L-selectin is constitutively expressed on the surface of most leucocytes and is important for tethering and subsequent rolling of leucocytes on endothelial cells, facilitating their migration into secondary lymphoid organs (e.g. naive T cells) and sites of inflammation (e.g. neutrophils). Previous studies have shown that the 17-amino-acid L-selectin cytoplasmic tail is important for its function in cell adhesion and, hence, identifying binding partners will provide insight into how L-selectin is regulated in leucocytes. This review describes currently known binding partners of the L-selectin tail and how their associations affect L-selectin function.
- transendothelial migration
- ezrin/radixin/moesin (ERM) protein
- signal transduction
Roles of selectins in leucocyte TEM (transendothelial migration)
Immune surveillance of the human body is predominantly carried out by leucocytes, which undergo TEM to exit the bloodstream and enter the surrounding tissues. When leucocytes are recruited to blood vessel walls, they undergo a series of adhesive interactions with the endothelium that become increasingly stable, ultimately resulting in their arrest against the rapidly flowing bloodstream . The primary stages of TEM involve a group of cell adhesion molecules known as the selectins. The selectins (L-, E- and P-selectin) regulate initial capture (or tethering) and subsequent rolling of leucocytes along endothelial cells that line vascular networks surrounding secondary lymphoid organs (such as lymph nodes) and sites of inflammation . L-selectin is constitutively expressed in leucocytes and is located on the tips of microvilli, whereas E-selectin is expressed de novo in activated endothelial cells. P-selectin is stored in preformed cytosolic storage granules (called Weibel–Palade bodies), which fuse with the plasma membrane after the activation of both platelets and endothelial cells. Leucocyte tethering and rolling allows leucocytes to sense their local environment for the presence of stimuli such as pro-inflammatory cytokines and chemokines that are presented on the surface of endothelial cells. Stimulation of leucocytes by these stimuli leads to the rapid activation of leucocyte integrins, mediating firm arrest and subsequent TEM .
Structure of selectins
Structures of all the three selectin extracellular domains are similar: a C-type lectin domain, followed by an epidermal growth factor-like domain, and a series of sequence consensus repeats, which varies in number from one selectin to the other . Unlike E- and P-selectin, L-selectin harbours a well-characterized membrane-proximal cleavage site that is proteolysed after leucocyte activation, for example using chemoattractants or phorbol esters such as PMA, leading to shedding of the extracellular domain. The transmembrane and cytoplasmic domains of L-, E- and P-selectin are the most divergent regions and this suggests that they are probably regulated differently. Identifying novel binding partners of the selectins' cytoplasmic tails could therefore provide valuable insights into how they are regulated.
L-selectin tail binding partners
The L-selectin tail is highly basic and consists of only 17 amino acids (Figure 1). A number of reports have demonstrated that the cytoplasmic tail of L-selectin can regulate L-selectin shedding, microvillar positioning and the tethering/rolling behaviour of leucocytes. The tail of L-selectin is reported to interact with at least three different proteins: α-actinin, CaM (calmodulin) and members of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins.
The C-terminal region of the L-selectin tail has been reported to interact with α-actinin  (Figure 1). Leucocytes expressing L-selectin lacking the last 11 amino acids (that lack the α-actinin-binding site) have reduced tethering and rolling efficiencies in vitro . Another report demonstrates that this L-selectin deletion mutant is unable to interact with the cytoskeleton, suggesting that α-actinin acts as a link between L-selectin and the cortical actin cytoskeleton, which could be important for tethering and rolling . However, this L-selectin deletion mutant is still localized on microvillar tips, suggesting that other cytoskeletal proteins apart from α-actinin are required to anchor L-selectin to microvilli .
CaM has been shown to interact with the L-selectin tail in resting leucocytes . Stimulation of leucocytes with PMA induces release of CaM from L-selectin tail and concomitant shedding of L-selectin. Mutagenesis of two membrane-proximal amino acids within the L-selectin tail that abrogate interaction with CaM can lead to a higher turnover of soluble (cleaved) L-selectin (Figure 1) . Furthermore, incubation of resting leucocytes with CaM antagonists (that disrupt L-selectin tail–CaM interaction) can induce shedding of L-selectin in the absence of PMA stimulation. However, a subsequent report has demonstrated that CaM antagonists can induce shedding of unrelated membrane proteins, indicating that this mechanism of regulation is not specific to L-selectin . These results have led to the hypothesis that CaM negatively regulates shedding, whereby its interaction with L-selectin tail induces a conformational change in the extracellular domain that renders the cleavage site resistant to proteolysis. In contrast, its dissociation from the L-selectin tail renders the cleavage site susceptible to proteolysis.
