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

• Published in: The Journal of biological chemistry 2002-09, Vol.277 (38), p.35378-35385
• Main Authors: Stephen J. Royle, Laura K. BobanoviÄ, Ruth D. Murrell-Lagnado
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Cells, Cultured; Endocytosis; Fluorescent Antibody Technique; Humans; Molecular Sequence Data; Mutagenesis; Rats; Rats, Wistar; Receptors, Purinergic P2 - chemistry; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2X4; Tyrosine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M204844200

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 Y XX GL. 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 μ2 subunit of adaptor protein-2 or the Y XX GL 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 Y XX GL 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.