Abstract
Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X(2) subunit C-terminal domain as bait we isolated the beta-amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X(2) C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X(2) splice variant P2X(2(b)), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X(2) subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X(2) and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X(2) receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X(2) receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.
Highlights
Second, P2X2, P2X4, and P2X7 receptors become increasingly permeable to organic cations (e.g. N-methyl-D-glucamine) and fluorescent dyes during prolonged or repeated exposure to extracellular ATP (4 – 6)
The most abundant cDNA isolated was represented by 29 clones (5 independent cDNAs; Fig. 1C) with overlapping sequences identical to the adapter protein Fe65 from the rat [31] (Fig. 1C)
We have identified the adaptor protein Fe65 as a native binding partner of the P2X2 receptor subunit by Y2H screening of a rat brain cDNA library
Summary
P2X2 Constructs—Yeast two-hybrid (Y2H) screening of the cDNA sequence corresponding to the full cytoplasmic domain of P2X2 comprising amino acids 355– 472 (P2X2CD) was amplified by PCR using full-length P2X2 cDNA The sequence coding for the P2X2 splice variant P2X2(b) cytoplasmic domain comprising amino acids 355– 403 (P2X2(b)CD) was amplified by PCR using the P2X2(b) cDNA For recombinant protein expression the sequences coding for the P2X2CD and P2X2(b)CD were amplified by PCR with oligonucleotides containing BamHI and NotI sites, the digested PCR products were cloned in-frame with gluthatione S-transferase (GST; pGEX-4T-1 vector; Amersham Biosciences) or thioredoxin (Trx; pET-32a(ϩ); Novagen). For recombinant protein expression the sequence coding for amino acids 218 – 479, 197–255, and 40 –100 were amplified by PCR and cloned in-frame both into pGEX-4T-1 and pET32a(ϩ) vectors employing the same strategy as with the P2X2CD. Detection was performed by species-specific horseradish peroxidase-labeled secondary antibodies (Bio-Rad), and visualized using ECL detection kit (Amersham Biosciences)
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