The fibroblast growth factor (FGF) (
)gene family is
currently comprised of nine members(1, 2) ; the two
prototypes, FGF-1 (acidic) and FGF-2 (basic), lack a classical signal
sequence to direct their secretion through the conventional endoplasmic
reticulum (ER)-Golgi apparatus. However, their biological activities
are mediated by their interaction with high affinity tyrosine kinase
receptors on the cell surface(1, 2) . Because of the
important role played by the FGF prototypes in angiogenic and
neurotrophic processes, both during development and in the
adult(1, 2) , it is important to understand the
mechanism responsible for their secretion.
We have previously
reported that FGF-1 is released from FGF-1-transfected NIH 3T3 cells
after heat shock(3) . The secreted form of the protein is a
biologically inactive homodimer that can be reduced in the
extracellular environment to obtain a functional FGF-1 monomer (4) . Cysteine residues are necessary for FGF-1 release since a
FGF-1 Cys-free mutant is not secreted in response to heat
shock(4) . Moreover, the FGF-1 release pathway is independent
of the ER-Golgi-mediated pathway since pretreatment of cells with
brefeldin A, a drug known to block membrane transport from the ER to
the cis-medial Golgi (5) prior to heat shock,
augmented rather than decreased the level of FGF-1 secreted in response
to temperature stress(4) . Furthermore, methylamine and
verapamil, drugs known to inhibit the exocytotic (6) and
multidrug resistance (7) pathways, respectively, also failed to
interfere with the release of FGF-1 (4) .
In an effort to
gain further insight into this unconventional mechanism of secretion,
we attempted to determine which cysteine residue(s) is essential for
the dimerization of FGF-1 prior to its release. Because intracellular
FGF-1 exists primarily as a cytosol-associated protein (8) and
a partially unfolded form of FGF-1 is able to interact with acidic
phospholipid-containing liposomes(9) , we analyzed the ability
of FGF-1 to bind acidic and neutral phospholipids. We report that FGF-1
is able to associate specifically with phosphatidylserine and the
phospholipid-binding domain is located between FGF-1 residues 114 and
137. We also report that residue Cys
is critical for the
release of FGF-1 in response to heat shock. These data suggest that the
domains responsible for FGF-1 homodimer formation and phospholipid
binding are structurally independent. This property may therefore
enable FGF-1 to dimerize and utilize the carboxyl-terminal region to
interact with membrane/organelle-associated phosphatidylserine and/or
other phospholipid-binding proteins during the secretion pathway.
EXPERIMENTAL PROCEDURES
Materials and Reagents
Human recombinant FGF-1
was purified as described previously(10) . FGF-1 was
radiolabeled with Na
I (Amersham Corp.) using the
Bolton-Hunter reaction in which 4 µg of protein was incubated with
1 µg of chloramine T and 0.7 mCi of NaI for 30 s, and
the reaction was stopped with 10 µg of metabisulfite. The protein
was processed over a 0.4-ml heparin-Sepharose column and eluted with
Tris-buffered saline (TBS; 24 mM Tris-HCl, pH 7.4, containing
136 mM NaCl and 2 mM KCl) containing 1.5 M
NaCl. The specific activity was approximately 250-300
mCi/mM. Human recombinant
(3-[I]iodotyrosyl) epidermal growth factor
(EGF) was purchased from Amersham Corp. L-
-Phosphatidyl-L-serine (PS), L--phosphatidylcholine (PC), L--phosphatidylinositol (PI), L--phosphatidylethanolamine (PE), and L--phosphatidyl-DL-glycerol (PG) were purchased
from Sigma. Synthetic peptides corresponding to FGF-1 residues
114-137 (KKHAEKNWFVGLKKNGSCKRGPRT) and 15-29
(FNLPPGNYKKPKLLY) were synthesized using an Applied Biosystems (Foster
City, CA) model 431A peptide synthesizer and purified by reversed-phase
high pressure liquid chromatography. Amino acid analysis was conducted
using a Waters Associates Pico-Tag system and amino acid sequencing was
done using an Applied Biosystems model 477A protein sequencer.
