Avoid common mistakes on your manuscript.
Purinoceptors, receptors for nucleosides and nucleotides, have been identified on the plasma membranes of many cell types [1]. The early hints and recent evidence for localization of purinoceptors on intracellular sites, including lysosomes [2–4], mitochondria [5, 6] and in nuclei where they open ion channels and appear to influence mRNA activity [7, 8], offers up a whole new aspect of purinergic signalling.
Purinergic signalling, ATP acting as an extracellular signalling molecule, was proposed in 1972 [9]. Separate families of purinergic receptors were recognised, named P1 receptors for adenosine and P2 receptors for ATP and ADP [10]. Two subtypes of P2 receptors were shown in 1985, based on pharmacology [11] and in the early 1990s P1, P2X and P2Y receptor subtypes were cloned and characterised: four subtypes of P1 receptors (A1, A2A, A2B and A3), seven subtypes of P2X ion channel receptors (P2X1-7) and eight subtypes of P2Y G protein-coupled receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) [1, 12].
Cloning of receptors made it possible to generate polyclonal antisera for immunohistochemical studies of their expression and distribution [13]. Many papers using this technique were published [14–17]. As expected, the receptors were located on the plasma membranes of cells, but sometimes there was also intracellular immunostaining. This was often dismissed by referees as artefacts. However, later studies have revealed that intracellular localization of purinoceptors is genuine. For example, it was suggested that uptake of P2X1 receptors into smooth muscle cells of the rat vas deferens was responsible for desensitization ([18] and see [19]). P2Y2 receptor internalization via the clathrin-mediated pathway was observed in HEK293 cells using receptors tagged with green fluorescent protein (GFP), to be colocalized with endosomes and lysosomes [2]. GFP was also used to show internalization of P2Y1 receptors in HEK-293 cells [20, 21]. Ser352 and Ser354 in the carboxyl terminus of human P2Y1 receptors were shown to be needed for internalization in MDCK cells [22]. P2X3 receptors transfected into HEK-293 cells and expressed endogenously in dorsal root ganglion sensory neurons undergo rapid constitutive endocytosis, targeting the late endosomal/lysosomal system [23]. The role of internalised P2X7 receptors on lysosomes in macrophages in the killing of mycobacteria is discussed in a review [24]. A Rab5-dependent pathway was described for internalisation of P2X4 receptors in HEK-293 cells [25]. The internalised P2X4 receptors are located on lamellar bodies, lysosomes, vesicles and vacuoles in HEK-293 cells, hippocampal neurons and alveolar type II cells [3, 4]. P2X4 receptor channel activity was directly measured in intact lysosomes in HEK-239 cells [26]. Both ATP and P2X4 receptors were present in lysosomes and the lysosomal P2X4 receptors were activated by ATP at the luminal side in a pH-dependent manner. The lysosomal P2X4-mediated responses were potentiated by ivermectin, but were insensitive to suramin and PPADS, as for the plasma membrane P2X4 receptors. Mitochondrial calcium transport was shown to be regulated by P2Y1- and P2Y2-like mitochondrial receptors from rat liver cells [5]. ATP, acting on the nuclear envelope, was reported to open ion channels in both Xenopus oocytes [27, 28] and patch-clamped isolated mouse liver nuclei [29]. It was later reported that P2X7 receptors were expressed in the outer nuclear membranes of rat hippocampal inhibitory neurons [7]. P2X7 receptor immunoreactivity was also shown on the nuclear membrane of guinea pig visceral smooth muscle cells [30]. Studies of the expression of P2X-like receptors in amoeba showed that most of the immunostaining was on the membranes of intracellular vacuoles, required for osmoregulation, rather than on the plasma membrane [31–33], encouraging further investigations of intracellular localization of P2 receptors in mammals.
A recent paper showed immunostaining of P2X6 receptors within the nucleus of cultured hippocampal neurons [8]. It was shown that once inside the nucleus, the P2X6 receptor interacts with the splicing factor 3A1, which results in a reduction of the mRNA splicing activity, which is relevant in the ageing process.
These findings open up a whole new aspect of purinergic signalling and new studies will be of much interest.
