Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells.

The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2b-Fc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity.

Efficient and inexpensive method for purification of heparin binding proteins.

Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

Protein partners in the life history of activated fibroblast growth factor receptors.

Fibroblast growth factor receptors (FGFRs) are a family of four transmembrane (TM) receptor tyrosine kinases (RTKs) which bind to a large family of fibroblast growth factor (FGF) ligands with varying affinity and specificity. FGFR signaling regulates many physiological and pathological processes in development and tissue homeostasis. Understanding FGFR signaling processes requires the identification of partner proteins which regulate receptor function and biological outputs. In this study, we employ an epitope-tagged, covalently dimerized, and constitutively activated form of FGFR1 to identify potential protein partners by MS. By this approach, we sample candidate FGFR effectors throughout the life history of the receptor. Functional classification of the partners identified revealed specific subclasses involved in protein biosynthesis and folding; structural and regulatory components of the cytoskeleton; known signaling effectors and small GTPases implicated in endocytosis and vesicular trafficking. The kinase dependency of the interaction was determined for a subset of previously unrecognized partners by coimmunoprecipitation, Western blotting, and immunocytochemistry. From this group, the small GTPase Rab5 was selected for functional interrogation. We show that short hairpin (sh) RNA-mediated depletion of Rab5 attenuates the activation of the extracellular-regulated kinase (ERK) 1/2 pathway by FGFR signaling. The strategic approach adopted in this study has revealed bona fide novel effectors of the FGFR signaling pathway.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forster resonance energy transfer suggest weak interactions between fibroblast growth factor receptor 3 (FGFR3) transmembrane domains in the absence of extracellular domains and ligands.

Lateral dimerization of membrane proteins has evolved as a means of signal transduction across the plasma membrane for all receptor tyrosine kinases (RTKs). The transmembrane (TM) domains of RTKs are proposed to play an important role in the dimerization process. We have investigated whether the TM domains of one RTK, fibroblast growth factor receptor 3 (FGFR3), dimerize in lipid vesicles in the absence of the extracellular domains and ligands. We have performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with peptides produced via solid-phase peptide synthesis that correspond to the TM domain of FGFR3. We have carried out Forster resonance energy transfer (FRET) measurements using two donor-acceptor pairs, fluorescein/rhodamine and Cy3/Cy5, as a function of peptide concentration and donor-to-acceptor mole ratios. Our results suggest that FGFR3 TM domains form sequence-specific dimers in lipid bilayers. However, the dimerization propensity of FGFR3 TM domain is much weaker than the dimerization propensity of glycophorin A (GpA), the well-characterized "membrane dimer standard". We discuss our findings in the context of cell signaling across the plasma membrane and diseases or disorders that occur due to single amino acid mutations in the TM domain of FGFR3.

Fibroblastic growth factor receptor (FGF-R) expression during uterine involution in goat.

To investigate the possible participation of fibroblastic growth factors (FGFs) in endometrial involution, 20 multiparous goats, slaughtered on days 0, 1, 4, 10, 16 and 22 postpartum (pp), were used. Samples of different parts of the previous pregnant horns were taken and processed using streptoavidin-biotin-peroxidase complex method to analyse FGF receptor (FGF-R) expression. The percentage of positive cells in luminal epithelium, superficial and deep glands and stroma was evaluated. Epithelial, glandular and stromal cells exhibited FGF-R immunoreactivity. No differences between caruncular and inter-caruncular epithelium were observed and staining was most evident in the superficial glands. The greatest degree of FGF-R expression was seen on days 10 and 16 pp, coinciding with epithelial and stromal cellular regeneration. These results suggest that caprine uterine involution is associated with variations in the expression of FGF-R.

A novel proliferation-associated variant of CFR-1 defined by a human monoclonal antibody.

The germline coded human monoclonal IgM antibody 103/51 was isolated from a gastric carcinoma patient. This antibody binds to a 130-kd membrane molecule and has a mitotic effect on tumor cells in vitro. To characterize the target, we sequenced the protein and showed that the antibody binds to the cysteine-rich fibroblast growth factor receptor (CFR)-1, which is highly homologous to MG-160 and the E-selectin-ligand (ESL)-1. The epitope was determined by glycosidase-digestion experiments to be an N-linked carbohydrate side chain. Immunohistochemistry was used to investigate the tissue distribution of CFR-1. Different healthy tissues were tested and only the collecting tubes of the kidney, the Golgi apparatus, and the glomerular and fascicular zones of the adrenal gland stained positive. However, on malignant tissue the receptor is overexpressed in nearly all tested stomach cancers (12 of 15) and other tested carcinomas (13 of 15). Most interestingly, the receptor is also present in Helicobacter pylori gastritis and gastric dysplasia, but absent on uninflamed stomach mucosa. This restricted tissue pattern indicates that antibody 103/51 reacts with a membrane-bound variant of CFR-1, which is mainly expressed on transformed cells and precursor lesions and is essential for proliferation processes. The possible activity of antibody 103/51 as an activating ligand in these proliferative changes of gastric epithelial mucosa is discussed.