Plasmid
Construction
FGF-1
and FGF-1 Cys-free
in the eukaryotic expression vector pMEXneo and FGF-1, FGF-1 Cys-free,
and FGF-1 C131S in the prokaryotic expression vector pET3c were
obtained as described previously(4) . There are three different
constructs in which two of the three Cys residues present in the human
FGF-1 sequence (Cys, Cys
, and
Cys
) were alternatively replaced by Ser. These FGF-1
mutants, each containing a single Cys residue, were inserted into the
pMEXneo expression vector (11) and named FGF-1 Cys (C97S/C131S), FGF-1 Cys (C30S/C131S), and FGF-1
Cys (C30S/C97S), respectively. The FGF-1 Cys (C97S/C131S) construct, in which only the Cys of the
protein was conserved, was obtained as follows. The FGF-1
Cys-free/pMEXneo plasmid was purified using a Qiagen column (Qiagen,
Inc.) and digested with BamHI/EcoRI restriction
enzymes (Boehringer Mannheim); the 867-base pair band was purified by
agarose gel electrophoresis and subcloned into BamHI/EcoRI-digested
FGF-1/pMEXneo, gel-purified prior to ligation.
The ligation was conducted with T4 ligase (Life Technologies, Inc.),
and the reaction product was analyzed by the dideoxy sequencing method
(Sequenase, U. S. Biochemical Corp.). The FGF-1 Cys (C30S/C131S) construct, in which residue Cys was
conserved, was obtained by ligation of the 867-base pair fragment from
the FGF-1 C131S/pET3c plasmid by digestion with BamHI/EcoRI into the BamHI/EcoRI
compatible ends of the FGF-1 Cys-free/pMEXneo plasmid. Purification of
plasmids, digestion, electroelution of bands, ligation, and analysis of
the product were conducted as described previously(4) . For the
FGF-1 Cys (C30S/C97S) construct, in which residue
Cys was conserved, the mutated sequence was cloned first
into the pET3c vector(4) . To obtain the FGF-1 C30S/C97S/pET3c
construct, FGF-1/pET3c and FGF-1 Cys-free/pET3c were digested with the
restriction enzyme, SphI. The 640-base pair fragment,
gel-purified from FGF-1 Cys-free/pET3c, was ligated into the SphI-compatible ends of FGF-1/pET3c. Purification, ligation,
and analysis of the product were conducted as described
previously(4) . The FGF-1 C30S/C97S/pET3c construct was
digested with BamHI/EcoRI, and the 867-base pair
fragment was gel-purified and subcloned into the BamHI/EcoRI-compatible ends of FGF-1
Cys-free/pMEXneo.
Transfection, Heat Shock, and Immunoblot
Analysis
NIH 3T3 cells were maintained in DMEM, 10% (v/v) fetal
bovine serum (FBS). When the cells were 60-70% confluent, fresh
medium was added, and the transfection was conducted using the calcium
phosphate precipitation method with either 5 µg of Qiagen-purified
FGF-1 Cys, FGF-1 Cys, or FGF-1 Cys DNA as described(4) . The medium was changed with fresh
DMEM containing 10% (v/v) FBS after 24 h, and 48 h after transfection
the selection was started with DMEM, 10% (v/v) FBS containing 800
µg/ml geneticin. The medium containing the antibiotic was changed
every 2nd day, and geneticin-resistant colonies were isolated and grown
separately in DMEM containing 10% (v/v) FBS and 400 µg/ml
geneticin. The individual NIH 3T3 cell transfectants were grown to
confluence and subjected to heat shock (42 °C, 2 h) as described
previously(4) . Conditioned media were collected, and latent
FGF-1 was activated with 0.1% (w/v) dithiothreitol and fractionated by
heparin-Sepharose chromatography, and the 1.5 M NaCl elution
fractions were resolved by 12.5% (w/v) SDS-polyacrylamide gel
electrophoresis and subjected to FGF-1 immunoblot analysis as
described(4) .
