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First published online 28 June 2005
doi: 10.1242/jcs.02451

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Non-canonical YXXG endocytic motifs: recognition by AP2 and preferential utilization in P2X4 receptors

Stephen J. Royle^1,2, Omar S. Qureshi¹, Laura K. Bobanovi¹, Philip R. Evans², David J. Owen³ and Ruth D. Murrell-Lagnado^1,*

¹ Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK
² MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK
³ Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Hills Road, Cambridge, CB2 2XY, UK

View larger version (42K): [in a new window]   Fig. 1. Schematic diagram of constructs used in this study. (A) P2X receptors are thought to assemble as homo- or hetero-trimers. Extracellular domains are not shown to aid visualization. (B) A single P2X4 subunit has intracellular N- and C-termini and two transmembrane domains. The position of epitope AU5 at residue 76 is shown as a blue circle. Diagrams of CD8 chimeras, CD8 (left), CD8-4C (middle) and CD8-4C (right). (C) Primary sequences of the C-termini of the constructs shown in B. (D) Sequence alignment of C-terminal regions of P2X subunits 1-6 from Rattus norvegicus, full or partially conserved residues are shown in bold. Total number of residues of each subunit is shown to the right, P2X2 and P2X5 have been truncated. Transmembrane helices in C and D are indicated by underlined regions. P2X4 is the only P2X family member that has a YXXG motif in its C-terminus. Other receptors and channels with a YXXG motif include the inward rectifier K+ channel Kir2.3, the adenosine receptor A2a and the cannabinoid receptor CB-2 (Royle et al., 2002).

View larger version (46K): [in a new window]   Fig. 2. The C-terminus of P2X4 alone can mediate internalization from the plasma membrane. (A) Representative confocal images of NRK cells transfected with either CD8-reporter or CD8-4C-reporter constructs, P2X4(AU5) wild-type, or Y378S-mutant receptors. Cells were live-labeled with anti-CD8 or anti-AU5 for 30 minutes at 37°C. Cell surface (red) and internalized (green) receptors were visualized using Cy3- and FITC-conjugated secondary antibodies, before and after permeabilization, respectively. Bars, 10 µm. (B) Histogram, showing the proportion of protein detected at the cell surface (red) or in internal compartments (green) after labeling (n=18-54). **, P<0.01.

View larger version (49K): [in a new window]   Fig. 3. In the CD8-4C chimera, both YXXV and YXXGL motifs are functional. (A) Representative confocal images of CD8-4C chimeras with point mutations in the intracellular domain following live-labeling with anti-CD8 as described in Materials and Methods. Bars, 10 µm. (B) Histogram, showing the proportion of protein detected at the cell surface (red) or in internal compartments (green) after labeling (n=61-76). **, P<0.01.

View larger version (100K): [in a new window]   Fig. 4. P2X4-GFP mutants Y372A, Y378A and Y378F, but not Y372F or V375A, show internalization defects. Representative confocal micrographs of P2X4-GFP and related mutants (Y372A, Y372F, V375A, Y378A and Y378F) expressed in HEK293 cells. Nucleic acids are shown in red. Internalization-deficient mutants (Y372A, Y378A, Y378F) have greater plasma membrane expression. Bar, 10 µm.

View larger version (7K): [in a new window]   Fig. 5. Tyr372 is essential for function. Typical inward currents elicited by application of ATP (100 µM) at untagged P2X4 receptors and related mutants (Y372A, Y372F, Y378A) expressed in HEK293 cells. Y372F is functional, but Y372A is not. Note the large response of Y378A mutant, reflecting the abundant surface distribution.

View larger version (88K): [in a new window]   Fig. 6. Interaction of a non-canonical tyrosine-based endocytic motif with µ2-adaptin. (A) Structure of the signal-binding domain of µ2 complexed with a peptide corresponding to P2X4 amino acids 375-384. (B) Electron density (mFo-DFc) map of the P2X4 peptide bound to µ2; level is 2.56. (C) Overlay of the P2X4 endocytic motif (YXXGL, green) with that of TGN38 (YXXL, gold), to compare binding of non-canonical and canonical tyrosine-based endocytic motifs. (D) Close-up view of the second hydrophobic pocket of µ2 to compare the entry and co-ordination of the Y+4 and Y+3 leucine residues of YXXGL and YXXL, respectively.

View larger version (83K): [in a new window]   Fig. 7. YXXG and YXX motifs are interchangeable in the P2X4 receptor. P2X4-GFP with residues YEQGL replaced by YQRLN (YQRL, above) was indistinguishable from wild type (see Fig. 5, WT). Mutation of Y378A abolished internalization (AQRL, below). Cells were co-stained for DNA (red). Bar, 10 µm.

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