Expression and glycosylation studies of human FGF receptor 4.

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.

Identification and functional characterization of the human and murine fibroblast growth factor receptor 4 promoters.

Fibroblast growth factor receptors (FGFRs) play crucial roles in signal transduction of adult tissues and during embryonic development. To study the transcriptional control, we isolated and characterized the promoter of human FGFR4. Two transcription initiation sites were identified. The deletion analysis in different cell types defined a core promoter reaching from -9 to -198, lacking TATA and CCAAT boxes but displaying high GC content (77%) in a stretch of 300 bp upstream of the major mRNA start. This region harbors multiple binding motifs for transcription factors. Moreover, the region between -1085 and -1140 contains a potential repressor element, which downregulates transcriptional activity. To identify conserved regulatory elements, we isolated and analyzed also the murine FGFR4 promoter. Only one transcription start was identified using RNase protection assays. Sequence alignment of human and mouse shows a striking similarity in the core promoter region of both genes, encompassing conserved transcription factor binding sites and a splice acceptor site. Furthermore, the region containing the putative repressor element is also conserved suggesting a functional role for gene expression.

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4.

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with (3)H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF.

Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues.

This report describes a systematic analysis of the expression of the fibroblast growth factor receptor (FGFR) multigene family (FGFR1, FGFR2, FGFR3, and FGFR4) in archival serial sections of normal human adult tissues representing the major organ systems, using immunohistochemical techniques. Polyclonal antisera specific for FGFR1, FGFR2, FGFR3, and FGFR4 and a three-stage immunoperoxidase technique were employed to determine the cellular distribution of these receptors at the protein level. The expression profiles for the tissue-specific cellular localization of the FGFR multigene family demonstrated wide-spread and striking differential patterns of expression of individual receptors in the epithelia and mesenchyme of multiple tissues (stomach, salivary glands, pancreas, thymus, ureter, and cornea) and co-expression of FGFR1-4 in the same cell types of other tissues. The wide-spread expression of FGFR1-4 in multiple organ systems suggests an important functional role in normal tissue homeostasis. Differences in the spatial patterns of FGFR gene expression may generate functional diversity in response to FGF-1 and FGF-2, both of which bind with equally high affinity to more than one receptor subtype. In vivo, this may lead to functional differences that are crucial for the regulation of normal physiological processes and are responsible for the pathological mechanisms that orchestrate various disease processes.

Latent transforming growth factor-beta complex in Chinese hamster ovary cells contains the multifunctional cysteine-rich fibroblast growth factor receptor, also termed E-selectin-ligand or MG-160.

Transforming growth factor-beta (TGF-beta) is secreted as latent high molecular mass complexes from producer cells. The N-terminal precursor remnant, also called latency-associated peptide (LAP), forms a non-covalently linked complex with TGF-beta and confers the latency to TGF-beta. In human platelets and certain other cell types, latent TGF-beta binding protein-1 (LTBP-1) is disulphide-linked to LAP, and forms complexes of more than 230 kDa. In addition, LTBP-2 and -3, which are structurally similar to LTBP-1, can be part of latent TGF-beta complexes. In Chinese hamster ovary (CHO) cells transfected with the TGF-beta1 cDNA, a major part of the latent TGF-beta secreted into the medium is a 100-kDa small latent complex containing TGF-beta and LAP. In addition, we found two other forms of latent TGF-beta complexes, i.e. a 220-kDa complex containing LTBP-1, and a 220-kDa complex containing a 140-kDa protein. Purification of the 140-kDa component, termed latent TGF-beta complexed protein-1 (LTCP-1), followed by amino acid sequencing and cDNA cloning from a CHO cell cDNA library, revealed that it is a hamster counterpart of a previously identified, multifunctional protein known as chicken cysteine-rich fibroblast growth factor (FGF) receptor, mouse E-selectin-ligand and rat MG-160 (a 160-kDa membrane sialoglycoprotein of the Golgi apparatus). Immunoprecipitation of LTCP-1 and TGF-beta1 from CHO cells stably transfected with TGF-beta1 precursor cDNA revealed that the expressed protein forms a complex with LAP, and that a major part of the complex is secreted. Northern blot analysis showed that mRNA for LTCP-1 was expressed in large amounts in testis, ovary and placenta, but less abundantly in other tissues. These results suggest that TGF-beta, produced in certain cell types, may form a complex with LTCP-1, which may have different properties compared with other latent TGF-beta complexes. It remains to be investigated whether the complex formation between LTCP-1 and TGF-beta1 also occurs in other cells, whether the association between them occurs in the Golgi complex, and whether it affects the interaction of LTCP-1 with FGF or E-selectin.

A natural kinase-deficient variant of fibroblast growth factor receptor 1.

A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation.

The nuclear trafficking of extracellular fibroblast growth factor (FGF)-1 correlates with the perinuclear association of the FGF receptor-1alpha isoforms but not the FGF receptor-1beta isoforms.