Solid Phase Phospholipid Binding
Assay
Phospholipid binding assays were performed using
polystyrene flat bottom wells (Dynatech Labs) as
described(12) . Briefly, PS, PC, PI, PE, and PG were dissolved
in ice-cold methanol to the desired concentrations (ranging from 1 to
10 µg/ml) and sonicated twice at 4 °C for 5-10 s, and 100
µl of the individual phospholipid was used to coat each well. Wells
coated with methanol alone were used as a control, and the wells were
kept at 25 °C for 18 h to evaporate the methanol. Nonspecific
binding was minimized by incubation of the phospholipid-coated wells
with TBS, containing 0.5% (w/v) gelatin, for 1 h at 37 °C. The
wells were washed three times with TBS containing 0.05% (v/v) Tween 20,
and the binding assay was performed in TBS. For the heparin competition
experiments, TBS containing 4 units/ml heparin (The Upjohn Co.), pH
7.4, was used as binding buffer. The labeled protein, I-FGF-1 or I-EGF, was added to the binding
buffer, and the wells were incubated for 1 h at 37 °C, aspirated,
and washed three times with TBS containing 0.05% (v/v) Tween 20. The
amount of radiolabeled protein bound to the phospholipid was
quantitated by an automatic 
counter. When the phospholipid
binding assay was performed under conditions of increased ionic
strength, 25-150 mM NaCl was added to the binding buffer
prior to the addition of I-FGF-1. Similarly, the
synthetic peptides were also added to the well prior to the addition of I-FGF-1 during the synthetic peptide competition
experiments.
RESULTS AND DISCUSSION
We have previously reported that FGF-1 is released from
FGF-1-transfected NIH 3T3 cells in response to heat shock as a latent
homodimer, and we were able to utilize a FGF-1 Cys-free mutant to
demonstrate the functional importance of Cys residues(4) .
Because two of the three Cys residues (Cys and
Cys) present in the human FGF-1 sequence are conserved
among all known species of FGF-1(1) , we sought to establish
whether a critical Cys residue exists for FGF-1 homodimer formation
during heat shock-induced FGF-1 secretion. Using the recombinant circle
polymerase chain reaction, we created three FGF-1 mutants in which two
of three Cys residues were converted to Ser (Fig. 1A).
Stable NIH 3T3 transfectants for each of these mutants were obtained,
and their expression levels were examined by immunoblot analysis. As
shown in Fig. 1B, the level of FGF-1 mutant expression
was comparable with the expression of FGF-1 in the wild-type NIH 3T3
transfectants. These transfected cells were individually examined for
their ability to release FGF-1 in response to heat shock. As shown in Fig. 1B, extracellular FGF-1 was observed in the
conditioned medium from FGF-1 wild-type and FGF-1 Cys NIH
3T3 transfectants but not in the media conditioned by heat shock from
either the FGF-1 Cys-free, FGF-1 Cys, or FGF-1
Cys transfectants. These data suggest that
Cys, positioned near the NH
-terminal nuclear
translocation signal in FGF-1(8) , is critical for the entry of
the protein into the heat shock-induced FGF-1 secretion pathway.
Figure 1:
Release of FGF-1
cysteine mutants in response to heat shock. NIH 3T3 cell FGF-1 and
FGF-1 Cys-free transfectants were obtained as described
previously(4) . NIH 3T3 transfectants expressing FGF-1
Cys, FGF-1 Cys, and FGF-1 Cys were prepared as described under ``Experimental
Procedures.'' A, structural alignment of the FGF-1 Cys
mutants and their release in response to heat shock. B, FGF-1
immunoblot analysis of the NIH 3T3 FGF-1, FGF-1 Cys-free, FGF-1
Cys, FGF-1 Cys, and FGF-1 Cys transfectants from lysates (L) and conditioned medium (CM) prepared as described under ``Experimental
Procedures.'' An arrow marks the position of the FGF-1
monomer.
Because brefeldin A potentiates rather than inhibits the release of
FGF-1 in response to heat shock(4) , we considered the
possibility that FGF-1 may be associated with Golgi-derived membranes.
Phospholipids are known components of plasma and organelle membranes;
thus, several phospholipids were analyzed for their ability to interact
with FGF-1. The acidic phospholipids, PS, PI, and PG, and the neutral
phospholipids, PC and PE, were evaluated in solid phase phospholipid
binding assays to assess the ability of the protein to bind
phospholipid-coated polystyrene wells. In these assays, I-FGF-1 was able to associate with PS but not with either
PI, PG, PC, or PE (Fig. 2A). In contrast, I-EGF, also an acidic protein of similar
size(13) , was unable to interact with any of the phospholipids
examined (data not shown). The association between I-FGF-1 and PS was saturable and dependent on the
concentration of PS with half-maximal binding occurring at
approximately 4 µg/ml (Fig. 2B).
