©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of B61, the Ligand for the Eck Receptor Protein-Tyrosine Kinase (*)

(Received for publication, August 25, 1994; and in revised form, December 22, 1994)

Haining Shao Akhilesh Pandey K. Sue O'Shea (1) Michael Seldin (2) Vishva M. Dixit (§)

From the  (1)Department of Pathology Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109 and the (2)Department of Medicine and Microbiology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M.(1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T. D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M.(1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidylinositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific backcross analysis.


INTRODUCTION

Receptor protein-tyrosine kinases function as transducers, transmitting information from the extracellular environment into the cell, allowing for compensatory or adaptive alterations, a key to cellular homeostasis. Cell migration, proliferation, and differentiation can all be modulated by activation of receptor protein-tyrosine kinases. Binding of the ligand to the receptor protein-tyrosine kinase leads to oligomerization, autophosphorylation, and subsequent engagement of the signal transduction machinery.

Many receptor protein-tyrosine kinases have been shown to play a key role in development. For example, both epidermal growth factor and fibroblast growth factor receptors have been shown to influence epithelial and mesenchymal cell differentiation and proliferation(1) . The Trk family of receptor protein-tyrosine kinases are preferentially expressed in the developing nervous system, and their cognate ligands are neurotrophic factors that mediate neuronal cell differentiation and survival(1) .

Receptor protein-tyrosine kinases have an extracellular domain containing various signature motifs that allows categorization into subfamilies, a hydrophobic transmembrane domain, and a cytoplasmic domain that possesses tyrosine kinase activity. The Eph/Eck family is characterized by a cysteine-rich region and two fibronectin type III repeats in the extracellular domain and is the largest family of receptor protein-tyrosine kinases, with at least 12 members. Although some members of this family show widespread expression, the expression of many of the members, such as Elk and Cek5, is highly restricted to the nervous system(1) .

To begin to understand the function of this family of receptor protein-tyrosine kinases, an important first step is the characterization of their cognate ligands. The first such ligand for a family member, Eck, was recently identified as the cytokine-inducible immediate early response gene B61 that was originally cloned by differential hybridization from tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells(2, 3, 4) . The B61 gene product was shown to be a secreted 25-kDa protein found in the conditioned medium of tumor necrosis factor-alpha-treated endothelial cells(2) . However, due to the presence of a characteristic stretch of hydrophobic residues at the C terminus that was reminiscent of the signal for glycosylphosphatidylinositol (GPI) (^1)linkage, the possibility was raised that B61 may also exist anchored to the cell surface as a GPI-linked protein(2) . The GPI moiety becomes attached through an ethanolamine residue to a newly processed C terminus (that has had the hydrophobic signal cleaved) and can be released by phosphatidylinositol (PI)-specific phospholipase C(5) . A number of membrane proteins have been shown to be associated with the plasma membrane via a GPI anchor; among these are cell adhesion molecules, cell surface hydrolyses, and lymphoid antigens(5) . However, to date, no ligand for a receptor protein-tyrosine kinase has been shown to exist in a GPI-linked form.

In this study, we provide biochemical evidence to show that B61 is indeed a GPI-anchored cell surface protein. In addition, this anchor can be specifically cleaved by PI-specific phospholipase C, leading to release of B61 into the medium. Importantly, the GPI-linked membrane form was found to be biologically active as it induced the autophosphorylation of its receptor, Eck. Given the central role of receptor proteintyrosine kinases in development and to confirm co-expression of ligand and receptor in vivo, the expression of B61 and Eck was characterized by in situ hybridization during mouse development. Additionally, since the disruption of genes encoding receptor protein-tyrosine kinases or their respective ligands has been linked to a number of developmental anomalies(1) , the chromosomal localization of B61 in the mouse genome was established.


