XB-ART-16506J Biol Chem May 9, 1997; 272 (19): 12583-90.
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Early expression of a novel nucleotide receptor in the neural plate of Xenopus embryos.
Extracellular ATP functions as a neurotransmitter and neuromodulator in the adult nervous system, and a signaling molecule in non-neural tissue, acting either via ligand-gated ion channels (P2X) or G-protein-coupled receptors (P2Y). ATP can cause an increase in intracellular Ca2+ (Ca2+i) in embryonic cells and so regulate cell proliferation, migration, and differentiation. We have isolated a Xenopus cDNA encoding a novel P2Y receptor, XlP2Y, which is expressed abundantly in developing embryos. Recombinant XlP2Y responds equally to all five naturally occurring nucleoside triphosphates (ATP, UTP, CTP, GTP, and ITP), which elicit a biphasic Ca2+-dependent Cl- current (ICl,Ca) where the second phase persists for up to 60 min. XlP2Y also causes a continuous release of Ca2+i and a low level persistent activation of ICl,Ca in Xenopus oocytes through the spontaneous efflux of ATP. mRNAs for XlP2Y are expressed transiently in the neural plate and tailbud during Xenopus development, coincident with neurogenesis. This restricted pattern of expression and novel pharmacological features confer unique properties to XlP2Y, which may play a key role in the early development of neural tissue.
PubMed ID: 9139711
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: h4c4 p2ry1 p2ry4 p2ry6
Article Images: [+] show captions
|FIG. 1. Nucleotide and deduced amino acid sequence of XlP2Y. The best fit Kozak sequence for initiation of translation is indicated in bold, but the figure also includes 5 amino acids encoded from an upstream ATG. An inframe stop codon 37 nucleotides upstream of the putative initiating ATG is underlined, as are the seven putative transmembrane domains (solid bars). Boxes indicate the four extracellular cysteines (thin lines) thought to be involved in interchain disulfide bond formation and the single intracellular cysteine (bold lines) that may be a site for palmitoylation. Circles indicate the potential phosphorylation sites.|
|FIG. 2. SDS-PAGE analysis of XlP2Y translation product. XlP2Y was translated in the TNT-T3-coupled reticulocyte lysate system and analyzed on a 10% SDS-polyacrylamide gel. XlP2Y gave a translation product of approximately 56 3 103, which is close to the 61 3 103 predicted from its amino acid sequence. Transcription and translation of a luciferase DNA gave a product of approximately 61 3 103, and water gave no specific translation product.|
|FIG. 3. Alignment of the amino acid sequence of XlP2Y with P2Y1–7. The alignment was made using CLUSTAL W; only sequences between the highly conserved NH2-terminal cysteine and the end of TM VII were included in the analysis (amino acid numbers are indicated at the end of the alignment). Proteins aligned to XlP2Y are chick P2Y1 (X73268), human P2Y2 (U07225), chick p2y3 (X98283), human P2Y4 (X91852), chick p2y5 (L06109), human P2Y6 (X97058), and human P2Y7 (U41070). Gaps (-) were introduced to maximize the alignment, and only non-conserved residues are indicated for P2Y1–7. The 26 absolutely conserved amino acids are indicated (*), as are the four positively charged amino acids reported to play a role in P2Y2 receptor activation by ATP and UTP (@) and the seven putative transmembrane domains (bars) of XlP2Y. Note the highly conserved sequence in TM III (SILFLTCIS) and the strong homology between XlP2Y and the UTP receptors P2Y2 and P2Y4 in TM VII (YKVTRPLASANSC(I/L)DP(I/V)LY).|
|FIG. 4. Northern blot analysis of XlP2Y expression in Xenopus embryos. A, Northern blot of total RNA isolated from staged Xenopus embryos (27), demonstrating that XlP2Y transcripts are predominantly expressed during neurula stages. Stages: 1, fertilized egg; 6, 32 cells; 8, mid blastula; 10, early gastrula; 13, early neurula; 18, neurula; 21, neural tube closed; 25, early tailbud; 27, tailbud; 33, tailbud; 42, tadpole. B, Northern blot of total RNA isolated from timed embryos demonstrating that XlP2Y transcripts are first detected 7 h (stage 12) after the onset of gastrulation (0 hours, stage 10). C, Northern blot of total RNA showing that XlP2Y is predominantly expressed in dorsal tissues. 17A, stage 17 dorsal-anterior tissues; 17P, stage 17 dorsal-posterior tissues; 17V, stage 17 ventral tissues; Exo, stage 17 exogastrulae; Con, stage 17 controls; UV, stage 17 UV-irradiated embryos. Exogastrulae, UV-irradiated, and control embryos were obtained from the same batch of embryos. Note low levels of expression in ventral tissues. All blots were probed with histone H4 as a loading control.|
|FIG. 5. Spatial expression of XlP2Y in Xenopus embryos. Whole mount in situ hybridization of staged Xenopus embryos showing expression of XlP2Y in the neural plate and tailbud. A, stage 14. B, stage 17. C, stage 28 tailbud. Ant, anterior; Post, posterior; NP, neural plate.|
|FIG. 6. Electrophysiological properties of recombinant XlP2Y. A, membrane currents (Vh 5 240 mV) evoked by ATP (10–1000 nM, for 180 s) and recorded from a defolliculated oocyte injected with XlP2Y transcript. B, concentration-response curves for fast (M) and slow (L) components of biphasic currents evoked by ATP (10 nM to 100 mM) in defolliculated oocytes. The amplitude of evoked currents were normalized to responses evoked by ATP (1 mM). Each data point is the mean of three observations. C, membrane currents showing a time- and voltage-dependent increase in conductivity (IX) in a defolliculated oocyte injected with XlP2Y transcript. The amplitude of IX was inhibited when suramin (10–100 mM) was present in the superfusate. D, the I/V relationship of IX in the absence (M) and presence (10 mM, ; 30 mM, L; 100 mM, E) of the P2 antagonist, suramin.|
|p2ry4 (pyrimidinergic receptor P2Y, G-protein coupled, 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior down.|