Figure 2:
Solid phase I-FGF-1
phospholipid binding assay. A, phosphatidylserine (PS), phosphatidylcholine (PC),
phosphatidylethanolamine (PEA), phosphatidylinositol (PI), and phosphatidylglycerol (PG) diluted in
methanol at a final concentration of 8 µg/ml were used to coat the
wells. Wells coated with methanol alone served as a negative control.
Nonspecific binding was blocked with 0.5% (w/v) gelatin, and the wells
were washed with 0.05% (v/v) Tween 20. The binding of I-FGF-1 was performed as described under
``Experimental Procedures,'' and the data are reported as
counts/min bound per well. C, control. B, PS diluted
in methanol at a final concentration of 1, 3, 6, and 9 µg/ml was
used to coat the wells. Wells coated with methanol alone served as a
negative control. Nonspecific binding was blocked with 0.5% (w/v)
gelatin, and the wells were washed with 0.05% (v/v) Tween 20. The
binding of I-FGF-1 was performed as described under
``Experimental Procedures,'' and the data are reported as
counts/min bound per well.
An analysis of
the FGF-1 protein sequence for the presence of a consensus sequence for
phospholipid binding (14) revealed a putative structural motif
in the carboxyl-terminal region of FGF-1 between residues 112 and 132.
Because of the presence of several basic amino acids in this
sequence(1) , we anticipated that the interaction between PS
and FGF-1 may involve ionic bonds. Indeed, the interaction between I-FGF-1 and PS was reduced by increasing the ionic
strength of the binding buffer (Fig. 3A). We also
sought to determine whether this domain was indeed responsible for PS
binding. Using a synthetic peptide containing residues 114-137
from the FGF-1 sequence, it was possible to compete for the PS binding
of I-FGF-1 in the solid phase binding assay (Fig. 3B). In contrast, a second peptide corresponding
to FGF-1 residues 15-29, which includes the basic residues
involved in nuclear translocation(8) , was not able to compete
with I-FGF-1 for PS binding (Fig. 3B).
Interestingly, the crystallographic structure of FGF-1 ()demonstrates that Lys
and Lys
of the putative phosphatidylserine binding domain are positioned
around a sulfate molecule from the freezing medium(15) . Since
this sulfate may be interchangeable with phosphate, it is possible that
Lys and Lys may be responsible for the
binding of FGF-1 to the phosphate present in phosphatidylserine.
Moreover, because the heparin-binding domain has been localized to a
domain within the carboxyl-terminal region of FGF-1 (16) , we
asked whether heparin could compete with PS for I-FGF-1
binding. Indeed, heparin was able to inhibit the interaction between I-FGF-1 and PS (Fig. 3C).
Figure 3:
The heparin, ionic strength, and
structural dependence of the association between I-FGF-1
and phosphatidylserine. A, PS diluted in methanol at a final
concentration of 8 µg/ml was used to coat the wells. Wells coated
with methanol alone served as a negative control. Nonspecific binding
was blocked with 0.5% (w/v) gelatin, and the wells were washed with
0.05% (v/v) Tween 20. The binding was performed as described under
``Experimental Procedures'' except that increasing
concentrations of NaCl, ranging between 25 and 150 mM, were
added to the binding buffer before incubation with I-FGF-1. Data are reported as counts/min bound per well. B, PS diluted in methanol at a final concentration of 8
µg/ml was used to coat the wells. Wells coated with methanol alone
served as a negative control. Nonspecific binding was blocked with 0.5%
(w/v) gelatin, and the wells were washed with 0.05% (v/v) Tween 20. The
binding was performed as described under ``Experimental
Procedures'' except that peptide 114-137 and peptide
15-29 at 5, 10, and 50 ng/well were added to the binding buffer
prior to the addition of I-FGF-1. Data are reported as
counts/min bound per well. C, PS diluted in methanol at a
final concentration of 8 µg/ml was used to coat the wells. Wells
coated with methanol alone served as a negative control. Nonspecific
binding was blocked with 0.5% (w/v) gelatin, and the wells were washed
with 0.05% (v/v) Tween 20. The binding was performed with I-FGF-1 in TBS with and without 4 units/ml heparin, and
the data are reported as counts/min bound per well. 1,
control, PS-free with I-FGF-1; 2, PS with I-FGF-1 without heparin in TBS; 3, heparin and I-FGF-1 without PS; and 4, PS, heparin, and I-FGF-1 in TBS.
