The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

Abstract

During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM), and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli, and the CD44 TM domain evoked expression on the cell body, whereas the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate under flow but not under static conditions compared with cells with cell body-expressed receptors. Substitution of the IC domain of L-selectin caused diminished tethering but no change in surface distribution, indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body, respectively.Neutrophil invasion at sites of inflammation as well as lymphocyte recirculation from blood into secondary lymphoid tissue require that free-flowing leukocytes become activated and subsequently exit the bloodstream. At the molecular level, the cells undergo a closely orchestrated cascade of interactions with the endothelium, each step consisting of a characteristic pattern of receptors, their ligands, cytokines, and chemoattractants (1, 2).Distinct cell surface receptor segregation is a widely observed phenomenon with effects on cell proliferation, migration, and tumorigenesis (3). Leukocyte membrane protrusions known as microvilli serve as active grouping sites for a variety of surface receptors including L-selectin (4), ␣4-integrins (5), P-selectin glycoprotein ligand 1 (PSGL-1) 3 (6, 7), CD4, and the chemokine receptors CCR5 and CXCR4 (8). In contrast, the hyaluronan receptor CD44 (4) and the integrins ␣M␤2 (Mac-1) and ␣L␤2 (LFA-1) (9, 10) show expression on the planar cell body excluding microvilli.Both L-selectin and CD44 are type I integral membrane glycoproteins expressed on most circulating leukocytes. L-selectin knock-out mice show a profound defect of lymphocyte accumulation in secondary lymphatic tissue and of neutrophils at sites of inflammation (11). CD44 is involved in many physiological processes, including lymphocyte activation, adhesion, and proliferation (12). There is also increasing evidence that CD44 and L-selectin contribute to tumor metastasis (13,14).Under many physiologic conditions, L-selectin initiates the first step of the adhesion cascade (15,1...

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases

“…By contrast, leukocyte rolling velocity was not affected by the topographic distribution of L-selectin (34,35). Most probably, the impairment of leukocyte tethering observed in our study (Figs.…”

“…Previous studies performed with transfectants expressing chimeric L-selectin/CD44 molecules demonstrated that leukocyte tethering on endothelium (34) or PSGL-1 (35) is dependent on the distribution of L-selectin on leukocyte microvilli (34,35) and on the binding of L-selectin cytoplasmic domain to ERMs and actin cytoskeleton (35). By contrast, leukocyte rolling velocity was not affected by the topographic distribution of L-selectin (34,35).…”

“…Substitution of these residues with alanines strongly reduces leukocyte tethering efficiency and recruitment on PSGL-1 (32), suggesting that they may be necessary for microvillar positioning of L-selectin (32). However, more recent data showed that they are not sufficient (35) and that the transmembrane domain is also required to allow L-selectin sorting to microvilli (35). As observed for L-selectin, alanine substitution of Arg-337 and Lys-338 on PSGL-1 also strongly reduced tethering efficiency and leukocyte recruitment on selectins (Fig.…”

“…The lack of significant effect of SRK mutations on rolling velocity is consistent with the hypothesis that velocity is mainly determined by receptor off-rate (33,34), which here depends on the interactions of PSGL-1 extracellular domain with P-selectin. Cell capture and subsequent cell recruitment on selectins is mainly dependent on microvillar distribution of the receptor (34,35) or on its ability to bind ERMs and actin (35). Because PSGL-1 positioning on leukocyte microvilli is not dependent on its cytoplasmic domain (11), we performed supplementary experiments with 32D leukocytes to determine whether deletion or mutations in the EBS interfere with leukocyte tethering and rolling on L-, P-, or E-selectin.…”

Characterization of Filifactor alocis and its immune evasion strategies employed against human neutrophils.

“…An extensive process referred to as 'Rolling-Adhesion -Transmigration' is responsible for neutrophil migration out of circulation to sites of infection or inflammation. The 'Rolling' step refers to the initial attachment of neutrophils to the endothelial cells and occurs when cells are stimulated with cytokines produced during an infection such as TNF-α, IL-1β, and interleukin 7 (IL-7) (74,(97)(98)(99). This chemokine production stimulates expression of selectins (P-and E-selectin), integrins such as intracellular adhesion molecules (ICAMs) and vascular cell adhesion molecules (VCAMs) on the luminal surface of the endothelial cells which can bind to the selectin and integrin ligands located on the neutrophils (74,98,100).…”