MATERIALS AND METHODS

Mouse Chromosomal Localization

C3H/HeJ-gld and Mus spretus (Spain) mice and ((C3H/HeJ-gld times M. spretus) F1 times C3H/HeJ-gld) interspecific backcross mice were bred and maintained as described previously(6) . M. spretus was chosen as the parent in this cross because of the relative ease of detection of informative restriction fragment length variants in comparison with crosses using conventional inbred laboratory strains. Genomic DNA isolated from mouse organs by standard techniques was digested with restriction endonucleases and 10 µg resolved by agarose gel electrophoresis, transferred to Nytran membranes (Schleicher & Schuell), and hybridized to [alpha-P]dCTP-labeled full-length B61 cDNA probe (2) under high stringency conditions, all as described previously(7) . Gene linkage was determined by segregation analysis with other published markers(8) . Gene order was determined by haplotype analysis that minimizes cross-over frequency between all genes that were determined to be within a linkage group. This method resulted in determination of the most likely gene order(9) .

Tissue Preparation and in Situ Hybridization

CD1 male mice were mated with virgin females, and embryonic day 1 was established by the presence of a vaginal plug. Embryos snap-frozen in OCT were cut as 8-µm sections and collected on acid-washed silane-treated slides. Sections were stored at -80 °C prior to use.

In situ hybridization was carried out as described previously with modifications(10) . Sections were fixed in fresh 4% paraformaldehyde, rinsed in PBS, digested in 10 mg/ml proteinase K for 5 min, and refixed in 4% paraformaldehyde. Following placement in 0.25% acetic anhydride containing 0.1 M triethanolamine for 10 min, sections were rinsed in PBS and dehydrated through an ethanol series essentially as described previously(11) .

A B61 cDNA substrate for in vitro transcription (encompassing nucleotides 246-512) (^2)was generated by PCR using rat B61 cDNA as template and an upstream oligonucleotide primer 5`-cactgaattcttattaaccctcactaaaGATTA CGAGGACGACTCTGTGG-3` (T3 promoter in lower case) and a downstream oligonucleotide primer cactgaattctaatacgactcactatagTTCAGGCACTGGGTTT CCTGA (T7 promoter in lower case). Similarly, an Eck substrate for in vitro transcription representing nucleotides 941-1202 was generated by polymerase chain reaction from a mouse Eck cDNA (^3)template (kindly provided by Dr. J. Ruiz, Harvard University) using an upstream oligonucleotide primer cactgaattctaatacga ctcactatagAAGTCTGAGGCATCTGAGAGCC (T7 promoter in lower case) and a downstream primer cactgaattcttattaaccctcactaaaGCGCAGCACTGTTCACAA GTG (T3 promoter in lower case). [S]UTP (DuPont NEN) uniformly labeled single-stranded RNA transcripts were obtained by in vitro transcription using either T3 or T7 RNA polymerase according to the manufacturer's instructions (Promega). Probes were purified by ethanol precipitation and used for in situ hybridization as described previously(11) .

Transfection and PI-specific Phospholipase C Treatment

The human embryonic kidney cell line 293T and the cervical carcinoma HeLa cell line were grown in Eagle's minimal essential medium supplemented with 10% bovine calf serum, penicillin (100 units/ml), streptomycin (100 units/ml), and 0.2 mM glutamine. 293T cells were transfected with either a B61 expression construct in the episomal vector pCEP4 (Invitrogen), which contained a full-length cDNA encoding B61(2) , or vector alone using the calcium phosphate method (12) and selected in the presence of 100 µg/ml hygromycin (Pharmacia Biotech Inc.). Cells were incubated in the presence of PI-specific phospholipase C (purified from B. thuringiensis(13) , kindly provided by Dr. M. G. Low, Columbia University, New York) at 0.5 unit/ml for 1 h at 37 °C. Cell lysates and media were cleared by centrifugation at 12,000 times g at 4 °C, lysed in SDS sample buffer containing beta-mercaptoethanol, and subjected to SDS-polyacrylamide gel electrophoresis.

