title: Identification of a novel di-leucine motif mediating K(+)/Cl(-) cotransporter KCC2 constitutive endocytosis.
abstract: The neuron-specific potassium-chloride cotransporter 2 (KCC2) plays a crucial role, by controlling chloride extrusion, in the development and maintenance of inhibitory neurotransmission. Although it is now well established that activity-dependent mechanisms can down regulate KCC2 gene expression, the role of post-translational mechanisms in controlling KCC2 expression, specifically at the cell-surface, are poorly understood. We therefore set out to identify the mechanisms and motifs regulating KCC2 endocytosis, one important pathway that may control KCC2 membrane expression. Using a fluorescence-based assay, we show KCC2 when expressed in HEK293 cells is constitutively internalized via a dynamin- and clathrin-dependent pathway. Consistent with this, we demonstrate KCC2 from adult mouse brain associates in vivo with the clathrin-binding adaptor protein-2 (AP-2) complex. Using an endocytosis reporter system, we identify the presence of an autonomous endocytosis motif in the carboxyl cytoplasmic terminus of KCC2. By site-directed mutagenesis we define this novel KCC2 endocytic motif as a non-canonical di-leucine motif, (657)LLXXEE(662). Finally by mutating this motif in the context of full-length KCC2 we demonstrate that this novel KCC2 endocytic motif is essential for the constitutive internalization of KCC2 and for binding to the AP-2 complex. Subsequent sequence analysis reveals this motif is highly conserved between the closely related K(+)/Cl(-) family members that mediate chloride efflux, but absent from the more distant related cotransporters controlling chloride influx. In conclusion, our results indicate constitutive internalization of KCC2 is clathrin-mediated and dependent on the binding of AP-2 to this novel endocytic motif. Furthermore, that this process appears to be an evolutionarily conserved mechanism amongst functionally homologous cotransporters.
authors.affiliation: Department of Anatomy & Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: Beibei
authors.initials: B
authors.lastname: Zhao
authors.forename: Adrian Y C
authors.initials: AY
authors.lastname: Wong
authors.forename: Ayesha
authors.initials: A
authors.lastname: Murshid
authors.forename: Derek
authors.initials: D
authors.lastname: Bowie
authors.forename: John F
authors.initials: JF
authors.lastname: Presley
authors.forename: Fiona Kay
authors.initials: FK
authors.lastname: Bedford
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D033962:Adaptor Protein Complex 2; D002966:Clathrin; D006388:Hemagglutinins; D007930:Leucine; D011993:Recombinant Fusion Proteins; D027981:Symporters; D000096922:K Cl- Cotransporters