References
Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492
Tulapurkar ME, Schäfer R, Hanck T, Flores RV, Weisman GA, González FA, Reiser G (2005) Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence. Cell Mol Life Sci 62:1388–1399
Qureshi OS, Paramasivam A, Yu JC, Murrell-Lagnado RD (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci 120:3838–3849
Xu J, Chai H, Ehinger K, Egan TM, Srinivasan R, Frick M, Khakh BS (2014) Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin. J Gen Physiol 144:81–104
Belous A, Wakata A, Knox CD, Nicoud IB, Pierce J, Anderson CD, Pinson CW, Chari RS (2004) Mitochondrial P2Y-Like receptors link cytosolic adenosine nucleotides to mitochondrial calcium uptake. J Cell Biochem 92:1062–1073
Belous AE, Jones CM, Wakata A, Knox CD, Nicoud IB, Pierce J, Chari RS (2006) Mitochondrial calcium transport is regulated by P2Y1- and P2Y2-like mitochondrial receptors. J Cell Biochem 99:1165–1174
Atkinson L, Milligan CJ, Buckley NJ, Deuchars J (2002) An ATP-gated ion channel at the cell nucleus. Nature 420:42
Díaz-Hernández JI, Sebastián-Serrano A, Gómez-Villafuertes R, Díaz-Hernández M, Miras-Portugal MT (2015) Age-related nuclear translocation of P2X6 subunit modifies splicing activity interacting with splicing factor 3A1. PLoS One 10:e0123121
Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509–581
Burnstock G (1978) A basis for distinguishing two types of purinergic receptor. In: Straub RW, Bolis L (eds) Cell membrane receptors for drugs and hormones: a multidisciplinary approach. Raven, New York, pp 107–118
Burnstock G, Kennedy C (1985) Is there a basis for distinguishing two types of P2-purinoceptor? Gen Pharmacol 16:433–440
Burnstock G (2007) Purine and pyrimidine receptors. Cell Mol Life Sci 64:1471–1483
Oglesby IB, Lachnit WG, Burnstock G, Ford APDW (1999) Subunit specificity of polyclonal antisera to the carboxy terminal regions of P2X receptors, P2X1 through P2X7. Drug Dev Res 47:189–195
Chan CM, Unwin RJ, Bardini M, Oglesby IB, Ford APDW, Townsend-Nicholson A, Burnstock G (1998) Localization of P2X1 purinoceptors by autoradiography and immunohistochemistry in rat kidneys. Am J Physiol 274:F799–F804
Llewellyn-Smith IJ, Burnstock G (1998) Ultrastructural localization of P2X3 receptors in rat sensory neurons. Neuroreport 9:2545–2550
Gröschel-Stewart U, Bardini M, Robson T, Burnstock G (1999) Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia. Cell Tissue Res 296:599–605
Gröschel-Stewart U, Bardini M, Robson T, Burnstock G (1999) P2X receptors in the rat duodenal villus. Cell Tissue Res 297:111–117
Ennion SJ, Evans RJ (2001) Agonist-stimulated internalisation of the ligand-gated ion channel P2X1 in rat vas deferens. FEBS Lett 489:154–158
Mueller A (2007) Internalization: what does it tell us about pharmacokinetic and pharmacodynamic properties of an antagonist? Br J Pharmacol 152:1145–1146
Tulapurkar ME, Zündorf G, Reiser G (2006) Internalization and desensitization of a green fluorescent protein-tagged P2Y1 nucleotide receptor are differently controlled by inhibition of calmodulin-dependent protein kinase II. J Neurochem 96:624–634
Reiner S, Ziegler N, Leon C, Lorenz K, von Hayn K, Gachet C, Lohse MJ, Hoffmann C (2009) β-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites. Mol Pharmacol 76:1162–1171
Qi AD, Houston-Cohen D, Naruszewicz I, Harden TK, Nicholas RA (2011) Ser352 and Ser354 in the carboxyl terminus of the human P2Y1 receptor are required for agonist-promoted phosphorylation and internalization in MDCK cells. Br J Pharmacol 162:1304–1313
Vacca F, Giustizieri M, Ciotti MT, Mercuri NB, Volonté C (2009) Rapid constitutive and ligand-activated endocytic trafficking of P2X3 receptor. J Neurochem 109:1031–1041
Qu Y, Dubyak GR (2009) P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways. Purinergic Signal 5:163–173
Stokes L (2013) Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin. Purinergic Signal 9:113–121
Huang P, Zou Y, Zhong XZ, Cao Q, Zhao K, Zhu MX, Murrell-Lagnado R, Dong XP (2014) P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. J Biol Chem 289:17658–17667
Mazzanti M, Innocenti B, Rigatelli M (1994) ATP-dependent ionic permeability on nuclear envelope in in situ nuclei of Xenopus oocytes. FASEB J 8:231–236
Shahin V, Danker T, Enss K, Ossig R, Oberleithner H (2001) Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes. FASEB J 15:1895–1901
Assandri R, Mazzanti M (1997) Ionic permeability on isolated mouse liver nuclei: influence of ATP and Ca2+. J Membr Biol 157:301–309
Menzies J, Paul A, Kennedy C (2003) P2X7 subunit-like immunoreactivity in the nucleus of visceral smooth muscle cells of the guinea pig. Auton Neurosci 106:103–109
Fountain SJ, Parkinson K, Young MT, Cao L, Thompson CR, North RA (2007) An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum. Nature 448:200–203
Ludlow MJ, Durai L, Ennion SJ (2009) Functional characterization of intracellular Dictyostelium discoideum P2X receptors. J Biol Chem 284:35227–35239
Parkinson K, Baines AE, Keller T, Gruenheit N, Bragg L, North RA, Thompson CR (2014) Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel. Nat Cell Biol 16:87–98
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Burnstock, G. Intracellular expression of purinoceptors. Purinergic Signalling 11, 275–276 (2015). https://doi.org/10.1007/s11302-015-9455-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11302-015-9455-6