Molecular characterization of free fatty acid receptors FFAR2 and FFAR3 in the domestic cat

Abstract G protein‐coupled receptors 41 and 43 were identified and characterized as free fatty acid receptors (FFAR) 3 and 2, respectively. FFAR2 and FFAR3 mediate short‐chain fatty acids (SCFAs) as signalling molecules. The present study aimed to give molecular characterization of FFAR2 and FFAR3 in the domestic cat. High homology with that in other mammals was revealed by cDNA cloning of cat FFAR2 FFAR3. We analyzed the tissue distribution of cat FFAR2 and FFAR3 mRNA using quantitative polymerase chain reaction. The inhibition of intracellular cAMP concentrations was observed in cells transfected with cat FFAR2 or FFAR3 and treated with SCFAs. The activation of nuclear factor of activated T cells‐luciferase reporter was only observed in cat FFAR2 transfected cells but not in FFAR3. Split luciferase assay (NanoLuc Binary Technology; NanoBiT) for FFAR2 or FFAR3 and Arrestin‐3/β‐arrestin‐2 revealed acetate‐/propionate‐induced recruitment to cat FFAR2 or FFAR3 in CHO‐K1 cells. Our results indicate that FFAR2 and FFAR3 are functional receptor proteins that are expressed in cat tissues and show differential distribution patterns.

. FFAR4 was also characterized as a long-chain fatty acid receptor that mediates gut glucagon like peptide-1 secretion in colonic intraepithelial neuroendocrine cells (Hirasawa et al., 2005).
Human GPR42 is considered a pseudogene, and it differs from GPR41/FFAR3 by only six amino acid residues (Brown et al., 2003).
Using quantitative polymerase chain reaction (qPCR), cat FFAR1 and FFAR4 were cloned and their cDNAs analysed for mRNA expression patterns (Habara et al., 2015). FFAR1 mRNA was found to be expressed in the duodenum, liver and pancreas, and high levels of FFAR4 mRNA were expressed in adipose tissue. In the cat, FFAR1 and FFAR4 mRNA are differentially controlled by mRNA expression mechanisms. The present study aimed to give molecular characterization to FFAR2 and FFAR3 in the domestic cat.

| cDNA cloning of cat FFAR2 and FFAR3
Cat tissue total RNA was purchased from Zyagen. This cat tissue was obtained from veterinary clinic and hospitals or certified animal tissue banks in USA. Tissues are freshly harvested under strict regulation by veterinarian during surgical operation or from animals donated for scientific research with the owner consent.

| Quantitative realtime PCR (Q-PCR) for cat FFAR2 and FFAR3
Total RNA was reverse-transcribed using the PrimeScript reverse transcription (RT) reagent kit with a gDNA Eraser kit (Takara). Genomic DNA was removed from the total RNA sample before cDNA synthesis.
The qPCR was performed as follows: predenaturation at 94°C for 2 min, 40 cycles of denaturation at 94°C for 10 s and annealing and extension at 60°C for 35 s. After the qPCR reaction, melting curve analysis was performed to check the specificity of the qPCR product. Quantitative analysis was conducted using a series of plasmid DNA dilutions. Expression levels of 18S ribosomal RNA were used as internal controls and measured by qPCR using primers for 18S-S (5′-GTAACCCGTTGAACCCCATT-3′) and 18S-A (5′-CCATCCAATCGGTAGTAGCG-3′).
Lowercase letters indicate the vector overlap regions for InFusion HD cloning into the EcoRI-NotI site of pcDNA3.1/V5-HisB (Invitrogen) using the InFusion HD cloning kit (Clontech Takara) and sequenced.

