Mer Receptor Tyrosine Kinase Research Articles

Gas6 and protein S

Gas6 and protein S are two homologous secreted proteins that depend on vitamin K for their execution of a range of biological functions. A discrete subset of these functions is mediated through their binding to and activation of the receptor tyrosine kinases Axl, Sky and Mer. Furthermore, a hallmark of the Gas6-Axl system is the unique ability of Gas6 and protein S to tether their non receptor-binding regions to the negatively charged membranes of apoptotic cells. Numerous studies have shown the Gas6-Axl system to regulate cell survival, proliferation, migration, adhesion and phagocytosis. Consequently, altered activity/expression of its components has been detected in a variety of pathologies such as cancer and vascular, autoimmune and kidney disorders. Moreover, Axl overactivation can equally occur without ligand binding, which has implications for tumorigenesis. Further knowledge of this exquisite ligand-receptor system and the circumstances of its activation should provide the basis for development of novel therapies for the above diseases.

TAM receptor function in the retinal pigment epithelium

The TAM receptor tyrosine kinase Mer is expressed by cells of the retinal pigment epithelium (RPE), and genetic studies have demonstrated that Mer is essential for RPE function. RPE cells that lack Mer exhibit a severely compromised ability to phagocytose the distal ends of photoreceptor (PR) outer segments, which leads to the complete postnatal degeneration of photoreceptors and to blindness. Although in vitro experiments have implicated Gas6 as the critical TAM ligand for this process, we find that Gas6 mutant mice have a histologically intact retina with no photoreceptor degeneration. We further find that, in addition to Mer, RPE cells also express another TAM receptor – Tyro 3 – and that both of these receptors are instead activated independently by the Gas6-related ligand Protein S. This protein is also expressed by RPE cells. Finally, we demonstrate that loss of Mer function is accompanied by a substantial down-regulation in Tyro 3 as well. These observations indicate that both Mer and Tyro 3 act in mouse RPE cells and suggest that their biologically relevant ligand in these cells is Protein S.

MERTK polymorphisms associated with risk of haematological disorders among Korean SLE patients

The MER receptor tyrosine kinase (MERTK) gene is critical for the efficient clearance of apoptotic cells and has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the genetic polymorphisms in MERTK to evaluate it as a potential candidate gene for a host genetic study of SLE and clinical manifestations in patients with SLE. By resequencing the coding and flanking regions of the MERTK gene in 24 unrelated Koreans, 37 polymorphisms were identified. Based on gene position, minor allele frequency and inter-single-nucleotide polymorphism (SNP) linkage disequilibrium, six of these polymorphisms were selected for subsequent genotyping and association analysis with the risk of SLE and haematological disorders in 350 Korean SLE patients and 330 controls. Although no significant associations with the risk of SLE were found, logistic regression analyses revealed that variants +465C > G (P = 0.05) and +130215insdelT (P = 0.0005) were significantly associated with decreased risk of leucopenia in SLE patients. Further, +465C > G, +95616G > A, +123157A > G and the haplotype ht1 also showed significant associations (P = 0.006-0.05) with a decreased risk of lymphopenia in SLE patients. Our findings suggest that polymorphisms in MERTK might be one of the genetic risk factors for presenting leucopenia and lymphopenia in SLE patients.

Gas6/Axl Signaling Activates the Phosphatidylinositol 3-Kinase/Akt1 Survival Pathway to Protect Oligodendrocytes from Tumor Necrosis Factorα-Induced Apoptosis