Metabolic Labeling and Immunoprecipitation

B61 or vector-transfected 293T cells were incubated with 50 µCi/ml of [^3H]ethanolamine (Amersham Corp.) in EMEM and 10% dialyzed fetal bovine serum for 20 h at 37 °C. Following removal of the conditioned medium, the cell layer was washed with PBS and lysed in PBS containing 1% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, and a protease inhibitor mixture (5 µg/ml leupeptin, 5 µg/ml aprotinin, 50 µg/ml soybean trypsin inhibitor, and 5 µg/ml pepstatin). Following centrifugation at 12,000 times g for 30 min, the supernatants were subjected to immunoprecipitation with 1 µg/ml of an anti-B61 antibody 3E.6 (a mouse monoclonal antibody raised against recombinant human B61 protein). (^4)Immune complexes were precipitated by the addition of 50 µl of a 50% slurry of protein A-Agarose (Life Technologies, Inc.) and incubation at 4 °C overnight. After three washes with lysis buffer, the beads were pelleted, resuspended in sample buffer containing beta-mercaptoethanol, boiled, and resolved on a 15% SDS-polyacrylamide gel. For immunoblotting with the antiphosphotyrosine antibody, treated HeLa cells were immunoprecipitated with an anti-Eck polyclonal antibody (3, 4) at 1 µg/ml and then processed as described above, except that 1 mM sodium orthovanadate was included in the lysis buffer.

Western Blot Analysis

Total cell lysates from PI-specific phospholipase C-treated cells or immunoprecipitates were resolved on SDS-polyacrylamide gels and transferred to nitrocellulose using an electroblotting apparatus (LKB Multiphor, Pharmacia). For detection of B61 or Eck, the membrane was blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% nonfat dry milk for 1 h followed by incubation with the anti-B61 monoclonal antibody (3E.6) at a concentration of 0.1 µg/ml for 1 h at room temperature. For demonstrating phosphorylation of the Eck receptor protein-tyrosine kinase, the membrane was blocked with 1% bovine serum albumin in TBST for 1 h and incubated with 4G10 anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, New York) at a concentration of 1 µg/ml for 1 h at room temperature. After washing with TBST 4 times, the membranes were incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad). For anti-Eck Western blot, the anti-Eck polyclonal antibody was used at a concentration of 1 µg/ml followed by incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). Membranes were washed extensively and developed by chemiluminescense (ECL, Amersham Corp.) according to the manufacturer's instructions.

Co-culture and Autophosphorylation Assay

HeLa cells that constitutively express high levels of the Eck receptor protein-tyrosine kinase were plated in 100-mm dishes and grown to 80% confluence as responder cells. Approximately 4 times 10^6 B61 or vector-transfected 293T cells (stimulator cells) were detached by incubation in PBS containing 1 mM EDTA for 1 min, washed twice with PBS, and resuspended in 3 ml of PBS. Vector-transfected 293T cells were directly added to the attached HeLa cells, while B61-transfected 293T cells were either added directly to the attached HeLa cells or placed into cell culture inserts (FALCON, Lincoln Park, NJ), which were then co-cultured with the HeLa cells. All incubations were carried out for the identical time period of 10 min at room temperature. Following incubation, plates were rinsed three times with PBS to remove nonadherent stimulating cells, and the adherent HeLa cells were lysed in a buffer containing 1% Nonidet P-40, 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, and the protease inhibitor mixture (as described above). Lysates were clarified by centrifugation, and the supernatants were incubated with anti-Eck polyclonal antibody (3, 4) at a concentration of 1.5 µg/ml for 4 h followed by incubation with protein A-Agarose beads for 2 h at 4 °C. Precipitated immune complexes were resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to nitrocellulose, and immunoblotted with the 4G10 anti-phosphotyrosine antibody at a concentration of 1 µg/ml or the anti-Eck antibody at a concentration of 1 µg/ml.


RESULTS AND DISCUSSION

Chromosomal Localization

To determine the chromosomal location of the mouse B61 gene, a panel of DNA samples from an interspecific backcross that had been characterized for genetic markers throughout the genome was analyzed. The genetic markers included in this map span between 50 and 80 centiMorgans on each mouse autosome and the X chromosome(14, 15, 16) . Hybridization of the B61 cDNA probe to BamHI-digested DNA from the two parental mice (C3H/HeJ-gld and (C3H/HeJ-gld times M. spretus) F1) defined informative restriction fragment length variants (data not shown). Each of the restriction endonuclease-digested DNAs from the ((C3H/HeJ-gld times M. spretus) F1 times C3H/HeJ-gld) interspecific backcross mice displayed either the homozygous or heterozygous F1 pattern when hybridized with the cDNA probes (Fig. 1A).


