Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.     

The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.     

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.     

Association of intercellular adhesion molecule-1 (ICAM-1) with actin- containing cytoskeleton and alpha-actinin

We have studied the cytoskeletal association of intercellular adhesion molecule-1 (ICAM-1, CD54), an integral membrane protein that functions as a counterreceptor for leukocyte integrins (CD11/CD18). A linkage between ICAM-1 and cytoskeletal elements was suggested by studies showing a different ICAM-1 staining pattern for COS cells transfected with wild-type ICAM-1 or with an ICAM-1 construct that replaces the cytoplasmic and transmembrane domains of ICAM-1 with a glycophosphatidylinositol (GPI) anchor. Wild-type ICAM-1 appeared to localize most prominently in microvilli whereas GPI-ICAM-1 demonstrated a uniform cell surface distribution. Disruption of microfilaments with cytochalasin B (CCB) changed the localization of wild-type ICAM-1 but had no effect on GPI-ICAM-1. Some B-cell lines demonstrated a prominent accumulation of ICAM-1 into the uropod region whereas other cell surface proteins examined were not preferentially localized. CCB also induced redistribution of ICAM-1 in these cells. For characterization of cytoskeletal proteins interacting with ICAM-1, a 28-residue peptide that encompasses the entire predicted cytoplasmic domain (ICAM-1,478-505) was synthesized, coupled to Sepharose-4B, and used as an affinity matrix. One of the most predominant proteins eluted either with soluble ICAM-1,478-505-peptide or EDTA, was 100 kD, had a pI of 5.5, and in Western blots reacted with alpha-actinin antibodies. A direct association between alpha-actinin and ICAM-1 was demonstrated by binding of purified alpha-actinin to ICAM-1,478-505-peptide and to immunoaffinity purified ICAM-1 and by a strict colocalization of ICAM-1 with alpha-actinin, but not with the cytoskeletal proteins talin, tensin, and vinculin. The region of ICAM-1,478-505 interacting with alpha-actinin was mapped to the area close to the membrane spanning region. This region contains several positively charged residues and appears to mediate a charged interaction with alpha-actinin which is not highly dependent on the order of the residues.     

Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein alpha-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin-mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin-carbohydrate bond.     

Host focal adhesion protein domains that bind to the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) attach to the plasma membrane of infected host cells and induce diarrhea in a variety of farm animals as well in humans. These bacteria inject a three-domain protein receptor, Tir (translocated intimin receptor), that is subsequently inserted into the plasma membrane. EPEC induce the host cell to form membrane-covered actin-rich columns called pedestals. Focal adhesion constituents, alpha-actinin, talin, and vinculin, are localized along the length of the pedestals and we have previously reported they bind the two cytoplasmic domains of Tir, (Tir I and Tir III) [Freeman et al., 2000: Cell Motil. Cytoskeleton 47:307-318]. In the present study, various constructs were made expressing different regions of these three focal adhesion proteins to determine which domains of the proteins bound Tir I. Three different assays were used to detect Tir I/host protein domain interactions. In co-precipitation assays, His-Tir I bound to the 27-kDa region of alpha-actinin; to four different domains of talin; and to the N-terminal domain of the vinculin head and the vinculin tail domain. A yeast two-hybrid analysis of Tir I and the various focal adhesion fusion proteins revealed a region near the C-terminus of talin was the only domain to interact with Tir I. Finally, to assess direct binding interactions, biotinylated Tir I was used in overlay assays and confirmed the binding of Tir I with the 27-kDa region of alpha-actinin, the four regions of talin, and the vinculin tail. These binding interactions between hostfocal adhesion proteins and EPEC Tir may facilitate the adhesion of EPEC to the host cell surface.     

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.     