Growth arrest-specific protein 6 (gas6) activity is mediated through the receptor tyrosine kinase family members Axl, Rse, and Mer, all of which are expressed in human oligodendrocytes. In this study, we examined whether recombinant human (rh) gas6 protects oligodendrocytes from growth factor (insulin) withdrawal or tumor necrosis factor-alpha (TNFalpha) cytotoxicity. In addition, we examined whether the effect was caspase-dependent, which receptor mediated the protective effect, and whether survival required Akt1 activation. Oligodendrocyte viability was assessed by O4 staining and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling. Addition of rhgas6 to insulin-depleted cultures resulted in a significant increase in oligodendrocyte viability. Rhgas6 and caspase inhibitors also reduced active caspase-3 immunoreactivity relative to TNFalpha-only-treated cultures. In cultures treated with TNFalpha (100 ng/ml), the oligodendrocyte survival rate was 18% compared with cultures treated with TNFalpha and rhgas6 (64%) or the caspase inhibitors IETD-fmk [z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone] (65%) and zVAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) (63%). Increased phosphoAkt (Ser473) immunoreactivity was detected 15 min after administration of gas6 and TNFalpha to oligodendrocyte cultures but not in TNFalpha-treated cultures. The gas6 protective effect was abrogated by the Axl decoy receptor Axl-Fc, by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one], and in Akt1(-/-) oligodendrocytes. Oligodendrocyte cultures established from wild-type and Rse(-/-) mice, but not from Axl(-/-) mice, were also protected from TNFalpha-induced cell death when maintained in rhgas6. We conclude that gas6 signaling through the Axl receptor and the PI3 kinase/Akt1 survival pathway protects oligodendrocytes from growth factor withdrawal and TNFalpha-mediated cell death.

Ectopic Expression of the Proto-oncogene Mer in Pediatric T-Cell Acute Lymphoblastic Leukemia

The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription-PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy.

Heterogeneous Nuclear Ribonucleoprotein P2 Is an Autoantibody Target in Mice Deficient for Mer, Axl, and Tyro3 Receptor Tyrosine Kinases

Deficiencies in clearance of apoptotic cells predispose to the development of autoimmune disease. This is evident in mice lacking the receptor tyrosine kinases Tyro3, Axl, and Mer. Deficient mice exhibit an increased abundance of apoptotic cells in tissues and manifest diverse autoimmune conditions. To test these mice for the presence of autoantibodies to apoptotic cells, we generated spontaneous splenic B cell hybridomas and used a novel microscopy screen to detect Ab binding to apoptotic Jurkat cells. From hybridomas secreting IgG Abs reactive with apoptotic cells, we selected one that recreated the major serum specificity for apoptotic cells. The Ab LHC7.15 bound to an Ag that is differentially distributed between the nucleus and the cytoplasm in live and apoptotic cells. In late apoptotic cells, the Ag coalesces into aggregates that bleb from the cell surface. Immunopurification of the Ag, followed by mass spectrometry, identified a protein of 69 kDa whose partial sequence matched heterogeneous nuclear ribonucleoprotein P2. This multifunctional protein binds DNA, RNA, and several known ribonucleoprotein autoantigens. Our observations indicate that a ribonucleoprotein complex, formed and translocated to the cell surface in apoptosis, represents a potent stimulus for breaking tolerance and inducing systemic autoimmunity in mice with defective clearance of cell remnants.

Expression of Mer and Axl receptor tyrosine kinases in pediatric astrocytoma patients

1555 Background: Tumors of the central nervous system (CNS) are the second most common cause of pediatric cancer and are the leading cause of cancer related deaths. Great strides have been made in ...

Ectopic Expression of Mer Receptor Tyrosine Kinase in Childhood T Cell Acute Lymphoblastic Leukemia.