Figure 1: Chromosomal localization of B61. A, segregation of the B61 locus on mouse chromosome 3 in ((C3H/HeJ-gld times M. spretus) F(1) times C3H/HeJ-gld) interspecific backcross mice. Filledboxes represent the homozygous C3H pattern, and openboxes represent the F1 pattern. The mapping of the reference loci in this interspecific cross have been previously described(13, 14, 15) . For B61, informative BamHI restriction fragments were defined in the current study (C3H/HeJ-gld: 17.0 and 4.8 kilobases; M. spretus: 11.0 and 8.0 kilobases). B, the name of each locus corresponds to the following genes: Cd1, cluster designation-1; Pklr, pyruvate kinase liver and red cell; Gba, beta-glucocerebrocidase, Fcgr1, Fcg high affinity receptor; Cacy, calcyclin; D3Tu51; DNA segment Turbugen 51; Cd2, cluster designation 2: Atp1a1, Na,K-ATPase 1a1 chain; Ngfb, nerve growth factor B; Tshb, thyroid-stimulating hormone B chain; Ampd-1, AMP deaminase-1; Nras; neuroblastoma Ras proto-oncogene.



Comparison of the haplotype distribution of the B61 gene restriction fragment length variants among markers previously defined in this cross allowed the gene to be mapped to a specific position on mouse chromosome 3 (Fig. 1B). The best gene order (9) ± the standard deviation (8) indicated the following gene order from proximal to distal on mouse Chr 3: Cd1/Pklr/Gba-0.9 ± 0.1 centiMorgans - B61/Fcgr1/Cacy/D3Tu51 - 1.2 ± 0.6 centiMorgans - Ampd-1/Ngfb/Tshb/Nras/Atp1a1/Cd2. This chromosomal assignment does not correlate with any known translocation or other chromosomal aberration associated with a developmental disorder.

Developmental Expression of B61 and Its Receptor Eck

Since an authentic ligand and its receptor should co-localize in tissues, the expression of the Eck receptor protein-tyrosine kinase and its cognate ligand B61 was examined in developing and adult mouse tissues. Expression of both B61 and Eck as determined by in situ hybridization peaked during the last third of gestation, with high levels of expression on day 19. B61 was expressed at relatively high levels in the developing lung and in the epithelium of the gut (Fig. 2, A-C). There was scattered expression in ossifying membranous bones of the face, in salivary gland, and in the central nervous system. In the thymus, B61 expression was low and appeared to be associated largely with connective tissue septae.


Figure 2: Localization of B61 and Eck in the mouse embryo. Parasagittal sections illustrating the pattern of expression of B61 (A-C) and of Eck (D-F) on day 19 of development. A, B61 is expressed at high levels in the lung (Lu), salivary gland (S), and gut (G) at this stage of development. There is scattered expression in the bones of the face and slight labeling of the perichondrium of forming vertebrae (arrowheads). The developing musculature of the tongue (T) also expresses low levels of B61. There is little expression in the spinal cord (Sc), thymus (Th), or heart (H). B, higher magnification view of the lung illustrating the high, uniform expression of B61 and slight expression in the perichondrium of the forming vertebral bodies. C, higher magnification view of the forming gut, illustrating the expression of B61 in the epithelium. D, low magnification photomicrograph of a day 19 fetus illustrating the high expression of Eck in salivary gland, thymus, lung, gut, and ossification centers in vertebral bodies (arrowheads) and bones of the face. Higher levels of expression were also present in the skin and in the epithelial lining of the esophagus. Eck expression was found to be low in the spinal cord. E, higher magnification view illustrating the high expression of Eck in the lung, the epithelium of the esophagus (E), and in the centers of ossification in the vertebral bodies. F, high magnification of the gut region illustrating the high expression of Eck in the epithelium. Scale bars = 100 µm.



The pattern of expression of Eck was similar in that it peaked at later gestational stages and was present at high levels in the lung, salivary gland, and gut (Fig. 2, D-F). In addition, Eck was expressed at especially high levels in the developing thymus. It was also expressed in skin, in ossifying bones of the face, and in vertebral bodies. There was little Eck expressed in the central nervous system at later stages of development.

