Identification of a traﬀicking motif involved in the stabilization and polarization of P2X receptors.

Extracellular ATP-gated channels (P2X receptors) define a third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, endocrine and exocrine glands. P2X subunits have two membrane-spanning domains and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic carboxy termini of P2X subunits that is necessary for their surface expression: mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD4) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X 2 subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.


Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors.
Séverine Chaumont-Dubel, Lin-Hua Jiang, Aubin Penna, R Alan North, A.

Baranski
To cite this version: P2X receptors are extracellular ATP-gated ion channels; they define a third major class of ligand-gated channels together with the glutamate and the nicotinic super-families (1). P2X receptors are involved in synaptic transmission in central and peripheral nervous system; but, in contrast to other ligand-gated channels, they are also widely expressed in peripheral tissues where they participate in physiological processes as diverse as smooth muscle contraction, secretion, and bone resorption.
ATP induced currents have been recorded from a large number of different cell types (2) but the molecular identity of P2X receptors responsible for these currents often remains elusive because of limitations of pharmacological tools to discriminate the different subtypes of P2X channels.
Seven P2X subunits genes (P2X 1 -P2X 7 ) are found in the human genome; this number seems to be an accurate estimation of the extent of the P2X family in mammalian species, although in zebrafish two additional subunits appear to exist (2,3). The basic structural determinants of P2X channels been established by numerous molecular studies (4). P2X subunits have a membrane topology with two transmembrane domains linked by a large extracellular loop, N-and C-termini are localized intracellularly (5,6). To form a channel P2X subunits are thought to associate as a homo-or hetero-oligomers, composed of three subunits (7,8) organized in a head-to-tail orientation around a central pore (9). This model is consistent with studies which show that the permeation pathways is associated with transmembrane regions (10)(11)(12), and with the fact that the gating of the channel is in part due to movements of the subunits relative to the each other (12). Conserved charged residues in the extracellular loop have been implicated in ATP binding (13,14), and desensitization is tightly linked to transmembrane domains and intracellular regions localized immediately beneath the plasma membrane (15,16).
Signals that regulate intracellular trafficking of P2X receptors are not well understood.
In P2X 4 subunits, an atypical endocytosis motif responsible for the rapid recycling of the receptor has been identified in the carboxy terminus of the channel (17,18); however, the existence of subcellular trafficking in other P2X subunits signals has not been reported. Yet evidence suggests that additional trafficking signals are present in P2X subunits. For example, transiently expressed P2X 6 subunits are not properly addressed to the cell surface presumably because of a glycosylation defect (19), and the presence of a retention signal in the rat P2X 5 subunit has been suggested to explain its poor functional expression (20). Different mutations that alter surface expression of P2X subunits have also been reported. Disrupting some of the conserved disulfide bridge of the extracellular loop of P2X 1 subunits alters their normal trafficking to the cell surface (21); similarly, the deletion of the three glycosylated asparagines of the P2X 2 subunits induces an intracellular retention and a complete loss of function of the receptors (5,22). However, these mutations are likely to alter the folding of the protein, and the trafficking defects might rather be due a failure to pass the quality check test that normally take place in the endoplasmic reticulum than to a trafficking defect per se. In the human P2X 7 receptor a dibasic amino acid motif (23) and a polymorphism (Ile 568 to Asn) (24) both located in the Cterminal tail of the protein have been shown to be necessary for the proper trafficking of the channel. No mechanistic explanations have been put forward to explain these trafficking defects.
In the present study we have identified a motif in the C-terminal tail (C-Term) 1 of P2X subunits that is conserved in almost all subunits known to date. Using a chemiluminescence assay (25,26) to measure the cell surface expression of P2X subunits we show that in HEK cells and in neurons this motif is involved in the stabilization at the plasma membrane of all P2X subunits tested (P2X 2 , P2X 3 , P2X 4 , P2X 5 and P2X 6 ). We also provide evidence that this motif might be important for the polarized expression of P2X 2 receptors in neurons.

