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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-acetatestimulated Raw 264.7 macrophages resulted in the binding of mu 1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-mu 1A and the GST-mu 1A C-terminal domain, but not the GST-mu 1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357, 359KK360, and 362KK363) 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 mu 1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of mu 1A associated with anti-L-selectin immunoprecipitates. However, fulllength GST-mu 1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of mu 1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo. Molecular docking of the L-selectin tail to mu 1A was used to identify the mu 1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates mu 1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.