Assays.
For luciferase reporter assays, the HEK293T cells were plated into 24-well plates. Then SRE-luc or NFAT-luc vector combined with Renilla luciferase expression plasmid (pTK) was cotransfected with expression vectors for various isoform of pGPR120 or pcDNA3.1 control plasmid. Then, 24 h after transfection, the cells were changed to the medium without FBS and maintained for at least 18 h for SRE-luc or NFAT-luc analysis. Then cells were treated with the appropriate ligand for 6 h. After treatment, cells were harvested and luciferase activity in cell extracts was determined using a luciferase assay system according to standard methods in a Dual-GLO reporter assay system (Progema, Madison, USA). Luciferase values were normalized by the Renilla values. Transfection experiments were performed in duplicate and repeated at least three times [25]. (Sigma) for 10 min. After treatment, cells were washed with PBS and lysed in lysis buffer (Beyotime, Shanghai, China). Lysed samples were centrifuged for 10 min at 10,000 rpm at 4 ∘ C, and the supernatant was subjected to SDS-PAGE and immunoblotting. Twenty mg of proteins/lane was separated on a 10% polyacrylamide and precast SDS gel (Bio-Rad) followed by transfer on polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 2 h with 5% skim milk powder (Sigma) and incubated overnight with the anti-phosphorylated ERK1/2 and antitubulin at a 1 : 2000 dilution. After 3 washes, the secondary antibody was added at a 1 : 10000 dilution and incubated at room temperature for 1 h. After 3 washes, the membrane was exposed by using WesternBrigh Peroxide (Advansta, California, USA) in imaging system (Carestream, New York, USA). The protein amount was normalized with the amount of tubulin as internal controls.

Statistical
Analyses. Data were presented as means ± SD and the difference between groups was determined by Student's -test.

Molecular Characterization and Evolution of Porcine
GPR120. The pGPR120 had 3 alternative transcripts, including a 1086 bp transcript (transcript 1), a 930 transcript (transcript 2), and a 960 bp transcript (transcript 3). The transcript 1 encoded wild type pGPR120 (pGPR120-WT) of 362 amino acids (aa), whereas transcript 2 and transcript 3 encoded 310aa and 320-aa isoforms of GPR120, respectively. The 310-aa isoform (encoded by pGPR120-V2) and the 320-aa isoform (encoded by pGPR120-V3) were the truncated isoforms, which did not contain fourth or fifth transmembrane domain, respectively (Figure 1(a)). Multiple sequence alignments and phylogenic trees comparisons of the CDS and deduced amino acid sequences of GPR120s from different species are shown in Figures 1 and 2. The pGPR120 shared higher homology (88% in CDS and 89.4% in amino acid sequence) with the human receptor sequences than they are to mouse and rat (85% and 85% in CDS; 88.4% and 87.0% in amino acid sequence), no matter for CDS or the amino acid sequence.

Transcriptional
Expression of Total pGPR120. The primer pair called GPR120-407 was used to detect the expression of total pGPR120 in various tissues as Figure 3(a) shows. Semiquantitative RT-PCR analysis showed that total pGPR120 was well detected in jejunum, duodenum, spleen, and adipose and negligibly expressed in other tissues (Figure 3(b)). To further check the expression of pGPR120 in the corresponding established cell lines related to the highly expressed tissues, we subsequently adopted a qPCR strategy to examine the expression of total pGPR120 in various cell lines and primary cultured cells. GPR120 was highly expressed in adipose tissue as well as adipogenic differentiated ADSC and DFAT at day 7. In addition, pGPR120 was also expressed in 3D4/2, IPECJ2, and PK15 (Figure 3(c)), which represent the porcine macrophage, kidney, and small intestine cells, respectively.

Transcriptional Expression of pGPR120 Spliced Variants.
To investigative the tissue expression of 3 spliced transcripts in the highly expressed tissues, we designed a primer called GPR120-Variant which can detect all of the transcripts and produce different length products for 3 spliced transcripts as Figure 4(a) shows. In Figures 4(b) and 4(c), the transcript coding pGPR120-WT was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. The pGPR120-V2 was found to be coexpressed in 3D4/2, IPECJ2, and PK15 and detected in ileum, jejunum, and duodenum, especially in spleen, but not in adipose. Another alternatively spliced transcript, pGPR120-V3, was only slightly expressed in spleen.

