SLIC‐1/sorting nexin 20: A novel sorting nexin that directs subcellular distribution of PSGL‐1

Abstract

P-Selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of leukocytes that serves as the major ligand for the selectin family of adhesion molecules and functions in leukocyte tethering and rolling on activated endothelium and platelets. Previous studies have implicated the highly conserved cytoplasmic domain of PSGL-1 in regulating outside-in signaling of integrin activation. However, molecules that physically and functionally interact with this domain are not completely defined. Using a yeast two-hybrid screen with the cytoplasmic domain of PSGL-1 as bait, a novel protein designated selectin ligand interactor cytoplasmic-1 (SLIC-1) was isolated. Computer-based homology search revealed that SLIC-1 was the human orthologue for the previously identified mouse sorting nexin 20. Direct interaction between SLIC-1 and PSGL-1 was specific as indicated by co-immunoprecipitation and motif mapping. Colocalization experiments demonstrated that SLIC-1 contains a Phox homology domain that binds phosphoinositides and targets the PSGL-1/SLIC-1 complex to endosomes. Deficiency in the murine homologue of SLIC-1 did not modulate PSGL-1-dependent signaling nor alter neutrophil adhesion through PSGL-1. We conclude that SLIC-1 serves as a sorting molecule that cycles PSGL-1 into endosomes with no impact on leukocyte recruitment.Supporting Information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2008/37777_s.pdf IntroductionThe initial tethering and rolling of leukocytes on activated endothelium and platelets is a key biological event mediated by P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein that functions as a unique high-affinity ligand for the selectin family of adhesion molecules (for reviews, see [1][2][3][4]). Structurally, PSGL-1 is a type I integral membrane protein consisting of an N-terminal selectin-binding domain followed by a region of multiple decameric repeats bearing O-linked glycans, a short transmembrane domain, and lastly a cytoplasmic tail [5][6][7][8][9][10]. Comparison of human and murine PSGL-1 reveals that the 69-amino acid intracellular domain of PSGL-1 is highly conserved. While overall identity between human and Abbreviations: EEA-1: early endosomal antigen-1 Á EGFP: enhanced green fluorescent protein Á ERM: ezrin/radixin/ moesin Á L-E/I: L cell monolayer expressing E-selectin and ICAM-1 Á PI: phosphatidylinositol Á PI 3-kinase: phosphatidylinositol 3-kinase Á PSGL-1: P-selectin glycoprotein ligand-1 Á PtdIns(3)P: phosphatidylinositol 3-phosphate Á PtdIns(3,4)P 2 : phosphatidylinositol 3,4-bisphosphate Á PtdIns(3,4,5)P 3 : phosphatidylinositol 3,4,5-trisphosphate Á PX domain: phox homology domain Á SLIC-1: selectin ligand interactor cytoplasmic-1 Á SNX20: sorting nexin 20 Á TM: transmembrane Ulrich Y. Schaff et al.

Structure and Membrane Binding Properties of the Endosomal Tetratricopeptide Repeat (TPR) Domain-containing Sorting Nexins SNX20 and SNX21

“…Previously it was reported that SNX20 could interact with the cytoplasmic domain of the PSGL-1 receptor, and it was observed that overexpression of SNX20 caused a redistribution of PSGL-1 from a predominantly cell-surface location to SNX20-decorated endosomal compartments (16). We attempted to confirm these findings using a CD8-PSGL-1 fusion reporter, where the cytoplasmic tail of PSGL-1 is fused to the lumenal and transmembrane domains of CD8.…”

“…SNX20 appears to be mainly expressed in macrophages and immune cells, whereas SNX21 appears to be expressed more broadly but with particularly high levels in fetal liver tissue (14,15). A single study of the SNX20 molecule identified it as an endosomal protein that could associate with the cytosolic domain of the transmembrane adhesion receptor P-selectin glycoprotein ligand 1 (PSGL-1) (16). Overexpression of SNX20 caused a redistribution of PSGL-1 from the cell surface into endosomal organelles, suggesting a role in PSGL-1 trafficking.…”

“…Previous studies of SNX20 indicated an endosomal localization of the protein in COS and CHO cells (16). Here we compared the localization of both SNX20 and SNX21 in HeLa cells using N-terminal myc-tagged myc-SNX20 and myc-SNX21 constructs (Fig.…”

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins

“…Although no specific function has been assigned to SNX31, a recent study revealed that mutations in the protein lead to melanoma (21). SNX27 is unique within the PX family, containing an N-terminal postsynaptic density 95/discs large/zonula occludens-1 (PDZ) domain that binds PDZ-binding motif (PDZbm)-containing cargo such as the β2-adrenergic receptor to influence alternative cargo recycling (18)(19)(20)(22)(23)(24)(25)(26). Thus a consensus is emerging that PX-FERM proteins are key regulators of endosome-to-cell surface recycling by binding PDZbm and/or NPxY/NxxY recycling signals.…”

Leukocyte ligands for endothelial selectins: specialized glycoconjugates that mediate rolling and signaling under flow

“…However, selectin ligand interactor cytoplasmic-1 does not participate in PSGL-1-mediated leukocyte adhesion and signaling in vivo. 34 mAbs to the N-terminal region of human or murine PSGL-1 block P-and L-selectin binding and abolish leukocyte rolling on L-selectin and P-selectin in vivo. 7,10,35,36 L-and P-selectin bind to the same or closely overlapping sites near the N-terminus of PSGL-1, whereas E-selectin appears to bind to at least one more site.…”