Postnatally, both Eck and B61 were expressed at high levels in the lung. In the thymus, however, there was a low level of B61 expression, while Eck continued to be expressed at high levels throughout the thymic tissue (Fig. 3, A and B). Exposure of similar sections to labeled sense strand controls resulted in a nonspecific, unpatterned scatter of grains over the tissue (Fig. 3, C and D). Taken together, these studies indicated that, with the exception of the thymus, the temporal spatial expression of Eck and B61 was largely overlapping and in keeping with a bonafide receptor-ligand pair. The discordance of expression in the thymus (Eck high, B61 low) suggests that a thymic equivalent of B61 may exist to activate thymocyte Eck.


Figure 3: Localization of B61 and Eck in the thymus. Sagittal sections through the thymus on postnatal day 15 illustrating the low level of B61 expression (A) compared with the high uniform expression of Eck (C). Arrowheads indicate the thymic capsule in A. Exposure of similar sections to S-labeled sense strand probes produced no specific labeling (B, D). Scalebars = 50 µm.



PI-specific Phospholipase C Releases B61 from the Cell Surface

B61 had previously been shown to be a tumor necrosis factor-alpha-inducible secreted protein in human umbilical vein endothelial cells. Protein sequence analysis, however, revealed B61 to possess a C-terminal hydrophobic region that shared significant similarity with the C-terminal hydrophobic domain of GPI-linked membrane proteins(5) . This domain functions as a signal for GPI linkage and is cleaved, allowing for linkage of the GPI anchor to the new C terminus via an ethanolamine residue. Since it is evident that GPI-linked proteins can be specifically labeled with [^3H]ethanolamine, together with the use of PI-specific phospholipase C, this can be used to definitively prove GPI linkage (5) . To demonstrate that B61 can exist as a GPI-linked membrane protein, 293T cells transfected with either a full-length B61 expression construct or vector alone were treated with or without PI-specific phospholipase C (the PI-specific phospholipase C preparation used was free of protease contamination). (^5)Presence of B61 was assessed by immunoblotting with an anti-B61 monoclonal antibody (3E.6). Exposure to PI-specific phospholipase C resulted in the release of B61 into the conditioned medium (Fig. 4A). Just as in the case of transfected cells, PI-specific phospholipase C was also found to be capable of cleaving endogenous constitutively expressed B61 from the surface of ZR-S human breast carcinoma cells (Fig. 4B). To confirm that B61 indeed contains covalently attached phosphatidylinositol, B61 and vector control-transfected cells were metabolically labeled with [^3H]ethanolamine for 24 h. B61 was immunoprecipitated from the cell lysates using the B61 monoclonal antibody (3E.6) and, as shown in Fig. 4C, a labeled protein of the predicted molecular weight for B61 was specifically precipitated from B61-transfected cells but not from vector control-transfected cells. Furthermore, incubation with PI-specific phospholipase C led to the release of [^3H]ethanolamine-labeled B61 from the cell surface. In summary, these data, taken together with previous work(2, 4) , suggest that B61 can exist as a GPI-linked protein and also potentially as a soluble secreted molecule that could be generated by proteolytic cleavage(4) .


Figure 4: PI-specific phospholipase C releases B61 from the cell surface. Cells were incubated in complete medium with or without PI-specific phospholipase C (0.5 unit/ml). B61 released into the medium from transfected 293T (A) or ZR-S human breast carcinoma (B) cells was detected by immunoblotting with the B61 monoclonal antibody (3E.6). C, B61 and vector control-transfected 293T cells were metabolically labeled with [^3H]ethanolamine (50 µCi/ml) for 20 h and cells incubated with or without PI-specific phospholipase C. Cell lysates were then immunoprecipitated with the B61 monoclonal antibody (3E.6), precipitates resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.