Signaling Properties of Isoforms Encoded by pGPR120 WT and V2.
To identify the molecular signaling of pGPR120s, pGPR120-WT and pGPR120-V2 expression plasmid were transiently transfected into HEK293T cells with the Ca 2+ or ERK1/2 reporter system [26]. As seen in Figure 5, under the treatment of the synthetic human and mouse GPR120 agonist TUG-891, signaling properties of pGPR120-WT and pGPR120-V2 encoded isoforms were detected as described in Section 2. The TUG-891 was used at the range of concentration from 10 −8 to 10 −4 M [27]. The pGPR120-WT showed well response to the TUG-891 (EC50 = 1.88 ± 0.52 M by NFAT reporter system and EC50 = 1.25 ± 0.36 M by SRE reporter system). Besides, the WT receptor also could be well activated by endogenous free fatty acids, including EPA, DHA, and ALA but not LA or PLA. However, the pGPR120-V2 encoded isoform remained inactivated either in the NFAT or in the SRE reporter systems.

Isoform Encoded by pGPR120-V2 Was Not a Dominant-
Negative Isoform of pGPR120. The alternative splicing can generate truncated GPCRs which may play a dominantnegative role in retarding the wild type receptor in the endoplasmic reticulum (ER) [28]. To explore whether pGPR120-V2 can act as a dominant-negative isoform of pGPR120-WT, we coexpressed pGPR120-V2 and pGPR120-WT at various ratios. The results showed that stimulation of TUG-891 significantly increased NFAT-Luc activity not only in cells cotransfected with pGPR120-WT and pcDNA3.1, but also in that cotransfected with pGPR120-WT and pGPR120-V2 ( Figure 6). The similar result was observed by using the SREdriven reporter system.

PUFAs Activates ERK1/2 Pathway via pGPR120-WT.
To further evaluate the effects of pGPR120-WT on ERK1/2 signaling by nutrients, pGPR120-WT expression plasmid or pcDNA3.1 control was transiently transfected into HEK293T cells. As Figure 7 shows, TUG-891 increased the phosphorylation level of ERK1/2 compared with the basal level, as well as various PUFAs. In Figure 7, the phosphorylation level of ERK1/2 activated by TUG-891 acted as a positive group and the basal group showed no response for activation of ERK1/2. Compared with TUG-891, n-3 PUFAs had the equivalent ability to increase phosphorylation of ERK1/2, whereas n-6 PUFAs (LA) had a lower ability to increase phosphorylation of ERK1/2. It was worth noting that though PLA increased the levels of ERK1/2 activity in cell transiently transfected with pGPR120-WT expression plasmid, PLA also showed the ability to induce the phosphorylation level of ERK1/2 in the cells transiently transfected with empty plasmid, in which other PUFAs were unable to induce the phosphorylation of ERK1/2.

