(Received for publication, October 12, 1995; and in revised form, December 27, 1995)
From the
Rse, Axl, and c-Mer comprise a family of cell adhesion
molecule-related tyrosine kinase receptors. Human Gas6 was recently
shown to act as a ligand for both human Rse (Godowski et al.,
1995) and human Axl (Varnum et al., 1995). Gas6 contains an
NH-terminal Gla domain followed by four epidermal growth
factor-like repeats and tandem globular (G) domains. The G domains are
related to those found in sex hormone-binding globulin and to those
utilized by laminin and agrin for binding to the dystroglycan complex.
A series of Gas6 variants were tested for their ability to bind to Rse
and Axl. The Gla domain and epidermal growth factor-like repeats were
not required for receptor binding, as deletion variants of Gas6 which
lacked these domains bound to the extracellular domains of both Rse and
Axl. A deletion variant of Gas6 containing just the G domain region was
shown to activate Rse phosphorylation. These results provide evidence
that G domains can act as signaling molecules by activating
transmembrane receptor tyrosine kinases. Furthermore, they provide a
structural link between the activation of cell adhesion related
receptors and the control of cell growth and differentiation by the G
domain-containing superfamily of proteins.
Specific signals that control the growth and differentiation of cells in developing and adult tissues often exert their effects by binding to and activating cell surface receptors containing an intrinsic tyrosine kinase activity. We recently reported the human and murine complementary DNA sequences of a receptor tyrosine kinase we termed Rse (Mark et al., 1994). A complementary DNA sequence encoding a protein identical to human Rse was reported independently and called Sky (Ohashi et al., 1994). Versions similar or identical to murine Rse have also been reported (Tyro3, Lai and Lemke, 1994; Brt, Fujimoto and Yamamoto, 1994; Etk2, Biesecker et al., 1995).
Rse has about 31% sequence identity with the receptor tyrosine kinases Axl (O'Bryan et al., 1991; Janssen et al., 1991) and c-Mer (Graham et al., 1994). The extracellular domains of the Rse/Axl/c-Mer family are composed of two immunoglobulin-like repeats followed by two fibronectin type III repeats. Together, these proteins define a class of receptor tyrosine kinases whose extracellular domains resemble neural cell adhesion molecules (reviewed by Rutishauser(1993) and Brummendorf and Rathjen(1993)).
While Rse mRNA is expressed preferentially in the adult brain, it is expressed at lower levels in a number of tissues including kidney, ovary, and testis and in a variety of hematopoietic cell lines (Mark et al., 1994; Lai and Lemke, 1994). Axl and c-Mer are also widely expressed, but the highest levels of Axl and c-Mer mRNA are detected in the heart and skeletal muscle (Graham et al., 1995) and testis, ovary, prostate, lung, and kidney (Graham et al., 1994), respectively.
Gas6 was initially identified
as a product of a gene whose expression is increased in fibroblasts
upon growth arrest (Manfioletti et al., 1993). We recently
identified Gas6 as a ligand for human Rse (Godowski et al.,
1995). Varnum et al. (1995) identified Gas6 as a ligand for
human Axl. Gas6 has 46% amino acid identity to Protein S (PS), ()an abundant serum protein and a negative regulator of the
coagulation cascade. Stitt et al.(1995) reported that PS, but
not Gas6, was a ligand for Rse. However, those conclusions were based
on the analysis of the interactions of human PS and bovine Gas6 with
murine Rse. While human PS does indeed bind to murine Rse, we found
that even high concentrations of human PS failed to activate human Rse
(Godowski et al., 1995). These results have recently been
confirmed by Ohsahi et al.(1995). Thus, there are no published
data indicating that PS is a physiologically relevant ligand for Rse.
Gas6 contains 678 amino acids, which may be divided into five
domains (Fig. 1A). The Gla domain is rich in
-carboxyglutamic acid (Gla) residues. The corresponding Gla domain
of PS mediates its Ca
-dependent binding to negatively
charged phospholipids (Dahlback et al., 1986; Hammond et
al., 1987). The loop region, containing thrombin-sensitive
cleavage sites in PS, but lacking such sites in Gas6, is followed by
four epidermal growth factor (EGF)-like repeats. EGF-like repeats are
found in a number of proteins which participate in diverse functions
such as coagulation and fibrinolysis, cell adhesion, cell growth, and
differentiation. These repeats are sufficient to bind to other receptor
tyrosine kinases, and are believed to participate directly in
protein-protein interactions (Cambell and Bork, 1993). The
COOH-terminal regions of Gas6 and PS are homologous to the steroid
hormone-binding globulin (SHBG) protein (Gershagen et al.,
1987; Hammond et al., 1987) and contain tandem
``globular'' or G domains (G1 and G2; Joseph and
Baker(1994)). G domains, first identified in laminin A chain, are
present in a superfamily of proteins that include basement membrane
proteins such as laminin A chain, agrin, merosin, and perlecan, as well
as the Drosophila regulatory proteins Crumbs, Fat, and Slit
(reviewed by Patthy and Nikolics(1993)).
