Veterinarni Medicina, 62, 2017 (08): 443–455 Original Paper doi: 10.17221/23/2017-VETMED Characterisation, localisation and expression of porcine TACR1, TACR2 and TACR3 genes A. Jakimiuk*, P. Podlasz, M. Chmielewska-Krzesinska, K. Wasowicz Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland *Corresponding author: [email protected] ABSTRACT: Substance P is involved in many physiological and pathophysiological processes. This functional diversity is mediated by three neurokinin receptor subtypes (NK1R, NK2R and NK3R) encoded by the TACR1, TACR2 and TACR3 genes, respectively. Despite the increasing interest in using pigs (Sus scrofa) to study human disease mechanisms, the sequences of these receptors are still unconfirmed or in the case of the NK1 receptor, not yet even unpredicted. We employed in silico analysis to define the localisation of the porcine tachykinin receptor genes, and to predict the structures and amino acid sequences of the respective proteins. A reverse transcription polymerase chain reaction (RT-PCR) assay was performed to analyse the expression of tachykinin receptor genes in different porcine tissues. The data show that the TACR1 gene is located on chromosome 3, TACR2 on chromo- some 14 and TACR3 on chromosome 8. All three genes encode proteins with structures that incorporate features of G-protein-coupled receptors with sizes of 407, 381 and 464 amino acids, respectively. The receptors display a high degree of similarity to other mammalian neurokinin receptors. The NK1R subtype is expressed in both the central nervous system and peripheral tissues, while NK2R expression seems to be localised mostly to peripheral tissues. The expression of NK3R is found mainly in the central nervous system. This report provides for the first time the results of a comprehensive analysis of the structure and distribution of porcine NK1R, as well as other porcine neurokinin receptors and their genes. We hope that our data may offer an invaluable foundation for the future studies on the function of diverse tachykinin peptides in the central nervous system and peripheral tissues. Keywords: NK1 receptor; NK2 receptor; NK3 receptor; pig; substance P; tachykinin receptors Tachykinins are a family of closely related peptides Substance P has been implicated in various be- that share a common C-terminal sequence, Phe-X- havioural responses and in the regulation of neu- Gly-Leu-Met-NH. They are widely distributed in ronal survival and degeneration (Hasenohrl et al. both the central nervous system and peripheral tis- 2000). It has been shown to be involved in a variety sues, and function as neurotransmitters and neuro- of neurodegenerative disorders such as Alzheimer’s modulators (Otsuka and Yoshioka 1993). For many (Sakurada et al. 1990), Parkinson’s (Halliday et al. years, the most well-known members of the tachy- 1990), and Huntington’s disease (Augood et al. kinin family were substance P, neurokinin A (NKA) 1996). Substance P also regulates cardiovascu- and neurokinin B (NKB). However, in 2000, a fourth lar (Hoover et al. 2000) and respiratory function mammalian tachykinin (hemokinin-1) was found in and is involved in activation of the emetic reflex the mouse. This discovery has shed light on a com- (Diemunsch and Grelot 2000). It participates in the pletely new and species-divergent group of novel neurotransmission of pain by mediating the process tachykinins (the endokinins) and more unexpect- of nociceptive transmission (Zubrzycka and Janecka edly, tachykinin gene-related peptides (Page 2005). 2000), stimulates haematopoiesis (Rameshwar et al. Supported by the KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal-Safe Food”, Ministry of Science and Higher Education, Poland (Decision No. 05-1/KNOW2/2015). 443 Original Paper Veterinarni Medicina, 62, 2017 (08): 443–455 doi: 10.17221/23/2017-VETMED 1993), motility and secretion in the gastrointestinal of porcine neurokinin receptors remain largely un- tract (Improta and Broccardo 2006). At the level known. In this paper, we report the distribution of of the genitourinary tract, it is involved in smooth neurokinin receptors in porcine tissues using the muscle contraction, and modulates release of in- RT-PCR technique. Using in silico analysis, we have flammatory mediators as well as steroid secretion designed specific primers for PCR, identified the by the gonads (Candenas et al. 2005). sequence of genomic DNA encoding the NK1 re- These disparate activities of substance P are me- ceptor as well as predicted the mRNA sequence for diated by three tachykinin receptor subtypes also NK2 and NK3 receptors. Finally, we have defined the known as neurokinin receptors (NK1R, NK2R and amino acid sequence of the neurokinin receptors. NK3R), which