Discussion
As a receptor of n-3 PUFA, GPR120 is involved in a wide range of biological events, including, but not limited to, antiinflammation and antiapoptosis in human and mouse [5,8].
In the current study, we cloned the pGPR120 and showed that pGPR120 had 3 transcripts. We showed that pGPR120-WT was well matched with human receptor sequence. In the present study, compared to the mouse and rat GPR120, the pGPR120-WT mRNA encodes a 362-aa protein that shared higher homology to the hGPR120. In addition, compared with human and mice, the similar expression patterns of pGPR120 in intestinal tissues maybe show the concordant functions such as inducing GLP-1 secretion [5,12,20] and inhibiting NF-B activity in intestinal cells [10]. The notable expressions of pGPR120 in 3D4/2 cells maybe imply the significant role of pGPR120 in macrophages such as antiinflammation in mouse [8]. However, pGPR120 showed the low expression in lung and the abundant expression in spleen while the hGPR120 and mGPR120 were expressed highly in lung and showed low expression level in spleen [5]. Like the hGPR120, the pGPR120 also had other alternatively spliced transcripts, which were called pGPR120-V2 and pGPR120-V3 in the current study. These 3 spliced transcripts have been published in database of mRNA sequence as NM 001204766, JQ437360, and JQ437361. However, in the current study, we showed that transcript 2, which corresponds to JQ437360, only had a slight expression level in spleen. Compared with the pGPR120-V3, the notable expression of pGPR120-V2 in the spleen as well as the porcine macrophage cell 3D4/2 was observed, naturally suggesting   that this alternative spliced isoform might act a role of inflammation in that tissue. Thus, the role of pGPR120-V2 in immunologic tissues and cells may be of great concern. The distinction between the same GPCRs from various species might cause the pharmacological variation [28]. Therefore, we study the pharmacology of 2 main isoforms of pGPR120s by assaying both [Ca 2+ ]i and ERK1/2 signaling. As expected, the specific agonist for human and mouse GPR120, TUG-891, can activate pGPR120-WT. However, the pGPR120-V2 almost showed negligible or no response to the small molecular agonist. It has been showed that, upon the stimulation of another human agonist GW9508, the hGPR120L also showed the poor response to the treatment while the hGPR120S can be well activated by this agonist [21]. As shown in the human GPR120 splice variants, the GPR120L inserts a splice in the key juxtamembrane between the TM domain 5 and IC3 regions, which leads to the less sensitivity of the longer receptors [21]. Compared with the pGPR120-WT, the 52 amino acids residues were deleted from the wild type amino acid sequence with the fourth transmembrane domains truncated, which may affect the normal functionality. Therefore, the truncated fragments of pGPR120, thus, suggest that the splice variant receptor may influence its receptor activity. Compared with results observed from other reports {Oh, 2010 #12}, the fold changes of the NFAT and SRE luciferase assay in the current study were lower [25]. This difference might be caused by the subtle alterations in sequence of GPR120 within different species. However, the EC50 values of treatments in the current study also range between the reliable scopes (1-10 M).
Alternative splicing acts as a main mechanism for regulating the expression of GPCR genes and allowing for the protein synthesis of truncated isoforms [28]. The truncated isoforms of some GPCRs play a pronounced role in associating with the detaining of the WT isoform in the endoplasmic reticulum (ER), which makes the attenuated expression of the WT isoform on the cell surface [28,29]. Thus, another goal of this research was to explore whether the pGPR120-V2 is dominant-negative isoform of pGPR120. However, in our research, results showed that pGPR120-V2 had no inhibitory effects on pGPR120-WT signaling. Hence, we suggested that pGPR120-V2 was not a dominant-negative isoform. Based on the tissues and cells distributions of pGPR120-V2, the truncated isoforms showed remarkable expression in 3D4/2 and PK15 cell lines. The high expression of pGPR120 maybe implies that pGPR120 functions in these tissues and cell lines; therefore, these cell lines could be models for further research, which we did not study in depth in the present studies. Moreover, whether alternative splicing of pGPR120 in spleen contributes to regulation of pGPR120-WT expression and thereby affects the pGPR120 signaling might be interested to study.
As previously mentioned, in human and mouse, activation of GPR120 under the stimulus of the PUFAs could stimulate the ERK1/2 phosphorylation signal transduction cascade [5,8,10,17]. Thus, GPR120 is named as the PUFA sensor and receptor [8]. In our studies, a series of n-3 (ALA, DHA, and EPA) and the agonist TUG-891 had similar ability in activation of ERK1/2. However, n-6 PUFA (LA) improved the phosphorylation of ERK1/2 at a lower level compared with n-3 PUFA and TUG-891, suggesting that the pGPR120-WT could also be activated by PUFAs, especially n-3 PUFA.
Other reports have showed that n-6 PUFAs increase ERK1/2 phosphorylation level and even improve the mRNA expression of GPR120 [10,30]. It is worth noting that both the n-3 and n-6 PUFAs increase ERK1/2 phosphorylation through a GPR120-dependent manner. However, PLA cannot activate either NFAT or SRE reporter gene but can increase ERK1/2 phosphorylation in cells transfected with GPR120 or control plasmid; therefore, PLA might activate ERK1/2 signaling in a GPR120-independent manner. These results match well with the results reported by Oh et al. [8]. In the study of Oh et al., they showed that the PLA increased phosphorylation level of ERK1/2 in GPR120-knockdown RAW264.7 cells and the PLA also showed no response to the SRE-luc activity in transiently transfected HEK293 cells.
Additionally, pGPR120-WT was also highly expressed in the adipose tissue and mature adipocyte, but not in the preadipocyte. This result matched well with the murine phenotype [12,13]. Knockdown of GPR120 by siRNA could reduce the expression of adipogenic marker genes and impair the triglyceride accumulation [12]. Consequently, it implies that GPR120 may be involved in porcine adipogenesis regulation.

Conclusion
In the current study, we cloned and characterized pGPR120 for the first time. We showed that pGPR120 was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose in pigs. Although pGPR120 has 3 spliced variants, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript of pGPR120. In addition, pGPR120 was also the only functional isoform, which was activated by synthetic agonist (TUG-891) and PUFAs, especially n-3 PUFA. Our study contributed to understanding the molecular regulation mechanisms of PUFA in pigs.