Figure 1:
Structure and Rse
binding activity of Gas6/PS chimeric variants. A and B, schematic representation (A) and expression and
binding of proteins to Rse-Fc (B). A, for Gas6, the boxes represent the Gla domain (amino acids 49-90), the
loop region (amino acids 91-117), the region containing four
EGF-like repeats (amino acids 118-278), and the region homologous
to sex hormone-binding globulin (amino acids 279-678). This
region contains tandem G domains (G1, amino acids 314-471; G2,
amino acids 502-671). The corresponding domains of PS and
chimeric proteins PG
and G
P
are also indicated. The black box represents the epitope
tag. B, expression and binding of proteins to Rse-Fc. Proteins
of the correct molecular size were detected in unfractionated (input)
conditioned media using anti-gD antibody. M, mock-transfected; lane 1, PS.gD; lane 2, Gas6.gD; lane 3,
P
G
.gD; lane 4,
G
P
.gD. Conditioned media from mock-transfected
cells or from cells expressing epitope-tagged proteins were incubated
with Rse-Fc or control Fc, as indicated. Complexes were captured with
Protein A and fractionated by SDS-PAGE. Following Western transfer,
epitope-tagged proteins were detected using the anti-gD antibody. The
input lanes represent 20% of the material used in the binding
assay.
As a first step in understanding the contributions of the Gla, EGF-like repeats and G domains of Gas6 to interaction with Rse and Axl, we analyzed the relative contributions of these domains in receptor binding and activation. Our data demonstrate the G domains of Gas6 are sufficient for receptor activation. These observations have implications for the mechanism by which other G domain-containing proteins may influence intercellular signaling.
Protein
interaction analysis using BIAcore(TM) instruments were performed on
research grade BIAcore CM5 sensor chips. Running buffer was PBS (10
mM sodium phosphate, pH 7.4, 150 mM sodium chloride)
with 0.05% Tween 20. The sensor chip was activated by injection of 20
µl of 1:1 mixture of N-ethyl-N`-(3-dimethylaminopropyl)carbodiimide
hydrochloride and N-hydroxysuccinimide at 5 µl/min flow
rate. 20 µl of Rse-Fc at 20 µg/ml in 10 mM sodium
acetate, pH 5.0, was injected over the sensor chip, followed by 30
µl of ethanolamine. A total of 3340 response units of Rse-Fc was
immobilized onto the sensor chip, corresponding to a density of 3.34
ng/mm. Conditioned media containing tagged Gas6 or deletion
variants expressed in 293 cells were concentrated in Amicon 10
Centriprep spin concentrators, and the buffer was changed to PBS with
0.05% Tween 20 using Pharmacia PD10 Sephadex columns. The
concentrations of tagged Gas6 or deletion variants were measured by
ELISA assays. In BIAcore protein interaction assays, 30 µl of
conditioned media containing tagged Gas6 or deletion variants was
injected onto the Rse-Fc sensor chip at a flow rate of 10 µl/min by
the Kinject method. Proteins were allowed to dissociate for 20 min in
the flow of PBS with 0.05% Tween 20. The sensor chip was regenerated by
a short pulse of 2 µl of 10 mM HCl, followed by 2 µl
of 10 mM NaOH before the next sample was injected. Sensorgrams
were analyzed with BIAevaluation 2.1 software from Pharmacia Biosensor
AB. Apparent dissociation rate constants (k
) and
association rate constants (k
) were obtained by
evaluating the sensorgram with A + B = AB type I fitting.
Equilibrium dissociation constant K
was calculated
as k
/k
.
