title: The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit μ1A via a novel basic binding motif.
abstract: L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of μ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-μ1A and the GST-μ1A C-terminal domain, but not the GST-μ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at thetrans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357,359KK360, and362KK363) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (369DD370) in the membrane-distal end of the L-selectin tail involved in μ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-μ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of μ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interactionin vivoMolecular docking of the L-selectin tail to μ1A was used to identify the μ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates μ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of atrans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.
authors.affiliation: From the Max Planck Institute for Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany, k.dib@qub.ac.uk.|the Center for Experimental Medicine, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom.
authors.forename: Karim
authors.identifier: 0000-0002-1777-1915
authors.initials: K
authors.lastname: Dib
authors.affiliation: the School of Pharmacy, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom.
authors.forename: Irina G
authors.initials: IG
authors.lastname: Tikhonova
authors.affiliation: the BHF Center for Research Excellence, Cardiovascular Division, Faculty of Life Sciences and Medicine, King's College London, London SE5 9NU, United Kingdom, and.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Ivetic
authors.affiliation: University Medical Center Göttingen, Department of Cellular Biochemistry, Georg-August University Göttingen, D-37073 Göttingen, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schu
mesh_terms: D033961:Adaptor Protein Complex 1 / Q000737:chemistry*; D033983:Adaptor Protein Complex mu Subunits / Q000737:chemistry*; D020816:Amino Acid Motifs; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D019041:L-Selectin / Q000737:chemistry*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D062105:Molecular Docking Simulation; D009000:Monocytes / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D040901:Proteomics; D000067996:RAW 264.7 Cells; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D033961:Adaptor Protein Complex 1; D033983:Adaptor Protein Complex mu Subunits; C499063:Ap1m1 protein, mouse; D019041:L-Selectin; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D012694:Serine; D005982:Glutathione Transferase; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse
keywords: adhesion; leukocyte; protein complex; receptor-interacting protein (RIP); signaling