Tyrosine kinases play an important role in normal cellular growth and differentiation. Deregulation of tyrosine kinase activity can result in cellular transformation leading to the development of human cancer. The Mer receptor tyrosine kinase, initially cloned from a human B lymphoblastoid cell line, is expressed in a spectrum of hematopoetic, epithelial, and mesenchymal cell lines. Interestingly, while the RNA transcript of Mer is detected in numerous T and B lymphoblastic cell lines, Mer RNA is not found in normal human thymocytes, lymphocytes or in PMA/PHA stimulated lymphocytes. We have developed an anti-human Mer monoclonal antibody to further study the ectopic Mer expression in lymphoblasts. The antibody detects several forms of the Mer protein including a fully glycosylated 205 kD Mer protein in monocytes and a less glycosylated 170–180 kD Mer protein in acute lymphoblastic leukemia (ALL) cell lines. We analyzed lymphoblasts from 16 T cell ALL patients diagnosed between July 1995 and July 2004 at The Children's Hospital, Denver for Mer protein surface expression. Of the T cell ALL patient samples, we found that 9/16 (56%) were positive for Mer protein surface expression. Although we were not able to statistically evaluate the clinical outcomes relative to Mer expression due to the study sample number, there was a statistically significant association between positive expression of Mer and lack of surface expression of CD3 (p = 0.035). We found that 7/9 (78%) of the Mer positive lymphoblasts lacked CD3 surface expression, while only 1/6 (17%) of the Mer negative samples lacked CD3 surface expression (see figure). Lymphoblasts that lack surface expression of CD3 represent an immature phenotype and have been associated with a decreased event free survival as compared with surface positive CD3 lymphoblasts. Further investigation of the ectopic expression of Mer in lymphoblasts may reveal the use of this novel Mer glycosylated protein as a prognostic marker and possibly a future therapeutic target in the treatment of childhood leukemia. [Display omitted]

Growth Arrest-Specific Gene 6 (Gas6) Deficiency in Mice Alleviates Graft-versus-Host Disease (GVHD) in the Liver.

GVHD is the single most important complication of allogeneic bone marrow transplantation (BMT), mediated by donor-derived alloreactive T cells. Gas6, a vitamin K-dependent protein, interacts with phospholipid membranes via its γ-carboxyglutamic acid-containing tail and binds to the receptor tyrosine kinases Tyro3, Axl and Mer via its carboxy-terminal globular domain. Gas6 has been implicated in reversible growth arrest, survival, proliferation, cell adhesion, hemostasis and erythropoiesis. Preliminary experiments have shown that Gas6 deficient (Gas6-/-) mice have a leukocyte trafficking defect into inflamed tissues. We hypothesized that donor Gas6-/- T cells might induce less GVHD than wild-type (WT) T cells in MHC-mismatched BMT. We therefore compared the ability of Gas6-/- FVB (H-2q) bone marrow cells to produce GVHD in lethally irradiated MHC haplo-mismatched Gas6-/- and WT B6 mice (H-2b). Gas6-/- and WT B6 received 9 Gy total body irradiation and 107 cells from bone marrow of FVB mice (H-2q) of the same genotype. (Gas6-/- B6 mice received Gas6-/- FVB cells and WT B6 mice were injected with WT FVB cells.) On Day 28, recipients were sacrificed and chimerism was confirmed by expression of H-2q haplotype in recipient bone marrow. Neither group had clinical evidence of GVHD. However, histologic studies revealed lesions in the livers of both genotypes, which were notably less severe in Gas6-/- mice. The size and number of portal space leukocyte infiltrates were reduced in Gas6-/- mice (40 ± 6.6 % of portal spaces infiltrated), compared to WT mice (76.8 ± 6.5 % of portal spaces infiltrated, P < 0.01). Apoptosis of liver cells was also reduced in Gas6-/- mice. We hypothesized that diminished leukocyte trafficking in the Gas6-/- mice may be due to defects in cytokine-induced expression of adhesion molecules. We therefore isolated primary capillary endothelial cells from Gas6-/- and WT mice, and assessed their response to TNF-α. In contrast to WT endothelial cells, Gas6-/- endothelial cells responded without increases in VCAM-1, ECAM-1, E-selectin, IL-1 or IL-6. This resistance to TNF-α was reversed when the Gas6-/- endothelial cells were co-treated with recombinant Gas6, suggesting that the effect of Gas6 on vascular endothelial function is critical for normal leukocyte trafficking in GVHD. Overall, these results provide strong rationale for considering Gas6 and/or its receptors as a potential therapeutic target to minimize GVHD.

