Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y13 receptor
Introduction
A wealth of compelling data supports a role for adenine (ATP, ADP) and uracyl (UTP, UDP) nucleotides as neurotransmitters/neuromodulators in diverse mammalian cells and tissues under both physiological and pathological conditions [1], [2]. Many of these effects are mediated by specific membrane G-protein-coupled P2Y receptors [3]. For example, ATP and UTP regulate salt and fluid transport through epithelia via the P2Y2, P2Y4 and P2Y6 receptors, which may hence represent potential targets for the therapy of cystic fibrosis and obstructive gut diseases [4], [5], [6], [7]. ADP activates platelet P2Y1 and P2Y12 receptors that play a crucial role in thrombus formation and stabilisation [8], [9]. ATP may also play a role in the immune system via the P2Y11 receptor [10]. The recent recognition of the UDP-glucose receptor (previously known as GPR105/KIAA0001 [11]) as the P2Y14 receptor [3] demonstrates that sugar-nucleotides may also serve as extracellular transmitters via the activation of specific members of this receptor family.
Eight distinct mammalian P2Y receptors (the P2Y1,2,4,6,11,12,13,14 subtypes) are currently recognised [3]. Their human orthologs and several (but not all) rodent orthologs have been reported [2]. Following the cloning of the rat and human P2Y12 receptor [12], it became clear that two phylogenetically- and structurally-distinct P2Y receptor subgroups can be identified [3], [10]. Group 1 encompasses specific adenine nucleotide-responding receptors (hP2Y1, hP2Y11), uracyl-nucleotides preferring receptors (hP2Y4, P2Y6), and receptors of mixed selectivity (P2Y2, rodent P2Y4) (ibidem). P2Y12, which is only distantly related to those subtypes, constitutes the first member of the second group. In 2001, Communi and coworkers identified human P2Y13 receptor (previously known as GPR86) as a new ADP receptor phylogenetically and structurally related to P2Y12[13]. The pharmacology of this second member of group 2 was also confirmed by Zhang and coworkers (who also identified its murine ortholog [14]), and its similarities with that of the hP2Y12 receptor have been further analysed recently [15]. P2Y14[3], [11] constitutes the third member of group 2. Receptors in the second group share the highest degree of sequence identity: for example, human P2Y12, P2Y13 and P2Y14 are 47–48% identical, with respect to a 29–46% identity of P2Y receptors in the first [3], [10]. P2Y12,13,14 also share several characteristic amino acid motifs in transmembrane (TM) 6 and 7 that are believed to play a role in binding to extracellular nucleotides. In particular, all cloned hP2Y receptors share a H-X-X-R/K motif in TM6 which is crucial for receptor activity [3]. In receptors of the first group (P2Y1,2,4,6,11), a Q/K-X-X-R motif in TM7 also seems to participate in ligand binding (ibidem). In human P2Y12,13,14 receptors, this motif is substituted with E-X-X-L, suggesting a different mode of agonist binding [3].
At present, very little is known on the pathophysiological roles of P2Y13 receptor. The abundance of human and mouse P2Y13 receptors is highest in brain and in spleen [13], [14], suggesting important roles in the nervous and immune systems. Model organisms such as rat and mouse provide powerful systems in which to study the function of novel proteins and may be also used to elucidate the biological roles of P2Y13 receptor. However, to make use of model systems, orthologous genes must be identified in these systems. While murine P2Y13 receptor has been cloned and characterised [14], no data regarding the expression and functional properties of rat P2Y13 receptor have been yet reported. The presence of a “putative” P2Y13 receptor in rat chromosome 2 has been very recently (October 23, 2003) predicted by automated computational analysis, by applying the gene prediction method GNOMON on a previously annotated genomic sequence. In the present study, by utilising specific RT–PCR primers external to the coding sequence of the putative open reading frame (ORF) of this genomic sequence, we have cloned this sequence from rat tissues, heterologously expressed it in 1321N1 cells, and showed that this rat protein is specifically activated by adenine nucleotides, as are the human and mouse P2Y13 receptors, although with some interesting pharmacological differences. In the present study, we also show that the tissue distribution of the newly-cloned rat protein receptor is similar (but not identical) to that previously reported for the human and mouse receptors [14], [15], with highest levels in spleen, liver, brain and kidney. In particular, rat P2Y13 receptor is expressed to significant levels in primary astrocytes from rat brain, where a yet-unidentified P2Y receptor mediating reactive astrogliosis had been previously reported [17], [18]. This is the first report demonstrating that the P2Y13 receptor is expressed and functional in rat tissues.
Section snippets
Identification of the putative rat ortholog of P2Y13 receptor in rat genome
The putative genomic sequence encoding for the rat P2Y13 receptor was identified by database querying (NBLAST, see also below) with the human P2Y13 sequence [13]. Database searching in HTGS revealed a similar rat sequence from clone CH230-189I10. Sequence determination of the rat clone confirmed an ORF consisting of 1011 nucleotides coding region (putative rat P2Y13 receptor) that encoded a full-length open reading frame (ORF) of 336 amino acids. This ORF was amplified by PCR from rat genomic
Cloning and sequence analysis
Interrogation of the rat genome database with the sequence of the human P2Y13 receptor revealed the presence of an highly homologous sequence in chromosome 2 WGS supercontig (GenBank accession no. NW_047625). This sequence contains a 1011 base open reading frame (ORF) encoding 336 aminoacid residues and is 79 and 87% identical to the human and mouse P2Y13 receptor, respectively. We designed specific oligonucleotide primers external to the putative ORF of this genomic sequence and utilised them
Discussion
In the present study, we report, for the first time, the cloning and pharmacological characterisation of the P2Y13 receptor from rat tissues. The newly-cloned rat receptor is a 336 amino acid protein with a 79 and 87% sequence identity to the human and mouse P2Y13 receptors, respectively, and is 23–49% identical to the other six cloned rat P2Y receptors. As also found for human [3] and mouse P2Y receptors, the newly cloned rat receptor shows highest identity with rP2Y12 and rP2Y14 (49 and 43%,
Acknowledgements
Authors are grateful to Prof. Marco Cattaneo, University of Milan, Italy and to Dr. Kenneth A. Jacobson, NIH, Bethesda, USA, for useful discussion, and to Dr. Gary Peters, Astra-Zeneca for kindly providing AR-C69931MX. This work was partially supported by the Italian Ministry of Education, University and Research (research programs of national interest, MURST-PRIN 2001) “Purinoceptors and neuroprotection” No. 2001052834 to Prof. Flaminio Cattabeni, and by F.I.RB. project 2001 “Ischemia
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These authors equally contributed to this work.