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We have developed a functional screen in yeast to identify ligands for receptor tyrosine kinases. Using this method, we cloned two Xenopus genes that activate the fibroblast growth factor (FGF) receptor. These encode novel secreted proteins, designated FRL1 and FRL2, distantly related to the epidermal growth factor and angiogenin/ribonuclease families, respectively. Both genes activate the FGF receptor in Xenopus oocytes as well as in yeast. Overexpression induces mesoderm and neural-specific genes in Xenopus explants; induction is blocked by a dominant negative inhibitor of the FGF receptor. FRL1 is broadly expressed during gastrulation and neurulation, while FRL2 is expressed principally in the axial mesoderm and brain at later stages. Our results indicate that despite their lack of similarity with FGF, FRL1 and FRL2 are ligands for the FGF receptor that play distinct roles in development.
Figure 1. Activation of the FGF Receptor in Yeast by Coexpression
of the FGF2 Gene
(A) Phosphotyrosinecontent of endogenousyeast proteins in response
to expression of FGF and its receptor. Yeast strains transformed with
the vectors (lanes 1 and 5), pG-FGFR (lanes 2 and 6) pG-FGFR plus
pFGF (lanes 3 and 7), and pG-FGFR plus pssFGF (lanes 4 and 6)
were cultured in glucose-containing media (lanes l-4) and in galactose-
containing media (lanes 5-8). Whole-cell extracts were prepared
by cell disruption with glass beads and were analyzed by immunoblotting
with anti-phosphotyrosine antibody.
(6) The detection of the FGF receptor activation in yeast cells by colony
immunoblotting. FGF and the FGF receptorwere coexpressed in yeast
and analyzed by colony immunoblotting with anti-phosphotyrosine antibody.
The FGF receptor was under the control of the GAL7 promoter
(G-FGFR) or the ACT7 promoter (A-FGFR). FGF with or without signal
sequence (ssFGF or FGF) was under the control of the GAL I promoter.
Yeast strains transformed by the indicated genes were grown on a
plate. Colonies were transferred to two membranes and cultured on
a glucose-containing or a galactose-containing plate.
Figure 2. The Activation of the FGF Receptor by FRL7 and FRLP in
Yeast
A yeast strain expressing the FGF receptor was cotransformed with
ssFGF, FRLl, and FRLPgenesunderthecontroloftheGALl promoter.
The transformants were streaked, and the colonies were transferred
to two membranes and cultured on a glucose-containing or a galactose-
containing plate. The membraneswere analyzed by colony immunoblotting
method with anti-phosphotyrosine antibody.
Figure 3. Ammo Acid Sequences of FRLl and FRLZ
(A) Sequence alignment of FRLI with mouse cripto. Stars indicate
identical amino acid residues. The putative signal sequence and the
C-terminal hydrophobic region of FRLl are underlined.
(B) Sequence alignment of FRL2 with bovine angiogenin and pancreatic
ribonuclease A. Asterisks indicate amino acid residues identical
among the three. The putative signal sequence and the C-terminal
hydrophobic region of FRLZ are underlined.
(C) Hydrophobicity plots of FRLI and FRL2 (Kyte and Doolittle, 1982).
(D) Phylogenetic analysis of FRL2 and some of the angiogeninlribonuclease
family members: human angiogenin (Kurachi et al., 1985),
mouse angiogenin (Bond and Vallee, 1990) rabbit angiogenin (Bond
et al., 1993) bovine angiogenin (Maes et al., 1988) mouse ribonuclease
A (Schuller et al., 1990) bovine ribonuclease A (Carsana et
al., 1988), and bullfrog ribonuclease (Titani et al., 1987).
Figure 4. The Activation of the FGF Receptor in Xenopus Oocytes by
FRLI and FRL2
Indicated RNAs were transcribed in vitro and injected into oocytes.
After 48 hr of incubation, oocytes were labeled with Wa2+ for 3 hr,
and released Ca2+ from oocytes was measured over 10 min.
Figure 5. FRLP Binding to the FGF Receptor
The fusion protein XFR-AP, composed of the extracellular domain of
the FGF receptor and alkaline phosphatase, was produced in COS
ceils. %-labeled FRL2 protein synthesized in the reticulocyte lysate
was mixed with the XFR-AP in the presence or absence of unlabeled
FGF. The proteins precipitated with anti-alkaline phosphatase antibody
were analyzed by autoradiography. The vector-transfected COS
cells was used as a control (vector). The receptor brings down the 18
kDa ligand at a level of 3.9x over background. Excess cold FGF
reduces this to 1.9x over background.
Figure 6. Effects of the Overexpression of FRLl and FRLZ on Animal
Caps and Embryos
(A) Morphological changes in animal caps. FRL7 RNA (5 ng) and FRLZ
RNA (1 ng) were injected into both blastomeres at the 2-cell stage.
Animal caps were isolated at stage 8, cultured for 20 hr at 16OC, and
photographed.
(B) The effect of FRLI and FRL2 on muscle actin and N-CAM expression.
Uninjected animal caps and animal caps injected with 1 ng and
5 ng of FRLI, 5 ng of FRLl plus 1 ng of HAV@, 5 ng of FRLl plus 1
ng of XFD, 1 ng of FRLZ RNA, 1 ng of FRLP plus 1 ng of HAVQ, or
1 ng of FRLP plus 1 ng of XFD RNA were cultured at 16OC for 20 hr.
RNA was isolated, and induction of muscle actin (m. actin) and N-CAM
genes was assayed by RT-PCR. EFl o was used as an internal control.
(C)Common phenotypes of the tailbud stage embryos overexpressing
FRLl and FRLP. FRLi or FRL2 RNA (1 ng) was injected into both
blastomeres at the 2-cell stage. The left side is anterior.
Figure 7. The Paracrine Signaling Assay
(A) The procedure. mRNA encoding FRLI or FRL2 (10 ng) was injected
into Xenopus oocytes. After 1 day of incubation, animal cap explants
isolated from Xenopus embryos at stage 8 were grafted onto the injected
or uninjected oocytes.
(B) The effect of FRLl and FRL2 on muscle actin and N-CAM expression.
RNA was isolated from the grafted explants when the sibling
embryos reached stage 25. Induction of muscle actin (m. actin) and
N-CAM genes was assayed by RT-PCR. EFI a was used as an internal
control.
Figure 8. The Temporal and Spatial Expression of FRL7 and FRLP
Genes during Embryogenesis
(A) Temporal expression. RNA was isolated from unfertilized eggs and
the indicated stages of embryos. The expression of the FRLl and FRLP
genes was assayed by RT-PCR. Ornithine decarboxylase (ODC) is
present as an internal control.
(B) Spatial expression. Stage 10 embryos were dissected into the dorsal
half (Dr) and the ventral half (Vn), or into the vegetal half including
the marginal zone (Vg) and the animal half (An), and RNA was isolated
from these tissues or from stage 10 embryos (Em). The expression
of the FRLl gene was assayed by RT-PCR. goosecoid (gsc) is known
to be expressed in the dorsal marginal zone (Cho et al., 1991). EFlu
is a ubquitously expressed control (Krieg et al., 1989).
(C) Whole-mount in situ hybridization in the tailbud stage embryo of
FRL2 RNA. Purple staining indicates the localization of the antisense
FRL2 probe.
unnamed (FRL2 protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