ERM proteins were first demonstrated to interact with the L-selectin tail using affinity chromatography of lymphocyte extracts . Moesin from cell extracts could bind to an L-selectin tail affinity column only when lymphocytes were pre-treated with PMA. In contrast, ezrin bound to the affinity column irrespective of cell stimulation with PMA, suggesting that interaction of moesin and ezrin with the L-selectin tail is regulated differently. Members of the ERM family of proteins share a high level of identity with one another, displaying up to 80% identity in their N-terminal domains . ERM proteins interact with the cytoplasmic tails of a number of cell adhesion molecules (such as CD44 and CD43) through their N-terminal domain and with F-actin through their C-terminal domain, thereby providing a link between plasma membrane receptors and the cortical actin cytoskeleton . ERM proteins regulate the assembly of actin filament-based membrane structures such as ruffles and microvilli (see Figure 2) .
We have recently identified two residues within the ERM-binding domain of the L-selectin tail, Arg-357 and Lys-362, which contribute to the interaction with the N-terminal domain of moesin (Figure 1) . Interestingly, Arg-357 is juxtaposed to a residue that is important for CaM binding, but whether CaM and ERM proteins can bind simultaneously to the L-selectin tail or compete for binding is not yet known. Pre-B cells expressing L-selectin tail with Arg-357 or Lys-362 mutated to alanine [referred as R357A (Arg-357→Ala) and K362A respectively] have reduced PMA-induced shedding compared with cells expressing wild-type L-selectin. In addition, R357A and K362A L-selectin showed lower levels of microvillar localization compared with wild-type L-selectin, and in cells expressing these mutants tethering efficiency was reduced by approximately 50%, suggesting that anchoring of L-selectin on microvilli is important for efficient ligand engagement and thus tether formation under flow conditions. The observed reduction in tethering efficiency could also be due to a weak linkage between ERM-binding mutants of L-selectin and the cortical actin cytoskeleton. Taken together, these results suggest that microvillar positioning of L-selectin is important both for tethering of leucocytes to counter ligands and shedding of the L-selectin extracellular domain, and that the interaction between ERM proteins and the L-selectin tail affects all of these by potentially regulating L-selectin association with actin filaments.
It is difficult to conceive that the small 17-amino-acid L-selectin cytoplasmic tail can bind all three of its known binding partners at the same time (Figure 2). It is possible that a sub-population of L-selectin interacts with each partner at any given time, and that these interactions are highly dynamic. Understanding the spatiotemporal regulation between the L-selectin tail and its binding partners will therefore be an important focus for the future. A recent report has demonstrated that some protein kinase C isoforms can phosphorylate and associate with the L-selectin tail . Post-translational modification of the L-selectin tail may be the key to regulating the recruitment and release of proteins from an L-selectin tail.
Many reports have described an interaction between L-selectin and the cortical actin cytoskeleton, but whether this involves α-actinin, the ERM proteins or both is yet to be determined. It is also currently unclear whether ezrin and moesin perform distinct functions in leucocytes. Biochemically, ezrin, but not moesin, can associate with the L-selectin tail in the absence of PMA stimulation, suggesting that they could affect different aspects of L-selectin function. For example, ezrin might regulate the microvillar positioning of L-selectin, whereas moesin might regulate shedding. Interestingly, amino acid residues that contribute to both ERM and CaM binding are juxtaposed, suggesting that these proteins occupy a similar site (see Figure 1). Could this mean that release of CaM from the L-selectin tail allows the binding of moesin to induce shedding? Both moesin- and ezrin-null mice have now been generated [15,16], and detailed analyses of leucocytes derived from these mice should reveal whether ezrin and moesin have different roles in regulating L-selectin.
Research Colloquia: Research Colloquia at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by M. Bouvier (Montreal, Canada), G. Milligan (Glasgow, U.K.), V. O'Donnell (Cardiff, U.K.), M. Brand (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.), M. Schweizer (Heriot-Watt University, Edinburgh, U.K.), R. Insall (Birmingham, U.K.), A. Ridley (Ludwig Institute for Cancer Research, London, U.K.) and M. Sutcliffe (Leicester, U.K.). The first eight papers featured in this Section were presented as a part of the GPCR Regulation and Signalling Research Colloquium, incorporating the GPCR–Ion Channel Interactions Pfizer-Sponsored Research Colloquium.
Abbreviations: CaM, calmodulin; ERM, ezrin/radixin/moesin; TEM, transendothelial migration
- © 2004 The Biochemical Society