The oligomeric status of syndecan-4 regulates syndecan-4 interaction with alpha-actinin

Syndecan-4, a cell surface heparan sulfate proteoglycan, is known to regulate the organization of the cytoskeleton, and oligomerization is crucial for syndecan-4 function. We therefore explored a possible regulatory effect of syndecan-4 oligomerization on the cytoskeleton. Glutathione-S-transferase-syndecan-4 proteins were used to show that syndecan-4 interacted specifically with alpha-actinin, but not paxillin, talin, and vinculin. Interestingly, only dimeric, and not monomeric, recombinant syndecan-4 interacted with alpha-actinin in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), and PIP2 potentiated the interaction of both the cytoplasmic domain syndecan-4 peptide and recombinant syndecan-4 proteins with alpha-actinin, implying that oligomerization of syndecan-4 was important for this interaction. Consistent with this notion, alpha-actinin interaction was largely absent in syndecan-4 mutants defective in transmembrane domain-induced oligomerization, and alpha-actinin-associated focal adhesions were decreased in rat embryo fibroblasts expressing mutant syndecan-4. Besides, this interaction was consistently lower with the phosphorylation-mimicking syndecan-4 mutant S183E which is known to destabilize the oligomerization of the syndecan-4 cytoplasmic domain. Taken together, the data suggest that the oligomeric status of syndecan-4 plays a crucial role in regulating the interaction of syndecan-4 with alpha-actinin.     

Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton

We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability to associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites. A detergent based extraction and Western blotting procedure demonstrated an association of E-selectin with the insoluble (cytoskeletal) fraction of endothelial monolayers that correlated with adhesion of leukocytes via an E-selectin-dependent mechanism. A mutant form of E-selectin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cell and supported leukocyte attachment (in a nonstatic adhesion assay) in a fashion similar to the native E-selectin molecule, but failed to become associated with the cytoskeletal fraction. To identify the cytoskeletal components that associate with the cytoplasmic domain of E-selectin, paramagnetic beads coated with the adhesion-blocking anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alpha-actinin, vinculin, filamin, paxillin, as well as focal adhesion kinase (FAK), were copurified by this procedure, however talin was not. When a mechanical stress was applied to H18/7- coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoskeletal mechanical linkage. COS-7 cells transfected with the tailless E-selectin failed to show resistance to the twisting stress. Taken together, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin through its cytoplasmic domain, a process which may have important implications for cell-cell signaling as well as mechanical anchoring during leukocyte- endothelial adhesive interactions.     

Vinculin binding site mapped on talin with an anti-idiotypic antibody.

Vinculin and talin are major adhesion plaque components which interact in vitro and presumably in vivo. The amino acid sequence of talin is now known so details of its domain structure can be mapped. We localized vinculin binding sites in the talin sequence by overlaying peptide maps of talin with an anti-idiotypic vinculin antibody that recognizes talin and with 125I-vinculin. A rabbit injected only twice with vinculin and producing anti-vinculin antibodies spontaneously generated a second antibody that recognizes talin. Vinculin and anti-vinculin antibodies specifically compete with this second antibody for binding to talin as determined by solid-phase binding and overlay assays. The antibody is thus most likely an anti-idiotypic antibody which mimics a region of vinculin that interacts with talin. The binding site of the anti-idiotypic antibody on talin was mapped to the 196 amino acids spanning residues 1653 to 1848. A second vinculin binding site identified with an 125I-vinculin blot overlay technique was located between residues 483 and 1652. The observation that talin has two immunologically distinct vinculin binding sites suggests that vinculin may have two different talin binding sites or one "complex" site with two interacting regions.     

Synthetic peptide GRGDS induces dissociation of alpha-actinin and vinculin from the sites of focal contacts

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were microinjected with fluorescently labeled alpha-actinin and/or vinculin and observed using video microscopy before and after the addition of 50 micrograms/ml GRGDS. As soon as 5 min after treatment, fluorescent alpha-actinin and vinculin became dissociated simultaneously from the sites of many focal contacts. The proteins either moved away as discrete structures or dispersed from adhesion plaques. As a result, the enrichment of alpha-actinin and vinculin at these focal contacts was no longer detected. The focal contacts then faded away slowly without showing detectable movement. These data suggest that the binding state of integrin has a transmembrane effect on the distribution of cytoskeletal components. The dissociation of alpha-actinin and vinculin from adhesion plaques may in turn weaken the contacts and result in rounding and detachment of cells.     

