Abstract
Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X(4) receptor. Three amino acids in the C terminus of P2X(4) (Tyr(378), Gly(381), and Leu(382)) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X(4) protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X(4) C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the mu2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X(4) receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X(4) receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.
Highlights
A cell can sense extracellular stimuli by using receptors at the surface to transduce these messages into electrical or chemical signals
We found that P2X4 protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X4 C terminus pulled down the adaptor protein-2 complex from brain extract
Do recycling ionotropic receptors link to the endocytic pathway via canonical YXXØ motifs? Regions of the AMPA receptor protein that are important for endocytosis have been identified [14], and an interaction with adaptor protein-2 (AP-2) has been shown for both AMPA and ␥-aminobutyric acid type A receptors [6, 8]
Summary
DNA Constructs—The construction of enhanced green fluorescent protein (GFP)- and epitope-tagged P2X4 and P2X2 receptors has been cyanate; CCV, clathrin-coated vesicle; MES, 4-morpholineethanesulfonic acid; GluR, glutamate receptor. For analysis of the subcellular distribution of GFP-tagged receptors, cells were fixed in 3% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS; 1.5 mM NaH2PO4, 8 mM Na2HPO4, and 145 mM NaCl (pH 7.3)) for 10 min at 4 °C. To detect surface and internalized receptors, AU5-labeled receptors at the surface of fixed non-permeabilized cells were stained with a Cy3 (indocarbocyanine)-conjugated anti-mouse secondary antibody for 2 h at room temperature. Cells were washed five times with PBS, permeabilized with 0.1% Triton X-100 in PBS, and stained with a fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody for 2 h at room temperature to visualize prelabeled internalized receptors. To examine the effects of ATP on receptor internalization, neurons were labeled as described above, washed five times with buffer, and subsequently incubated for 15 min at 37 °C with or without ATP (100 M) in extracellular solution (see “Electrophysiological Recordings”). All reagents were obtained from Sigma or Invitrogen
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