| GloSensor cAMP assay for cat FFAR2 and FFAR3
We monitored the changes to intracellular cAMP levels using a func-

| NFAT-luciferase reporter assay for cat FFAR2 and FFAR3
We performed nuclear factor of activated T cells (

| Tissue distribution of cat FFAR2 and FFAR3 mRNAs
To determine gene-and tissue-specific mRNA expression profiles of cat FFAR2 and FFAR3, we conducted qPCR analysis using cDNAs derived from adult cat tissues. High levels of FFAR2 mRNA expression were observed in bone marrow, colon and spleen, whereas high levels of FFAR3 mRNA expression were observed in the duodenum and lung (Figure 2).  (Table 1). Also, NFAT-luciferase activity was measured by acetate treatment because mammalian FFAR2 is known as promiscuous receptor that couples to both G αi/o and G αq . Acetate stimulated NFAT-luciferase reporter activity that transiently transfected cat FFAR2 with pEC50 value (6.141 ± 0.5932) but not FFAR3 (Figure 4).

| Desensitization of cat FFAR2 and FFAR3 by SCFAs
Ligands bind to GPCRs, which then mediate cell-signalling molecules such as cAMP, inositol phosphates and Ca 2+ (Srivastava, Gupta, Gupta, & Shukla, 2015). Because continuous cellular signalling by ligand is disadvantageous to cell physiology, GPCR desensitization is an essential process. To understand cat FFAR2 and FFAR3 desensitization, we analyzed the interactions between FFAR2 or FFAR3 and Arrestins 1-4 using a NanoBiT luciferase assay (data not shown).
Only Arrestin-3 and cat FFAR2 or FFAR3 demonstrated a specific interaction, because the luminescence level was more than 10-fold higher than that of FFAR2 or FFAR3 with HaloTag negative control. Arrestin-3 was classically identified as ß-Arerstin-2 previously.

