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Early expression of a novel nucleotide receptor in the neural plate of Xenopus embryos.
Bogdanov YD
,
Dale L
,
King BF
,
Whittock N
,
Burnstock G
.
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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.
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 17ventral 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.