We compared the ability of tagged
Gas6, PS, PG
, or G
P
to
bind to a soluble form of the extracellular domain of Rse termed
Rse-Fc. Conditioned media from human 293 cells expressing the variants
were incubated with Rse-Fc or, as a control, just the Fc portion of
IgG. Proteins that bound to the Fc fusion proteins were recovered from
the supernatant with protein A and tagged proteins were revealed by
Western blotting of the resolved precipitates. As observed previously
(Godowski et al., 1995), Gas6.gD bound to Rse-Fc. The epitope
tag at the carboxyl terminus did not appear to influence receptor
binding because Gas6.gD bound to Rse-Fc as efficiently as a version
that contains an amino-terminal epitope tag (Fig. 3) or
authentic, untagged Gas6 (data not shown). The binding was specific
because Gas6.gD did not bind to an irrelevant Fc protein, and PS.gD did
not bind to Rse-Fc. Interestingly, P
G
.gD,
containing the Gla domain and EGF-like repeats of PS and the G domains
of Gas6, bound to Rse-Fc. The complementary fusion protein
G
P
.gD did not bind to Rse-Fc. Thus, the ability
to bind to the extracellular domain of Rse correlated with the presence
of the G domains of Gas6.
Figure 3:
Structure and Rse binding activity of Gas6
deletion variants. A, schematic representation of Gas6. The
position of the NH-terminal deletion variants are indicated
by the arrowheads. B, expression and binding of
proteins to Rse-Fc (top) and Axl-Fc (bottom).
Proteins of the correct molecular size were detected in unfractionated
(input) cell supernatants using anti-gD antibody. M,
mock-transfected; lane 1, Gas6.gD; lane 2, gD.Gas6; lane 3, gD.Gas6
; lane 4,
gD.Gas6
; lane 5, gD.PS. Binding, SDS-PAGE, and
Western blot analysis are described in the Fig. 1legend. In
contrast to PS, the Gas6 derivatives were bound by Rse-Fc and Axl-Fc.
The binding was specific to the extracellular domains of Rse and Axl
because the epitope-tagged proteins were not precipitated by control
human Fc (Fc). The input lanes represent 20% of the material
used for binding.
While tagged Gas6 and PG
were capable of binding to the extracellular domain of Rse in
vitro, it was important to compare their ability to activate Rse
expressed on the surface of cells. We compared the ability of tagged
Gas6, PS, G
P
, and P
G
to
induce phosphorylation of Rse expressed in NIH 3T3 cells. Conditioned
media from mock-transfected 293 cells or from cells expressing the
variants were added to serum-starved NIH 3T3 cells expressing human
Rse. Consistent with the results obtained in the binding assay, both
Gas6.gD and P
G
.gD induced phosphorylation of
Rse while PS.gD and G
P
.gD did not (Fig. 2).
Figure 2:
Activation of receptor phosphorylation by
Gas6/PS chimeric proteins. Conditioned media from mock-transfected 293
cells, M, or containing 40 nM epitope-tagged PS, Gas6,
PG
, or G
P
were added to
serum-starved NIH 3T3.hRse cells at 37 °C for 5 min. Rse was
immunoprecipitated from cell lysates with antibody 19B, a polyclonal
antibody directed against the extracellular domain of Rse.
Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with
anti-phosphotyrosine antibodies (
-pTyr). The blot was
stripped and probed with antibody 19B (
-Rse) to control
for the amount of Rse on the blot. Sizes of the molecular weight
standards are indicated on the left (in
kilodaltons).
Human Gas6 has also been shown to
bind to and activate the Rse-related receptor Axl (Varnum et
al., 1995). We analyzed the ability Gas6 deletion derivatives to
interact with the extracellular domain of Axl. As observed with Rse-Fc,
Axl-Fc also efficiently bound both Gas6 and
Gas6
(Fig. 3B).
These results show that the G domains of Gas6 are sufficient to bind to the extracellular domains of both Rse and Axl and that the Gla, loop, and EGF-like repeats are not absolutely required for this interaction. We then attempted to express the individual G1 and G2 domains of Gas6 in 293 cells. However, we could not detect significant levels of these variants in conditioned medium of transfected cells (data not shown).
Figure 4: Kinetics for binding of Gas6 and deletion variants to Rse-Fc coupled to a BIAcore(TM) biosensor. Rse-Fc was coupled to the carboxymethylated dextran layer on the surface of the biosensor chip. Either purified Gas6 or buffer-exchanged media containing the indicated proteins were injected over the surface of the biosensor at 160 s. At 340 s, the injector loop was switched to buffer to follow dissociation. No change in RU was observed when buffer-exchanged media from mock-transfected cells were passed over the Rse-Fc chip (data not shown).