EXPERIMENTAL PROCEDURES
Insertions of extracellular tags on P2X receptors --Tags were introduced by overlapping PCR as described previously (8)  Mutagenesis --Mutations were introduced as described in (10)  Electrophysiological recordings --HEK293 cells were used to express the wild type and mutant P2X receptors with green fluorescent protein as described above. A plasmid ratio of 1:5 was adopted for co-expression of P2X 2 and P2X 3 subunits.
Whole cell recordings were carried out at room temperature using an EPC9 patch clamp amplifier (HEKA Elektronik, Germany) as detailed previously and briefly presented as current density (in pA/pF). All data are presented where appropriate as mean ± s.e.m.. In most subunits the residue preceding the conserved lysine is also a basic residue;

Identification of an intracellular motif implicated in
we therefore investigated if this motif YXXXK could be implicated in P2X receptor trafficking. To that aim we used P2X subunits in which epitope tags were introduced in the extracellular loop in a position that did not modify noticeably receptors properties (8). We next studied whether other residues in the vicinity of the YXXXK motif could also interfere with the surface expression of the P2X 2 subunit. Residues downstream of the second transmembrane region, from K360 to K369, were individually mutated to alanine and their membrane expression examined. As shown in Fig. 1D, none of these mutants showed any decreased membrane expression; nor did the mutation to alanine of the two leucines (L354, L355) located at the end of the second transmembrane domain (data not shown). These results indicate that the YXXXK motif plays an important role in the trafficking of the P2X 2 subunit to the plasma membrane.

Specificity of the YXXXK motif and functional characterization of mutant
subunits --Additional amino acids substitutions were introduced at the Y362 and K366 positions (Y to F; K to R, K to G and K to Q). All mutants were individually transfected in HEK cells and membrane expression quantified by chemiluminescent assay. Fig. 2A shows that all mutants had reduced surface expression ranging from 22 ± 6.2% (n = 3) for K366Q to 43 ± 3.5% (n = 3) for Y362F; even when the conservative substitutions Y362F and K366R were introduced surface expression of the receptor was still decreased. These values were not different from what was observed for membrane expression of Y362A and K366A.
We next investigated if the introduced mutation at both Y and K positions had any consequences on ATP-evoked currents in HEK transfected cells. Because a 20 to 40% residual membrane expression of these mutants was consistently observed, we expected to observe similar reduction of the ATP current densities. As shown in  Recordings from ATP-induced currents in HEK cells transfected with P2X 3 , P2X 4 and P2X 5 tyrosine and lysine mutants showed that none of them responded to ATP, even at concentrations up to 1 mM. By comparison, when wild type subunits were used in the same experiments, normal currents were observed (see figure 3B).
The ATP analog αβ-methylene-ATP (αβmeATP) was also ineffective to induce These results indicate that mutant P2X subunits are not retained in the ER and that their transport is not likely to be affected. Red-labeled secondary antibody after permeabilization. This approach has been used to monitor P2X 4 constitutive internalization in HEK cells as well as in neurons (17). As a positive control for receptor internalization, HEK cells transiently transfected with HA-tagged P2X 4 receptor cDNA were used. As shown in Fig. 6A The YXXXK motif enhances surface expression of CD4 --We next asked if the YXXXK motif of P2X receptor could stabilized the surface expression of unrelated membrane proteins. To that aim we fused wild type or mutated C-termini tails to CD 4 intracellular extremity (see Fig. 6B); surface expression of Wt CD 4 and CD 4 -P2X chimera were measured by chemiluminescence assay after transfection in HEK cells.
When the P2X 2 C-Term was fused to CD 4 its surface expression increased by 38% ± 7.3 (n = 6) as compared to Wt CD4 (Fig. 6B). However this increase was not observed in subunits where the YXXXK motif of the P2X 2 C-Term was disrupted by mutation of either the tyrosine or the lysine residues. In this case we even observed a reduction of the surface expression of the CD 4 -P2X 2 fusion protein (76% ± 16.8, n = 4) and 72% ± 7.3 (n = 4) for the tyrosine and the lysine mutants, respectively). P2X 2 C-terminus is 129 amino acids long and contains a 69 amino acids long alternative proline-rich exon that could be responsible for the reduction membrane expression of the mutated CD4-P2X 2 protein (30). We tested this hypothesis by using the Cterminal tail of the P2X 2b subunit which lacks this exon (30). Surface expression of the CD 4 -P2X 2b was increased to 138 % ± 4.3 (n = 4) when compared to CD4; the disruption of the YXXXK motif abolished this increase, and surface expression of CD4-P2X 2b was not different from Wt CD4 (105 % ± 12.4 (n = 3) and 102% ± 11.6 (n = 3) for the tyrosine and the lysine mutants, respectively). In a third experiment, the 43 amino acid long P2X 3 C-terminal tail was fused to CD4. We observed essentially the same result: CD 4 -P2X 3 surface expression was 126% ± 7.0 (n = 5) compared to CD 4 , whereas disruption of either of the two key residues of the YXXXK motif abolished this increased (surface expression was 99% ± 4.4 (n = 5) and 97% ± 5.2 (n = 5) for the tyrosine and the lysine mutants, respectively) (Fig. 5A). All together, these results clearly indicate that the YXXXK motif of P2X subunits enhances the surface expression of the unrelated CD4 membrane protein most likely by enhancing its stabilization at the plasma membrane. Our present results demonstrate that the YXXXK motif is responsible for appropriate surface expression of P2X 2 , P2X 3 , P2X 4 , P2X 5 and P2X 6 receptors.
Mutation to alanine of either conserved residues also leads to a loss of function of  (32).