Human vitamin K-dependent GAS6: gene structure, allelic variation, and association with stroke.

The product of the growth arrest-specific gene 6 (GAS6), a ligand for the Axl, Sky, and Mer tyrosine kinase receptors, is a vitamin K-dependent protein, structurally related to anticoagulant protein S. Gas6-deficient mice are protected against thrombosis, demonstrating the importance of this protein in the cardiovascular system. The present study was aimed at determining the human GAS6 intron-exon structure and analyzing the gene for the presence of allelic variants that could be associated with atherothrombotic disease. Online analyses allowed us to localize 15 GAS6 exons and to determine the sequence of their intron-flanking regions, in a chromosome 13 region spanning 43.8 kb of DNA. SSCP analysis of PCR-amplified GAS6 exons with their intron-flanking regions from a minimum of 12 control DNA samples, revealed the presence of eight different variants, which were confirmed to be single nucleotide polymorphisms (SNPs). Three of them (c.1263G>C, c.1332C>T, and c.1869T>C) are localized in exons 11, 12, and 14, and appear to be neutral since they do not modify the encoded amino acid. The other SNPs (c.280+170C>G, c.712+26G>A, c.713-155C>T, c.834+7G>A, and c.1478-94C>G) are in introns 3, 7, 8, and 12. A preliminary analysis of five of these SNPs in a group of 110 healthy controls and 188 patients with atherothrombotic disease has revealed statistically significant differences between controls and stroke patients in the allelic distributions of one of these variants (c.834+7G>A in intron 8). The SNP identification in GAS6 reported here would be very useful in future association studies aimed at determining the physiologic role of GAS6 in stroke and other human diseases.

Mechanisms of autoimmunity in mice lacking the mer membrane tyrosine kinase

There has been increased recent interest in the role of macrophages (Mp) and dendritic cells in systemic lupus erythematosus pathogenesis. The mer receptor tyrosine kinase, expressed on Mp and dendritic cells, mediates the binding of the former but not the latter to apoptotic cells. mer also is important in determining the cytokine profile that ensues after phagocytosis. Mice lacking mer develop antinuclear antibodies and rheumatoid factor. These autoantibodies appear to arise mostly from the splenic marginal zone. Stimulated spleen cells and peritoneal Mp from mer-deficient mice have increased expression of tumor necrosis factor alpha and IκBα, along with increased spontaneous expression of CD30L. The proinflammatory cytokine profile of mer-deficient mice may contribute to the immunogenicity of apoptotic debris. In vitro ligation of mer from normal Mp leads to diminished tumor necrosis factor alpha expression and increased IL-10 and IL-4. mer, which controls both phagocytosis and cytokine synthesis after exposure to apoptotic cells, may be an attractive target for therapeutic intervention in inflammatory and autoimmune disorders.

The mer receptor tyrosine kinase: expression and function suggest a role in innate immunity.

The mer receptor tyrosine kinase mediates phagocytosis of apoptotic cells and modulates cytokine production; it is also required for prevention of systemic autoimmune disease. Using a mer-specific antibody, we have confirmed the presence of mer on macrophages and now report its expression on NK cells, NKT cells, and dendritic cells (DC). We found that DC do not require mer for ingestion of apoptotic cells, as DC from mer-deficient mice phagocytose apoptotic cells normally. Mer was observed in splenic sections on cells outside follicular areas, probably representing DC and macrophages. Mer apparently participates in NKT-cell antigen-induced signaling, as NKT cells from mer-deficient mice evinced much lower cytokine production after in vivo alpha-galactosylceramide stimulation; this defect was intrinsic to the mer-deficient NKT cells. Taken together, these studies show mer expression on cells of the innate immune system. Mer, through its binding of lipid antigens, may not only mediate ingestion of apoptotic cells, but also signal events in NK cells, NKT cells, and DC.