TA B L E 1 Values of pEC50 for SCFA inhibition to cAMP accumulation in CHO-K1
FFAR2 is one of the most characteristic motifs of the rhodopsin GPCR, and it must be maintained in an inactive state (Fredriksson et al., 2003). An ERF motif, rather than an ERY/DRY motif, was conserved in all mammalian FFAR3, which appeared to have the same function, as the tyrosine residue mutations showed no or only a slight effect on receptor function (Rovati, Capra, & Neubig, 2007).
Different mRNA expression patterns were demonstrated in cat FFAR2 and FFAR3 in the examined tissues. We observed high levels of cat FFAR2 mRNA expression in immune tissues, such as bone marrow and spleen. Before the deorphanizing of FFAR2/GPR43, FFAR2 was considered as an immune signal transducer because of the expression in human leukocytes (Senga et al., 2003). FFAR2 was expressed in mouse bone marrow neutrophils as well, and it mediated neutrophil chemotaxis by SCFA (Vinolo et al., 2011). These reports may account for FFAR2's characteristic mRNA expression in cat bone marrow and spleen. In digestive organ and other tissues, FFAR2 and FFAR3 expression mechanisms were well characterized in some species. High levels of FFAR2 mRNA expression were observed in mouse stomach, colon, spleen, and adipose tissue, as well as human peripheral blood leukocytes and spleen (Hong et al., 2005;Nilsson, Kotarsky, Owman, & Olde, 2003). In chicken, FFAR2 mRNA was expressed in the testes, spleen, peripheral blood mononuclear cells, adipose tissue, duodenum, lung and liver (Meslin et al., 2015).
High levels of FFAR3 mRNA were expressed in the duodenum and lung in cats; kidney, colon and spleen in mice (Hong et al., 2005); and peripheral blood mononuclear cells, polymorphonuclear cells, lung, and adipose tissue in humans (Le Poul et al., 2003). FFAR2 and FFAR3 were also expressed in the rumen epithelium of young and adult cattle (Wang, Akers, & Jiang, 2012;Yang, Zhan, Ning, Jiang, & Zhao, 2020;Zhang et al., 2018). Sensing of SCFAs is an essential function for FFAR2 and FFAR3, and deficiency each of receptors result in chronic inflammation and obesity in mice (Ang et al., 2016;Kim, Kang, Park, Yanagisawa, & Kim, 2013;Yonezawa et al., 2013).
SCFAs produced from digestive organs are an indispensable energy source, and these must be sensed by FFAR2 or FFAR3 in ruminant and nonruminant animals. luminal content. However, butyrate strongly inhibited cAMP accumulation in FFAR3 as compared with FFAR2 ( Figure 3). Butyrate was reported to be a natural, strong ligand for both FFAR2 and FFAR3 in humans (Le Poul et al., 2003) and mouse (Nilsson et al., 2003), but butyrate may have a species-specific effect on FFAR2 because the inhibition of cAMP accumulation was not followed in a dose-dependent manner in cattle (Wang, Gu, Heid, Akers, & Jiang, 2009).
FFAR2 inhibits cAMP accumulation by SCFAs, while FFAR2 stimulates phospholipase C activity as a G αq receptor (Brown et al., 2003;Le Poul et al., 2003;Nilsson et al., 2003). Our results revealed the possibility that cat FFAR2 can act as G αi/o and G αq receptor for SCFA.
It may be suggested that FFAR2 and FFAR3 are classified as same SCFA biosensors, but it is clear that cats also differ in their ligands specificity and signalling pathway.
GPCR desensitization mechanisms are tightly regulated by GPCR kinase and arrestin. Following phosphorylation of GPCR by GPCR kinase, arrestin binds and mediates GPCR internalization to terminate G αi and G αq signal transductions by ligand (Peterson & Luttrell, 2017).
Arrestin is also known to work as a mediator as well as GPCR desensitization. For example, FFAR4/GPR120 and Arrestin 3 influence to anti-inflammation via transforming growth factor-ß activated kinase 1 (TAK1) and TAK1 binding protein (TAB1) (Oh et al., 2010). In FFARs, long-chain fatty acids dose-dependently promote the recruitment of Arrestin-2 and -3 to human FFAR1/GPR40 (Mancini et al., 2015;Qian et al., 2014). The recruitment of Arrestins has been confirmed under physiological conditions, chronic exercise-activated Arrestin-3 with FFAR4/GPR120, and decreased inflammatory responses in mice (Gaspar et al., 2019). In the present study, we examined the interaction between four types of cat arrestins with FFAR2 and FFAR3 using the NanoBiT assay, and our results revealed that both cat FFAR2 and FFAR3 interacted with Arrestin-3/β-arrestin-2.
Interestingly, not only did human FFAR2 and FFAR3 interact with Arrestin-3, but these receptors also formed a heterodimer in colon epithelial cells (Ang, Xiong, Wu, & Ding, 2018). In addition to G αi and G αq interaction, the heterodimer of FFAR2 or FFAR3 and Arrestin-3 mediated the ligand signal to p38 phosphorylation. It is unknown whether cat FFAR2 or FFAR3 and Arrestin-3 intervene in the signal transduction from SCFA in colon epithelial cells, but they may regulate some physiological events such as intestinal inflammation and metabolic regulation.

| CON CLUS IONS
In conclusion, we identified FFAR2 and FFAR3 in cats, which are characterized as G αi/o receptors and FFAR2 also act as G αq receptor.
We observed the highest mRNA expression of FFAR2 in the bone marrow and the highest mRNA expression of FFAR3 in the spleen.
The NanoBiT assay revealed that desensitization of cat FFAR2 and FFAR3 was induced by binding of Arrestin-3. FFAR2 and FFAR3 may play some roles in carnivorous cats, and it is suggested that both FFAR2 and FFAR3 are involved in lipid metabolism as SCFAs biosensors. for English-language review.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests related to this work.

AUTH O R CO NTR I B UTI O N
Koh Kawasumi: Formal analysis. Kozo Ohkusu-Tsukada: Formal analysis; Funding acquisition. Toshiro Arai: Project administration.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.356.