The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit μ1A via a novel basic binding motif

Karim Dib,Irina G Tikhonova,Aleksandar Ivetic,Peter Schu

doi:10.1074/jbc.m116.768598

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 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 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 in vivo 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 trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.

Highlights

L-selectin regulates leukocyte adhesion and rolling along the endothelium
To confirm the ␮1A/L-selectin tail interaction, we investigated the binding of full-length ␮1A as well as of ␮1A subdomains, expressed in E. coli as GST-fusion proteins, to L-selectin tail peptide. ␮1A consists of a N-terminal domain, positioned close to the membrane, and a C-terminal domain, which binds the canonical YXXØ sorting motif [17, 18, 22, 23]
We performed L-selectin cytoplasmic tail pulldown assays combined with a high sensitivity liquid chromatography (LC)/mass spectrometry (MS) platform to identify novel L-selectin tail-binding proteins in cell extracts from stimulated Raw 264.7 macrophages

Summary

To whom correspondence should be addressed

L-selectin mutants lacking the membrane-distal 11 amino acid residues cannot support leukocyte rolling along endothelial venules and adhesion [9] This defect in L-selectin function cannot be explained by changes in ligand binding and is instead due to a defect in the recruitment of binding partners to L-selectin cytoplasmic tail. The recruitment of Lck to the C-terminal tyrosine residue [13] and binding of PKC family members to the L-selectin tail [14] are essential for L-selectin outside-in signaling. L-selectin is phosphorylated in response to T lymphocyte receptor cross-linking or treatment with PMA [14] (an activator of PKC) or engagement of chemokine receptors [16] It was proposed, but not proven, that such phosphorylation may be essential for the dynamic association/dissociation of proteins to the L-selectin tail relevant to the regulation of L-selectin functions. We propose that the AP-1-dependent transport of L-selectin may not be constitutive but regulated by the dynamic interaction between L-selectin tail and ␮1A in specific cellular compartments

Results

Discussion

Experimental procedures