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 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.
Royle
Stephen J
SJ
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Bobanović
Laura K
LK
Murrell-Lagnado
Ruth D
RD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D014443:Tyrosine / Q000378:metabolism*
C548226:P2RX4 protein, human; C548228:P2rx4 protein, rat; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D014443:Tyrosine