The alternatively spliced fibroblast growth factor receptor (FGFR)-1 isoforms, FGFR-1alpha and FGFR-1beta, are characterized by the presence of either three or two Ig-like loops in the extracellular domain and are differentially expressed during embryonic development and tumor progression. We have previously shown that in cells irreversibly committed to DNA synthesis by FGF-1, approximately 15% of cell surface FGFR-1 traffics to a perinuclear locale as a structurally intact and functional tyrosine kinase (Prudovsky, I., Savion, N., Zhan, X., Friesel, R., Xu, J., Hou, J., McKeehan, W. L., and Maciag, T. (1994) J. Biol. Chem. 269, 31720-31724). In order to define the structural requirement for association of FGFR-1 with the nucleus, the expression and trafficking of FGFR-1 in FGFR-1alpha and FGFR-1beta L6 myoblast transfectants was studied. Although FGFR-1alpha was expressed as p145 and p125 forms, FGFR-1beta was expressed as p120 and p100 forms in the L6 myoblast transfectants. Tunicamycin and N-glyconase experiments suggest that these forms of FGFR-1alpha and FGFR-1beta are the result of differential glycosylation. However, only the p145 form of FGFR-1alpha and the p120 form of FGFR-1beta were able to bind FGF-1 and activate tyrosine phosphorylation. Pulse-chase analysis of FGFR-1 biosynthesis suggests that the p125 and p100 proteins are the precursor forms of p145 FGFR-1alpha and p120 FGFR-1beta, respectively. Because ligand-chase analysis demonstrated that FGFR-1beta L6 myoblast transfectants exhibited a reduced efficiency of nuclear translocation of exogenous FGF-1 when compared with FGFR-1alpha transfectants, the intracellular trafficking of the FGFR-1alpha and FGFR-1beta isoforms was studied using an in vitro kinase assay to amplify immunoprecipitated FGFR-1. Indeed, the appearance of the FGFR-1alpha but not FGFR-1beta isoform in the nuclear fraction of L6 myoblast transfectants suggests that the distal Ig-like loop in FGFR-1alpha mediates the differential nuclear association of FGFR-1alpha as a structurally intact and functional tyrosine kinase. Further, the FGFR-1beta L6 myoblast transfectants but not the FGFR-1alpha myoblast transfectants exhibited a pronounced morphologic change in response to exogenous FGF-1. Because this phenotype change involves the induction of a rounded cellular shape, it is possible that the FGFR-1alpha and FGFR-1beta may ultimately exhibit differential trafficking to adhesion sites.

Association of a 85-kDa serine kinase with activated fibroblast growth factor receptor-4.

Fibroblast growth factors (FGFs) transduce a variety of biological signals via four distinct tyrosine kinase receptors. We have characterized the phosphorylation of FGF receptor 4 (FGFR-4) and its association with a putative substrate, p85, using transfected L6 myoblast and NIH3T3 fibroblast cell lines. FGFR-4 was phosphorylated in vivo and in vitro mainly on serine and threonine residues in several peptides and to a lower degree on tyrosine residues. When analyzed further by in-gel kinase assay, immunoprecipitates of ligand-activated FGFR-4 contained a serine autophosphorylated polypeptide doublet of 85 kDa. Analysis of the major autophosphorylation site Y754F mutant of FGFR-4 showed that binding of p85 and its serine phosphorylation were independent of receptor autophosphorylation at this site. Okadaic acid treatment increased the basal autophosphorylation activity of p85 but decreased FGFR-4 tyrosine phosphorylation. In contrast, orthovanadate treatment increased the tyrosine phosphorylation of FGFR-4. These data show that a serine kinase is associated with activated FGFR-4 and suggest a role for serine phosphorylation in FGFR-4 function.

Ligand-induced polyubiquitination of receptor tyrosine kinases.

The platelet-derived growth factor beta-receptor undergoes polyubiquitination as a consequence of ligand binding. In the present study, we have examined the ligand-induced receptor ubiquitination also in the other receptor tyrosine kinase (structurally different) subfamilies by immunoblotting with anti-ubiquitin antiserum. In addition to the platelet-derived growth factor alpha- and beta-receptors, all the monomeric receptor tyrosine kinases examined, such as the receptors for epidermal growth factor (subfamily I), colony stimulating factor-1 (subfamily III), and fibroblast growth factor (subfamily IV), were found to be ubiquitinated after ligand stimulation. However, the insulin receptor (subfamily II), which is a tetrameric molecule, was not. These data suggest that the ligand-induced polyubiquitination of the receptor is a general phenomenon observed in most of the monomeric receptor tyrosine kinases.

Androgen and fibroblast growth factor (FGF) regulation of FGF receptors in S115 mouse mammary tumor cells.

We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen-increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent.

The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins.

P- and E-selectins belong to a family of Ca(2+)-dependent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca(2+)-dependent manner. This protein has a molecular mass of approximately 120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P-selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E-selectins.