Trafficking rescue of mutant P2X subunits --P2X receptors are oligomers
probably composed of three subunits (7) that assemble either homo-oligomers or hetero-oligomers. P2X 2/3 hetero-oligomeric channels has been extensively studied (28,29). Based on biochemical and functional evidence it has been recently proposed that P2X 2/3 receptor is formed by the head-to-tail association of one P2X 2 and two P2X 3 subunits (9  3, and data not shown). However, this question will be difficult to address experimentally since in co-expression experiments ATP does not discriminate between homo-oligomeric P2X 2 and hetero-oligomeric P2X 2/3 channels.

Mutation of the YXXXK motif destabilized surface expression of P2X 2 subunits
--Mutations of the YXXXK motif reduce the surface expression of all homomeric P2X tested. Such a reduction may have different causes such as a misfolding of the protein leading to its degradation, a reduction in the forward trafficking of the protein during its biosynthesis or a lack of stabilization of the receptors at the plasma membrane. The two first possibilities seem unlikely. Thus, we never noticed any decrease in the total cellular content of mutated subunits when compared with wild type subunits as assayed either by western blotting or by luminescence assay (see Fig. 1C); this rules out a misfolding and/or degradation of mutated P2X subunits. By comparison, a P2X 2 subunit that lacks glycosylation sites presents a trafficking defect (5), that can be attributed, at least in part, to a misfolding of the protein, because a reduction in the total cellular content of the non glycosylated channel is observed 2 . In addition, it is likely that a misfolded subunit would have not form an oligomeric receptor, nor retained any channel activity. Our results show that mutant P2X 2 subunits are able to co-immunoprecipitate and to some extent can be activated by ATP (see Fig. 2). The forward trafficking of mutant P2X receptors is not impaired. show that mutant P2X 2 subunits are significantly internalized over a period as short as 30 min whereas Wt P2X 2 shows very little endocytosis as already described (17).
This internalization of P2X 2 mutant subunits is not likely mediated by clathrinmediated endocytosis since these mechanisms are based on the recognition by the endocytic machinery of tyrosine based motifs. This is clearly not the case for mutant P2X subunits in which the deletion of a tyrosine residue promotes internalization.
The most likely interpretation for these results is that mutant P2X receptors are internalized though clathrin-independent or fluid-phase endocytosis. We believe that after their endocytosis mutant subunits are recycled to the plasma membrane; this could explain the residual surface expression that we observed for all mutant P2X subunits that were analyzed. Because we did not observe any difference in the total cellular content between mutant and Wt subunits, it is not likely that internalized P2X mutant receptors are directed to the lysosomal pathway. Clathrin-independent internalization and recycling of D2 dopamine receptor has been demonstrated, however the mechanism underlying this type of endocytosis remains unexplained (34).
We propose that the YXXXK motif stabilizes P2X receptors at the plasma membrane. This interpretation is supported by our experiments using CD4 protein fused either with P2X 2 or P2X 3 C-terminal. These CD4-fusion proteins display higher surface expression than Wt CD4, that return to normal level when the YXXXK motif is mutated. Because the YXXXK is not involved in the forward trafficking of P2X receptors it is most likely that it enhances CD4 residency time at the plasma membrane by promoting its stabilization (presumably by interactions with cytoskeletal proteins).
There The YXXXK motif is too degenerate to be used in data mining. We searched databases with a more stringent pattern (LI)-(LV)-X 8 -Y-X 3 -K in which the double hydrophobic residues helps to anchor the motif near the membrane (see Fig. 1). This   however, both mutated P2X 2 subunits were present in MAP2 negatives processes as indicated by arrows. Images are representative of results obtained from at least three independent cultures.