The role of genetic variability in the interaction between Fe65 and the P2X 2

receptor

The adapter protein Fe65 has, in humans, two isoforms named like1 and Fe65-like2 (Fe65L1 and Fe65L2). Fe65L1 was isolated as an interactor of APP (Guenette et al., 1996), while Fe65L2 was isolated from a cDNA library based on sequence homology to its rat orthologue (Tanahashi and Tabira, 1999). These two proteins contain the same three protein-protein interaction domains and, like Fe65, interact with APP through the PTB2 domain. The rat orthologue of Fe65L2 was early isolated from a rat-brain cDNA library

(Duilio et al., 1998), but rat Fe65L1 has not been described so far. In our Y2H screening of a rat-brain cDNA library for proteins interacting with the C-terminus of P2X2, besides Fe65, we isolated a cDNA that showed 96 % homology with human Fe65L1. We believe that this cDNA represent the rat orthologue of this protein, named APBB2. We further performed a direct Y2H assay to corroborate its ability to interact with P2X2, and we found that this protein indeed binds the C-terminus of the receptor, in a similar fashion that the Fe65 protein.

Binding partners for the WW domain of Fe65L1 are not yet known, but a recent systematic wide screen approach has addressed binding preferences from the WW domains of Fe65 and the two isoforms Fe65L1 and Fe65L2, from human (Hu et al., 2004). All the three proteins share the key conserved residues in its WW domain, including the Tyr-Tyr-Trp sequence in the β2 sheet, thus they were expected to bind ligands with a proline-rich motif of the classes II and III. That study confirmed predilection for both Fe65 and Fe65L1 to the central sequence PPPP/K or PPPP/R, while Fe65L2 may bind to a more degenerate sequence G/PPPP/R. In this thesis work we found that the rat orthologue of Fe65L1 is able to bind the PPPP sequence at the C-terminus of P2X2, similarly to Fe65. In contrast to these two proteins, the rat orthologue of Fe65L2, although being highly expressed in brain, was not isolated in the Y2H screening, which in principle suggest that it is not able to bind to the poly-proline motif PPPP. Sequence comparison of this domain between Fe65 and Fe65L1 shows 75% conservation, while comparing to Fe65L2 shows only a 60% homology (Figure 5.2).

Figure 5.2. Sequence homology for the WW domains for the rat Fe65 family. Residues shared among Fe65 and its isoforms are highlighted in grey, while amino acids in light blue represent positions conserved between Fe65L1 and Fe65L2.

Notably, the sequence identity between rat Fe65 and Fe65L1 rises to 100% if we just consider the residues of the β2 and β3 strands of the WW domain, the ones directly involved in binding to the ligand, while comparison with Fe65L2 drops identity to 75%. This is very likely to explain the different binding behavior of the members of the family, and points once more towards the role that adjacent residues to key hydrophobic amino acids play on defining ligand predilection of WW domains. Our results are thus the first evidence for functional diversity for the WW domains of the Fe65 family of proteins.

Alternative splicing on the C-terminus of the P2X2 subunit mRNA originates a shorter variant of this protein named P2X2(b), that bears a 69-amino acid deletion. The loss of the region from residues Val371 to Gln439 is due to a 207 bp DNA fragment spliced out directly from the carboxyl-terminal exon 11 due to the presence of a criptic splice site (Brandle et al., 1997). This splice variant is expressed at high levels in neonatal rat brain, and its transcript is distributed in the central and peripheral nervous systems similarly to the P2X2 subunit (Simon et al., 1997). Activity of the receptor is not affected on the spliced variant, since ATP-evoked currents in P2X2(b) microinjected X. laevis oocytes weresimilar in size to those seen for the P2X2(a) receptor (Simon et al., 1997), but the P2X2(b) subunit shows significant differences in desensitization kinetics and steady-state currents in the continuous presence of ATP (Brandle et al., 1997; Simon et al., 1997). In addition, it was recently suggested that alternative splicing of mouse P2X2 receptor may regulate multimerization, since interactions between C-termini and between C- and N-termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X2(b) subunits (Koshimizu et al., 2006).

Interestingly, we found by means of Y2H and pull-down assays that the P2X2(b)

subunit is not able to interact with the adapter protein Fe65, which was anticipated due to the absence of the first proline rich domain (393PPPP396) at the C-terminus of this receptor. We

have discussed above how the first proline motif of P2X2CD was sufficient and necessary for the interaction with the WW domain of Fe65, and the behavior of the splice variant confirms once more our findings.

It was reported that ATP-activated currents mediated by the P2X2(b) receptor desensitized faster than P2X2-mediated currents, whereas the splice variant desensitized almost completely. When Ca⁺² uptake was evaluated, the P2X2(b) subunit did not mediate a steady Ca⁺² influx in the presence of ATP, which may reflect functional differences between cells expressing either of the two P2X2 isoforms (Brandle et al., 1997). This evidence points towards a potential role of the adapter protein Fe65 as regulator of desensitization kinetics of the P2X2 receptor, feature that still needs experimental confirmation.

We demonstrate here that alternative splicing of the P2X2 receptor is indeed and evolutionary mechanism by which neurons have impaired this interaction, while retaining some specific functionality features of the receptor. An analogous situation has been described for the NMDA receptor subunit NR1, where the alternative splicing of the C-terminal sequence C1 regulates the association with Yotiao and Neurofilament 1 (Sheng and Pak, 2000). Yotiao is an NR1-binding protein potentially involved in cytoskeletal attachment of NMDA receptors. Thus, alternative splicing of the NR1 subunit can act through splice-specific interactors such as yotiao to differentially localize or anchor distinct subsets of NMDA receptors. Differential subcellular localization by virtue of subunit-specific or splice variant-specific interactions could be exploited to create glutamatergic synapses with distinct pharmacological and physiological properties within the same neuron (Rubio and Wenthold, 1997).

5.4. Fe65 is present in hippocampal excitatory synapses where colocalize with P2X₂