Cytoplasmic Domain of P-selectin Glycoprotein Ligand-1 Facilitates Dimerization and Export from the Endoplasmic Reticulum

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate ␤ 2 integrins to slow rolling on integrin ligands. Leukocytes from knock-in "⌬CD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface.During inflammation, reversible interactions of selectins with their glycosylated ligands mediate leukocyte rolling on vascular surfaces (1). Cooperative signaling upon engagement of selectin ligands and chemokine receptors causes rolling leukocytes to decelerate and arrest through interactions of integrin ␣ L ␤ 2 on leukocytes with intercellular adhesion molecule-1 (ICAM-1) 4 on endothelial cells. Leukocytes then use integrins to migrate across blood vessels in response to chemokine gradients or other signals (2). P-selectin is expressed on activated endothelial cells and platelets; E-selectin is expressed on activated endothelial cells, and L-selectin is expressed on leukocytes (1). Each selectin mediates leukocyte rolling by interacting with a subset of membrane glycoproteins that must be appropriately glycosylated and, for ligands binding to P-and L-selectin, sulfated. P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric, type I transmembrane mucin (1, 3, 4). It is the dominant ligand for P-and L-selectin on leukocytes (5-8), and it cooperates with other ligands to mediate rolling on E-selectin (9 -11). Dimerization of PSGL-1 enhances rolling on P-selectin (12). The subunits of the PSGL-1 homodimer are linked by a disulfide bond just outside the plasma membrane (13). Each extracellular domain has multiple Ser/Thr-linked O-glycans that cause it to adopt a highly extended conformation (14). PSGL-1 is localized on the tips of leukocyte microvilli (5) and has affinity for cholesterol-rich membrane domains or lipid rafts (15). It moves to the uropods as cells polarize after chemokine stimulation (16,17).The sequences of the transmembrane and cytoplasmic domai...

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

“…In the endoplasmic reticulum, cooperative interactions between transmembrane domains and between cytoplasmic domains facilitate the formation of PSGL-1 dimers. 29,30 Each noncovalent dimer is then stabilized by a single juxtamembrane disulfide bond. An export signal in the cytoplasmic domain promotes transfer of PSGL-1 from the endoplasmic reticulum to the Golgi apparatus, where O-glycans are added en route to the cell surface.…”

“…An export signal in the cytoplasmic domain promotes transfer of PSGL-1 from the endoplasmic reticulum to the Golgi apparatus, where O-glycans are added en route to the cell surface. 30 The cytoplasmic tail of PSGL-1 consists of 67 amino acids in mice and 69 amino acids in humans and may interact with different proteins. 11 In vitro, the cytoplasmic domain binds to ezrin/radixin/moesin (ERM) proteins, which in turn interact with actin filaments.…”

Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin αLβ2

“…2A). PSGL-1 forms dimers through noncovalent interactions of the transmembrane and cytoplasmic domains, which are stabilized by a single extracellular disulfide bond (21,22). Mutating the extracellular cysteine and replacing the trans- Table 1.…”

“…membrane domain with the transmembrane domain of CD43 generates a monomeric form of PSGL-1 (CD43 TMD PSGL-1) (21). Substituting the transmembrane domain of PSGL-1 with that of glycophorin A creates an alternative dimeric form of PSGL-1 (GpA TMD PSGL-1) (22). Each construct was stably expressed in transfected CHO cells.…”

“…Nevertheless, cytoplasmic domain binding to ERMs or other adaptors could anchor PSGL-1 to the cytoskeleton to facilitate signaling. PSGL-1 dimerizes through cooperative interactions of its transmembrane and cytoplasmic domains (21,22), which might enhance interactions with ERM proteins or other adaptors. Dimerization provides a structural basis for some membrane proteins to form microclusters and to activate SFKs associated with noncatalytic receptors (23,24).…”