Several
proteins have been described that have Ca
-dependent
and -independent phospholipid-binding domains, and those that bind
specifically to acidic phospholipids include members of the annexin
family(17) . Annexins have been implicated in membrane
trafficking, signal transduction, cell-cell and cell-matrix
interactions, inhibition of coagulation, and other activities,
including Ca-regulated exocytosis and membrane fusion (17) . Neuromodulin and neuroregulin (18, 19) are among the proteins sharing the putative
phospholipid-binding consensus sequence with FGF-1 and are
brain-specific protein kinase C substrates. Interestingly, several
functions have been proposed for these proteins, including an
involvement in neurotransmitter release(19) . Synaptotagmin, a
component of exocytotic vesicles whose functional domains as a
trafficking macromolecule are cytosol-oriented, has been described as a
Ca-dependent PS-binding protein (20) and is
also involved in the recycling of clathrin-coated endocytotic
vesicles(21) . While the ability of FGF-1 to interact with PS
appears to be Ca-independent and FGF-1 does not
contain a Ca-binding structural motif, it is likely
that FGF-1 is not able to bind Ca. However, FGF-1 is
well described as a Cu-binding
protein(10, 22) . It is also interesting that
Cu is able to oxidize the formation of FGF-1
homodimer(10) , and since the FGF-1 homodimer is released in
response to heat shock, Cu may be involved in the
regulation of the FGF-1 secretion pathway. This may be particularly
appropriate for the FGF-1 release mechanism since the domain
responsible for FGF-1 homodimer formation resides near the NH terminus while the domain involved in PS binding resides near the
COOH terminus of FGF-1.
Recently, Mach and Middaugh (9) reported that a thermally modified state of the FGF-1
protein is able to interact with negatively charged lipid vesicles at a
neutral pH. In this system, a change in protein structure from its
native, globular state to a partially unfolded ``molten
globular'' state is required to obtain the binding of FGF-1 to
unilamellar vesicles(9) . Interestingly, the temperature range
that is required to obtain the unfolded state (9) is consistent
with the temperature that is able to stimulate the release of
FGF-1(3) , and our data are also consistent with the
observation that FGF-1 is able to bind to acidic
phospholipids(9) , with a specific affinity for PS. Further,
the crystal structure for FGF-1 also predicts that residue Cys may not be accessible to solvent and may also require a
significant structural rearrangement for it to participate in
intermolecular disulfide bond formation. Indeed, our data argue that
heat shock may enable FGF-1 to unfold and thus not only provide access
of residue Cys for intermolecular disulfide bond formation
but may enable FGF-1 to disrupt membrane structural integrity attaining
characteristics of ``molten globules'' as described by
Bychkova et al.(23) and van der Goot et al.(24) for protein-membrane insertion and/or translocation.
Since ``molten globule'' character has been assigned to FGF-1
as an acidic phospholipid-dependent state(9, 25) , it
is likely that the ``molten globule'' character of FGF-1 may
be specific for PS.
It is intriguing that the PS-binding domain in
FGF-1 resides near the carboxyl-terminal region of the protein. It is
possible that under conditions of temperature stress, the FGF-1
secretion pathway is activated and thermally modified FGF-1 undergoes
dimerization through the Cys residue at position 30. Under these
conditions, FGF-1 may still be able to maintain its ability to interact
with the PS component of membranes through the carboxyl-terminal
region. The interaction of FGF-1 with membranes may stabilize the
partially unfolded state of the protein exposing domains that may be
involved in the association of FGF-1 with other phospholipid-related
molecules and/or other phospholipid-binding proteins. Alternatively,
the interaction of the FGF-1 homodimer with PS may be dependent upon a
structural rearrangement, which may provide solvent accessibility to
PS. Due to the prevalent representation of PS in the inner face of
plasma membranes it is likely that this cytosolic surface represents
the target of FGF-1-PS binding activity. However, the significance of
this protein-phospholipid interaction in relation to the transport of
FGF-1 through the plasma membrane during temperature-induced secretion
remains to be determined.
ABSTRACT
