(Received for publication, August 28, 1995; and in revised form, October 11, 1995)
From the
Fibroblast growth factor (FGF)-1 lacks a classical signal
sequence to direct its secretion yet utilizes high affinity cell
surface receptors to signal its heparin-dependent angiogenic and
neurotrophic activities. We have previously reported that FGF-1 is
released in response to temperature stress as a latent homodimer
through a pathway that is potentiated by the Golgi inhibitor, brefeldin
A (Jackson, A., Tarantini, F., Gamble, S., Friedman, S., and Maciag,
T.(1995) J. Biol. Chem. 270, 33-36). In an attempt to
further characterize this unconventional secretion mechanism, we sought
to define the Cys residue(s) critical for FGF-1 dimer formation and
release and to determine whether FGF-1 can associate with known
phospholipid components of organelle or plasma membranes, which may be
disturbed by brefeldin A. Utilizing FGF-1 Cys mutants, we were able to
demonstrate that residue Cys is critical for FGF-1 release
in response to heat shock. In addition, using solid phase phospholipid
binding assays we demonstrate that FGF-1 is able to specifically
associate with phosphatidylserine (PS). Heparin inhibits the
association between FGF-1 and PS, and synthetic peptide competition
assays suggest that the PS-binding domain of FGF-1 lies between
residues 114 and 137. These observations indicate that FGF-1 may be
able to associate with the PS component of organelle and/or plasma
membranes and that the domains responsible for FGF-1 homodimer
formation and PS binding are structurally distinct.
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.
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.