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 the -Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (RR, KK, and KK) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (DD) 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 interaction Molecular 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 a-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.

L-selectin: A Major Regulator of Leukocyte Adhesion, Migration and Signaling

“…The binding of L-selectin to μ1A—a 45 kDa protein of the clathrin-coated vesicle AP-1 complex—was identified in classic pull-down experiments and later verified by protein-protein interaction (121). Interestingly, binding was only witnessed in response to PMA-induced cell activation.…”

“…The tail of L-selectin is only 17 amino acids long, yet it has been documented to bind up to 6 intracellular proteins, which include: alpha-actinin (118), calmodulin (111), ezrin, moesin (119), protein kinase C (PKC) isozymes (120) and μ1alpha/AP-1 (121). Given the size of the L-selectin tail, not all of these proteins can bind simultaneously, but are likely to interact under tight spatio-temporal constraints—e.g., during tethering, rolling, firm adhesion, and TEM.…”

A head-to-tail view of L-selectin and its impact on neutrophil behaviour

“…μ1A of the AP-1 complex is the latest identified L-selectin binding partner and was isolated from “pull-down” experiments using extracts from Raw 264.7 mouse macrophages and the interaction validated with purified proteins (Dib et al 2017 ). Pre-stimulation of Raw cells with PMA increased the affinity of μ1A for L-selectin.…”

“…Single point mutations are therefore likely to unearth more meaningful data in respect of teasing out mechanisms associated with adhesion/shedding/signalling. The recent identification of the μ1A subunit of AP1 adaptin complex may add a fourth dimension to the current triad, delivery of translated and glycosylated L-selectin to the plasma membrane (Dib et al 2017 ). However, for the purposes of this review, understanding the triad of L-selectin regulation is restricted to the mature translated and glycosylated form presented at the plasma membrane of circulating neutrophils.…”

Differential regulation of synaptic AP-2/clathrin vesicle uncoating in synaptic plasticity

“…Differential AP-1/μ1A activation could play a role in the differential activation and thus different functions of γ2 and γ1 AP-1 complexes. In addition, the three μ1 isoforms are expected to be activated by different kinases 6 , 63 , 64 . Differential AP-2 activation could be achieved by specific targeting of kinases to the site of CME, e.g.…”