Identification of a Non-canonical Tyrosine-based Endocytic Motif in an Ionotropic Receptor*

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 P2X₄ receptor. Three amino acids in the C terminus of P2X₄ (Tyr³⁷⁸, Gly³⁸¹, and Leu³⁸²) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X₄ protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathioneS-transferase fusion of the P2X₄ C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the μ2 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 P2X₄ receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X₄ receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.