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 myristate acetate-stimulated Raw 264.7 macrophages resulted in the binding of mu1A of the clathrin-coated-vesicle AP-1 complex. Furthermore, full length GST-mu1A and the GST-mu1A C-terminal domain but not the GST-mu1A 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 di-basic residues (RR357, KK360, and KK363) in the membrane-proximal domain of L-selectin tail as well as a doublet of aspartic acid residues (DD370) in the membrane-distal end of L-selectin tail involved in mu1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of mu1A associated with anti-L-selectin immunoprecipitates. However, full length GST-mu1A did not bind to phospho-L-selectin tail or phospho-mimetic Ser364Asp L-selectin tail. Accordingly, we propose that phosphorylation of mu1A is required for interaction with L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo. Molecular docking of L-selectin tail to mu1A was used to identify the mu1A surface domain binding L-selectin tail and to explain how phosphorylation of L-selectin tail abrogates mu1A 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 L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.
- mu1a, L-selectin, Leukocytes
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- School of Medicine, Dentistry and Biomedical Sciences - Senior Lecturer
- Wellcome Wolfson Institute for Experimental Medicine