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.     

L-selectin and its biological ligands

This review considers the leukocyte adhesive receptor known as L-selectin. This protein, belonging to the selectin family of cell-cell adhesion receptors, mediates adhesion by virtue of a C-type lectin domain at its amino terminus. The protein was discovered as a lymphocyte homing receptor involved in the attachment of lymphocytes to high endothelial venules (HEV) of lymph nodes. Its widespread distribution on all leukocyte populations underlies a more general role in a variety of leukocyte-endothelial interactions. In the HEV interaction, cognate HEV ligands for L-selectin have been identified as two sulfated, sialylated, and fucosylated glycoproteins, known as GlyCAM-1 and Sgp90. These ligands have mucin-like domains which confer important properties for their proposed adhesive function. The carbohydrate features of these ligands, essential for their interaction with L-selectin, are reviewed. The existence of extralymphoid ligands for L-selectin is also discussed.Presented at the XXXV Symposium of the Society for Histochemistry, 29 September 1993, Gargellen, Austria     

Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the alpha-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS alpha-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.     

Structural and biophysical properties of the integrin-associated cytoskeletal protein talin

Talin is a large cytoskeletal protein (2541 amino acid residues) which plays a key role in integrin-mediated events that are crucial for cell adhesion, migration, proliferation and survival. This review summarises recent work on the structure of talin and on some of the structurally better defined interactions with other proteins. The N-terminal talin head (approx. 50 kDa) consists of an atypical FERM domain linked to a long flexible rod (approx. 220 kDa) made up of a series of amphipathic helical bundle domains. The F3 FERM subdomain in the head binds the cytoplasmic tail of integrins, but this interaction can be inhibited by an interaction of F3 with a helical bundle in the talin rod, the so-called “autoinhibited form” of the molecule. The talin rod contains a second integrin-binding site, at least two actin-binding sites and a large number of binding sites for vinculin, which is important in reinforcing the initial integrin–actin link mediated by talin. The vinculin binding sites are defined by hydrophobic residues buried within helical bundles, and these must unfold to allow vinculin binding. Recent experiments suggest that this unfolding may be mediated by mechanical force exerted on the talin molecule by actomyosin contraction.     

Identification of a repeated domain within mammalian alpha-synemin that interacts directly with talin

The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, alpha-actinin, and talin. We report herein a direct interaction of human alpha-synemin with the cytoskeletal protein talin by protein-protein interaction assays. The 312 amino acid insert (SNTIII) present only within alpha-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human alpha- and beta-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human alpha-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.     

Specific interaction of vinculin with alpha-actinin

Vinculin and alpha-actinin are cytoskeletal proteins present at focal contacts of the ventral surface of cultured fibroblasts. We labelled alpha-actinin with an acceptor fluorophore and vinculin with a donor. A mixture of vinculin and alpha-actinin showed a 28% quench, due to energy transfer, suggesting an interaction. Quench of vinculin was dependent on the concentration of alpha-actinin; Scatchard analysis gives a dissociation constant in the microM range. Quench was inhibited by excess unlabelled alpha-actinin, and by reaction of the acceptor protein with p-chloromercuribenzoate. We found that vinculin had a slightly greater elution volume in a gel filtration column equilibrated with alpha-actinin, indicating a higher effective Stokes radius due to the interaction of the two proteins.     

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L- selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino- terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L- selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.     

A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion

The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCR). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin conferred the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L- selectin and the EGF-like domain of P-selectin exhibited dual ligand- binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains had no detectable effect on receptor function or specificity. Thus, the EGF- like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF- like domains collectively forming a functional ligand recognition unit.