GPI-linked B61 Is Capable of Activating the Eck Receptor Protein-Tyrosine Kinase

Soluble recombinant B61 has recently been shown to be the Eck receptor protein-tyrosine kinase ligand(4) . To determine if the membrane bound GPI-linked form also had the ability to activate Eck, resulting in its autophosphorylation, a co-culture experiment was performed. Stimulator cells consisting either of B61-transfected 293T cells (expressing GPI-linked ligand) or vector-transfected cells were added to adherent subconfluent HeLa cells that constitutively express high levels of the Eck receptor protein-tyrosine kinase (responder cells)(3) . In order to demonstrate that the activation of autophosphorylation of the Eck receptor protein-tyrosine kinase required cell-cell contact and was not due to soluble B61 shed into the medium, cell culture inserts composed of 0.45 µM protein-permeable filters were used to separate stimulator and responder cells in the co-culture assay. Alternatively the stimulator cells were directly plated onto the responder cells allowing for cell contact. Following a short incubation (10 min), the stimulator cells were removed, and the responder HeLa cells were lysed and subjected to immunoprecipitation analysis using an anti-Eck antibody(4) , followed by an anti-phosphotyrosine Western blot to assess the phosphorylation status of the precipitated Eck receptor protein-tyrosine kinase. As shown in Fig. 5, only B61 transfectants directly plated onto the responder HeLa cells were able to elicit Eck autophosphorylation. Vector-transfected or B61-transfected cells separated from the responder cells by permeable cell culture inserts were unable to induce autophosphorylation of the Eck receptor protein-tyrosine kinase. Furthermore, conditioned medium from the co-culture assay contained no detectable soluble B61 as assessed by immunoprecipitation followed by immunoblot analysis (data not shown). Taken together, these results indicate that the membrane-expressed form of B61 is capable of engaging and activating its cognate receptor. Additionally, within the 10-min time period of the assay, we asked if surface-expressed B61 induced activation of Eck to a level comparable with that induced by an optimal concentration (10 µg/ml) of soluble B61 (expressed in recombinant fashion as an immunoglobulin chimera)(17) . As shown in Fig. 5, soluble B61 chimera at its maximal concentration was better able to induce Eck autophosphorylation, but the levels achieved by surface-expressed B61 were respectable, certainly within an order of magnitude. While this result is open to interpretation in terms of physiological relevance, as one does not know the normal concentration of either soluble or surface expressed B61 (which would be impossible to accurately gauge), it does, however, emphasize that the surface-expressed form is biologically active.


Figure 5: GPI-linked form of B61 activates the Eck receptor protein-tyrosine kinase. Subconfluent adherent HeLa cells were directly incubated with either vector-transfected or B61-transfected 293T cells, or the B61 transfectants were separated by cell culture inserts for the duration of the assay. Following removal of unattached cells or the cell culture inserts, HeLa cell lysates were immunoprecipited with an anti-Eck antibody, resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to nitrocellulose, and immunoblotted with either an anti-phosphotyrosine antibody or an anti-Eck antibody. For purposes of comparison, soluble B61 in the form of an Ig chimera was added directly to a separate well of HeLa cells for the same time period, and the resulting cell lysate immunoprecipitated as per the other samples.



In summary, B61, a tumor necrosis factor-alpha-, interleukin-1-, and lipopolysaccharide-inducible primary response gene is a ligand for the Eck receptor protein-tyrosine kinase and exists in both soluble and GPI-linked forms. The surface-bound form is functional in that it is capable of binding and activating Eck. This is the first example of a receptor protein-tyrosine kinase ligand that is GPI-linked. The widespread and developmentally regulated expression of B61 and Eck suggest an important role for this ligand-receptor pair during ontogeny.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL45351. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: University of Michigan Medical School, Dept. of Pathology, 1301 Catherine St., Ann Arbor, MI 48109-0602. Tel.: 313-747-0264; Fax: 313-764-4308; vishvadixit{at}mailqm.pds.med.umich.edu.

(^1)
The abbreviations used are: GPI, glycosylphosphatidylinositol; PI, phosphotidylinositol; PBS, phosphate-buffered saline.

(^2)
R. A. Lindberg, personal communication.

(^3)
J. C. Ruiz and E. J. Robertson, manuscript in preparation.

(^4)
H. Shao, L. B. Holzman, and V. M. Dixit, manuscript in preparation.

(^5)
M. G. Low, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. Larry B. Holzman for providing B61 cDNA and Martin G. Low for providing PI-specific phospholipase C. We also thank Fred Wolf, Dr. Rory M. Marks, Xiaokang Lu, and Liandi Lou for technical assistance and Ian M. Jones and Karen O'Rourke for help in the preparation of the manuscript. We also thank AMGEN for sharing the sequence of rodent B61 with our laboratory and for providing us with anti-Eck antibody.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.