TheGrowth Arrest-SpecificGene Product Gas6 Promotes the Survival of Human Oligodendrocytes via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+ oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-L-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway.

An RCS-like retinal dystrophy phenotype in mer knockout mice.

To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (mer(kd)) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene MERTK: Eyes of mer(kd) and C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG). The mer(kd) mice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to mer(kd) mice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained. Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

Phagocytosis and clearance of apoptotic cells is mediated by MER.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6.

Gas6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains g-carboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that Gas6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of Gas6 with PS, we examined the effect of Gas6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that Gas6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of Gas6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that Gas6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of Gas6. These findings suggest that Gas6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, Gas6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells.

A Novel Receptor Tyrosine Kinase, Mer, Inhibits TNF-α Production and Lipopolysaccharide-Induced Endotoxic Shock

AbstractThe regulation of monocyte function and the inhibition of TNF-α production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-α found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-α Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-α production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-κB nuclear translocation resulting in greater TNF-α production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-α secretion and attenuating endotoxic shock.

Cell Adhesion to Phosphatidylserine Mediated by a Product of Growth Arrest-specific Gene 6

Gas6, a product of a growth arrest-specific gene 6, potentiates proliferation of vascular smooth muscle cells and prevents cell death of vascular smooth muscle cells. It has been also demonstrated that Gas6 is a ligand of receptor tyrosine kinases Axl, Sky, and Mer. Gas6 contains gamma-carboxyglutamic acid residues, which are found in some blood coagulation factors and mediate the interaction of the coagulation factors with negatively charged phospholipid. In this study, we clarified that Gas6 specifically bound to phosphatidylserine and the binding was dependent on Ca2+ and gamma-carboxyglutamic acid residues. Furthermore, we found that U937 cells, which express Gas6 receptor on their surfaces, adhered to phosphatidylserine-coated enzyme-linked immunosorbent assay (ELISA) plate only in the presence of Gas6 and Ca2+. U937 cells also bound to ELISA plate coated with phosphatidylinositol, but the binding was independent of Gas6 and Ca2+. On the other hand, U937 cells did not adhere to phosphatidylcholine- or phosphatidylethanolamine-coated ELISA plate even in the presence of Gas6 and Ca2+. These findings suggest that Gas6 may play a role in recognition of cells exposing phosphatidylserine on their surfaces by phagocytic cells, which is supposed to be one of the mechanisms for clearing dying cells.

Identification of the Product of Growth Arrest-specific Gene 6 as a Common Ligand for Axl, Sky, and Mer Receptor Tyrosine Kinases

Axl, Sky, and Mer, members of an Axl/Sky receptor tyrosine kinase subfamily, are typified by the cell adhesion molecule-related extracellular domain. The product of growth arrest-specific gene 6 (Gas6), structurally homologous to the anticoagulant protein S, was recently identified as the ligand for Axl and Sky, but the ligand for Mer remained unknown. We have now obtained evidence that Gas6 can also function as a ligand for Mer. Co-precipitation analysis, using soluble receptors of Axl, Sky, and Mer (Axl-Fc, Sky-Fc, and Mer-Fc) composed of the extracellular domain of receptors fused to the Fc domain of immunoglobulin G1, clearly showed that Gas6, but not protein S, specifically bound to Axl-Fc, Sky-Fc, and Mer-Fc fusion proteins. Quantitative kinetic analyses using a BIAcore biosensor instrument revealed dissociation constants (Kd) of the binding of rat Gas6 to Axl-Fc, Sky-Fc, and Mer-Fc are 0.4, 2.7, and 29 nM, respectively. We also found that Gas6 stimulated tyrosine phosphorylation of Axl, Sky, and Mer receptors ectopically expressed in Chinese hamster ovary cells. Taken together, these findings suggest that Gas6 is a common ligand for Axl, Sky, and Mer, all known members of an Axl/Sky receptor subfamily.