(Received for publication, August 25, 1994; and in revised form, December 22, 1994)
From the
B61 was originally described as a novel secreted tumor necrosis
factor--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.
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--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-
-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) (
)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.
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) ()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 (
)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) .
Figure 1:
Chromosomal localization of B61. A, segregation of the B61 locus on mouse chromosome 3 in
((C3H/HeJ-gld M. spretus) F
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,
-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.
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.
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
[H]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.
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--, 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.