Research report
Evidence for alternative splicing of ecto-ATPase associated with termination of purinergic transmission

https://doi.org/10.1016/S0169-328X(99)00244-2Get rights and content

Abstract

Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPaseA; GenBank accession #Y11835). The splice variant (rEATPaseB; GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPaseB contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPaseA. Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.

Introduction

Extracellular purinergic signaling has a pivotal role in the regulation of many functions in vertebrate and invertebrate tissues 1, 3. In the central and peripheral nervous systems, ATP acts as a co-transmitter and neuromodulator in many major nerve types [5]. Signaling pathways for ATP and other nucleotides (ADP, UTP) are provided by a variety of ATP-gated ion channels (P2X receptors) and G protein-coupled nucleotide receptors (P2Y receptors). The concept of purinergic neurotransmission is also supported by evidence for neuronal synthesis, storage and release of ATP, inactivation of released ATP by ectonucleotidases, and purine reuptake via nucleoside transporters [4].

The principal ectonucleotidase pathway for termination of purinergic signaling is characterized by sequential hydrolysis of ATP to adenosine by ecto-ATPase (ATP hydrolysis), ecto-ATPDase (ATP and ADP hydrolysis) and ecto-5′-nucleotidase (AMP hydrolysis) [35]. Ecto-ATPase and ecto-ATPDase (ATP diphosphohydrolase, ecto-apyrase, mammalian CD39) fall within the E-type ATPase family, and provide the principal pathway for inactivation of released ATP [21]. These enzymes are clearly distinguished from intracellular ATPases by their extracellular active site, strict dependence on divalent cations (Ca2+ and Mg2+) for activity, insensitivity to inhibitors of F-, P-, and V-type ATPases and ability to hydrolyze a wide range of purine and pyrimidine nucleoside tri- and diphosphates [21]. E-type ATPases have been described in both membrane-bound [21]and soluble form 9, 25, 29including being co-released with neurotransmitter [29]. Ecto-ATPase has a very high preference for ATP over ADP, whereas ecto-ATPDase degrades both substrates similarly [21].

E-type ATPases share considerable sequence homology 6, 18, 24, 26. Membrane-bound ecto-ATPase and ecto-ATPDase are integral membrane proteins with two transmembrane domains and short cytoplasmic peptides at the amino- and carboxy-terminal ends. However, soluble ATPases do not appear to have a transmembrane domain at the C-terminus 7, 12, and express a large extracellular C-terminal end instead. For ecto-ATPases and ecto-ATPDases, the majority of the protein is extracellular, and contains N-glycosylation sites, cysteine residues (putative extracellular disulfide bonds) and four apyrase conserved regions presumably required for enzymatic activity [9].

Based on the criteria generally regarded as necessary for establishing ATP as a transmitter molecule [4], there is emerging evidence that ATP may play a key role in auditory sensory processing. ATP storage [34]and release [33]have been demonstrated in the cochlear tissues, and functional studies have provided clear evidence for an action of extracellular ATP via P2 receptors 14, 15, 16, 28. Ionotropic P2X and metabotropic P2Y receptors have been found in sensory, supporting and secretory cells of the cochlea, including the spiral ganglion neurones [10]. In addition, E-type ATPases have been identified biochemically as a mechanism for terminating purinergic signaling in the cochlea 30, 31, as previously reported for both neuronal and glial signaling in the central and autonomic nervous systems 17, 32, 36.

The present study provides molecular characterization of ecto-ATPase and ecto-ATPDase mRNA in rat cochlea, brain and other tissues. We identify a novel ecto-ATPase isoform (rEATPaseB) arising from alternative splicing which is co-expressed with rEATPaseA in a wide range of tissues and has internal regulatory sites which likely provide differential regulation of ATP signal levels.

Section snippets

Animals

Cochleae were obtained from adult Wistar rats euthanized using pentobarbitone (120 mg/kg). All procedures in this study were in accordance with the guidelines approved by the University of Auckland Animal Ethics Committee.

PCR screening, cloning and sequencing of ecto-ATPase and ecto-ATPDase in the rat cochlea

Polyadenylated RNA was isolated from all regions of the rat cochlea (n=10) using a guanidium thiocyanate-based extraction procedure followed by polyadenylation selection using a cellulose oligo(dT) matrix (QuickPrep® Micro mRNA purification kit, Pharmacia Biotech).

Ecto-ATPase mRNA expression in rat cochlea

Four PCR products of different sizes (593, 550, 486 and 400 bp, respectively) were detected in the initial PCR experiments (first ecto-ATPase primer set; Fig. 1). Sequence analysis of the cloned PCR products showed identity of clone B1 (593 bp) with rat brain ecto-ATPase (GenBank accession #Y11835) [12]. The 400 bp PCR product (clone B4) was found to be a novel isoform of the ecto-ATPase, the first direct evidence of alternative splicing in ecto-ATPase gene family. We designate the isoform

Discussion

This study presents the first direct evidence of alternative splicing in the ecto-ATPase gene family. Evidence for ecto-ATPase and ecto-ATPDase expression in cochlear tissues has been provided using RT-PCR. Two isoforms of ecto-ATPase mRNA and a single ecto-ATPDase mRNA were expressed in the cochlea. Both ecto-ATPase isoforms (rEATPaseA and rEATPaseB) were highly expressed in rat brain and other tissues. The novel isoform (rEATPaseB) contains a long cytoplasmic C-terminal tail with different

Acknowledgements

This study was supported by the Health Research Council of New Zealand, the Deafness Research Foundation of New Zealand, the Auckland Medical Research Foundation and the Lottery Grants Board. We thank Dr. David Muñoz for technical assistance in preparation of Fig. 5 and Dr. Leif Jarlebark for his helpful comments on the manuscript.

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