Figure 5:
Activation of Rse phosphorylation by Gas6
deletion derivatives. Conditioned media from mock-transfected cells, or
containing 40 nM Gas6.gD, gD.Gas6, gD.Gas6, or
gD.Gas6
were added to serum-starved NIH 3T3.hRse cells
at 37 °C for 5 min. SDS-PAGE, immunoprecipitation, and Western
blotting analysis are described in the Fig. 2legend. Molecular
sizes are indicated on the left (in
kilodaltons).
Figure 6:
Dose response of activation of Rse by Gas6
and deletion derivatives. The amount of phosphorylation of Rse in
response to the indicated concentrations of either Gas6,
Gas6, or Gas6
was determined using the
KIRA assay.
, gD.Gas6;
, gD.Gas6
;
,
gD.Gas6
.
Gas6 is secreted ligand with structural homology to members
of a superfamily of basement membrane proteins implicated in the growth
and differentiation of many cells. A series of Gas6 variants were
expressed to begin to define the relative roles of the Gla domain,
EGF-like repeats, and G domains in receptor binding and activation. Our
deletion studies show that the Gla and EGF-like repeats are not
absolutely required for receptor binding or activation, and that the G
domain region is sufficient for these activities. The on-rates and
off-rates for binding of either full-length Gas6, Gas6,
and Gas6
to the extracellular domain of Rse were
similar. This maps the receptor binding domain of Gas6 to the G
domain-containing region. Furthermore, it shows that the Gla and EGF
repeats contribute little to receptor binding.
We also quantitated
the effects of deletion of the Gla and EGF-like repeats of Gas6 on
activation of Rse phosphorylation. In the KIRA assay, we observed that
the EC for activation of Rse by Gas6
was
reduced approximately 2-fold compared to intact Gas6. Thus, while the
Gla and EGF repeats are not absolutely required for receptor
activation, they may contribute directly or indirectly to this process.
Furthermore, either the Gla or EGF-like domains may modulate Gas6
activity in vivo. For example, Gas6 binds to cell membranes in
a Ca
-dependent fashion. (
)Ca
-dependent binding to membranes and
phospholipids is characteristic of Gla domain-containing
-carboxylated proteins. The Gla domain of Gas6 may function to
promote the establishment of a local concentration gradient of the
ligand. Alternatively, the Gla domain might allow Gas6 to be
concentrated on the surface of receptor-bearing target cells.
The deletion analysis of Gas6 localized the receptor binding domain to a region that contains tandem G domains. First characterized as five repeating globular domains of approximately 180 residues found at the COOH terminus of laminin A chain (Patthy, 1991), G domains are found in a superfamily of basement membrane proteins as well as in integral membrane proteins such as Drosophila Crumbs, Fat, and Slit. The individual G domains of Gas6 are most closely related to those found in PS and SHBG and are more distantly related to those found in agrin and laminin. SHBG, which binds to both estrogens and androgens with high affinity, has been reported to regulate intracellular signaling in prostatic stromal cells by binding to cell surface receptors (Hryb et al., 1985; Hryb et al., 1989; Nakhla et al., 1994), although the specific receptors involved have not yet been identified. Interestingly, steroids are reported to regulate the ability of SHBG to bind to and activate its receptor. Steroid-free SHBG is required for binding to the receptor, but activation of the receptor requires subsequent binding of SHBG by a specific steroid ligand. Based on sequence similarity, and our observation that the SHBG-like region of Gas6 is sufficient to bind Rse and Axl, we speculate that SHBG will interact with a Rse-related molecule.
A number of G domain-containing proteins have been shown
to play a role in regulating cellular growth and development. Crumbs,
Fat, and Slit are involved in ectodermal differentiation processes such
as neurogenesis and epithelial polarization (Mahoney et al.,
1991; Rothberg et al., 1990; Tepass et al., 1990).
The ability of laminin to promote epithelial cell polarization and
neurite outgrowth are dependent upon the G domains. Similarly, the G
domains of perlecan have been suggested to mediate the neurite
outgrowth activity of perlecan-laminin complexes (Noonan et
al., 1991). Aggregation of acetylcholine receptors on skeletal
muscle fibers is induced by the binding of the G domains of agrin and
laminin to dystroglycan-, a component of the dystrophin receptor
(Gee et al., 1993; Campanelli et al., 1994; Gee et al., 1994;).
Our results demonstrate that the G domains of Gas6 are necessary and sufficient for a functional interaction with both Rse and Axl and provide direct evidence that G domains can activate receptor tyrosine kinases. Furthermore, they suggest that other G domain-containing proteins may exert their effects by binding to cell adhesion molecule-related proteins and subsequently activating intracellular signaling pathways.