all belong to the G protein-coupled receptor (GPCR) superfamily and are encoded by the TACR1, TACR2 and TACR3 genes, respective- MatERIAL AND METHODS ly. These receptors are glycoproteins with seven α-helical transmembrane segments, an extracel- The study was performed on female pigs (Sus lular amino-terminus and an intracellular carboxyl scrofa) of the Polish Large White breed weighing tail (Pin and Bockaert 1999). The conserved car- approximately 30 kg. The experiment was accepted boxyl terminal domain of tachykinins interacts with by the Local Ethical Commission of the University the tachykinin receptors, while the unique amino of Warmia and Mazury in Olsztyn, Poland (affili- terminal sequence dictates receptor specificity. ated to the National Ethics Commission for Animal Each tachykinin appears to preferentially activate Experimentation, Polish Ministry of Science and a specific tachykinin receptor, although at high li- Higher Education). Animals were deeply anaesthe- gand concentration, each tachykinin can activate tised with pentobarbital (Vetbutal, Biowet, Poland; each of the tachykinin receptors. Substance P has 30 mg/kg body weight i.v.). Sixteen different tissues a high affinity for NK1R, whereas NKA and NKB including skeletal muscle, stomach wall, spleen, kid- have been observed to bind preferentially to NK2R ney, adrenal glands, hippocampus, skin, lung, heart, and NK3R, respectively (Nakanishi 1991). spinal cord, hypothalamus, liver, pituitary gland, thy- Nowadays, pigs are increasingly being used as mus, ovary and jejunum were collected and placed laboratory animals, because of their close ana- into RNALater (Ambion, USA) overnight at 5 °C and tomical and physiological similarity to humans. As then frozen at –20 °C. Total RNA was isolated using a result, they are often used as models to study a the Total RNA Mini Kit (A&A Biotechnology, Poland) variety of disease mechanisms and different patho- according to the manufacturer’s instructions. physiological conditions in humans, such as obe- Primer design. All sequences required for de- sity and gastrointestinal dysfunctions (Spurlock signing specific primers were obtained from the and Gabler 2008; Pidsudko et al. 2011; Zhang et GenBank database. For this purpose, the Primer- al. 2013). Substance P was reported to be localised BLAST tool was used (http://www.ncbi.nlm.nih. in the porcine central nervous system (Barnes et gov/). The predicted nucleotide sequence of the al. 1993; Holm et al. 1993) and in the nerve fibres porcine NK1 receptor was based on Sus scrofa innervating many porcine organs (Schmidt et al. breed Ellegaard Gottingen minipig Contig39424, 1991; Happola et al. 1993; Czaja et al. 2001; Lamb whole genome shotgun sequence (GenBank ac- and Sparrow 2002; Podlasz et al. 2003; Pidsudko cession No. AOCR01039423.1) in alignment with et al. 2008; Podlasz and Wasowicz 2008; Kaleczyc the predicted Bos taurus tachykinin receptor 1 et al. 2010). In the pig, substance P can stimulate (TACR1) mRNA sequence (GenBank accession both endocrine and exocrine pancreatic functions No. XM_002691188.1). through NK-1 receptors (Schmidt et al. 2000). It also Primers for NK2 and NK3 receptors were de- has a modulatory effect on ovarian function (Pitzel signed using predicted Sus scrofa tachykinin re- et al. 1991). Pigs can be useful models for the study ceptor 2 (TACR2) mRNA sequence (GenBank of human cystic fibrosis as it has been shown that accession No. XM_001929073.5) and predicted Sus substance P induces liquid secretion from bronchial scrofa tachykinin receptor 3 (TACR3) mRNA se- submucosal glands of pigs (Trout et al. 2001). quence (GenBank accession No. XM_003482466.2). Despite the important physiological roles sub- Tissue distribution of NK1R, NK2R and NK3R stance P plays in pigs, the identities and functions transcripts. RT-PCR assays were performed to de- 444 Veterinarni Medicina, 62, 2017 (08): 443–455 Original Paper doi: 10.17221/23/2017-VETMED tect the expression of neurokinin receptor genes in human and mouse using the Clustal Omega online different porcine tissues. Based on the coding se- programme. The putative transmembrane domains quences obtained, gene-specific primers (Table 1) were predicted using the TMHMM 2.0 online pro- for NK1R, NK2R, NK3R and GAPDH (AF017079; tein topology prediction tool. Phylogenetic analysis served as an internal control) were designed to am- was computed using the MEGA6 program, in which plify cDNA fragments of 404, 393, 298 and 320 bp, the phylogenetic tree was constructed using the respectively. Reverse transcription (RT) was per- Neighbor-Joining method. formed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR for 10 min at 25 °C fol- lowed by 15 min at 50 °C and terminated