By using p65 synaptotagmin-1 and fibroblast growth factor (FGF)-1:-galactosidase (-gal) NIH 3T3 cell cotransfectants, we demonstrate that a proteolytic fragment consisting of the extravesicular domain of synaptotagmin-1 is released into the extracellular compartment in response to temperature stress with similar kinetics and pharmacological properties as FGF-1:-gal. Using a deletion mutant that lacks 95 amino acids from the extravesicular domain of synaptotagmin-1, neither synaptotagmin-1 nor FGF-1:-gal are able to access the stress-induced release pathway. Furthermore, the p40 extravesicular fragment of synaptotagmin-1 is constitutively released in p40 synaptotagmin-1 NIH 3T3 cell transfectants, and this release is potentiated when the cells are subjected to temperature stress. These data demonstrate that the p40 fragment derived from synaptotagmin-1 is able to utilize the FGF-1 non-classical exocytotic pathway and that the release of FGF-1 is dependent on synaptotagmin-1. FGF1 -1 and FGF-2 are the prototype members of a large family of related heparin-binding growth factors that regulate numerous biological processes including mesoderm formation, neurogenesis, and angiogenesis in vivo (1). The FGF prototypes are distinguished among the FGF family members by the absence of a classical signal peptide sequence to direct their secretion through the conventional endoplasmic reticulum (ER)-Golgi pathway. Because the FGF prototypes initiate their biological responses through the activation of high affinity receptors on the surface of target cells, it has been suggested that the secretion of both FGF-1 and FGF-2 may be regulated by novel release pathways. Indeed, the ligation of a signal peptide sequence to FGF-1 followed by somatic gene transfer, which results in the constitutive release of FGF-1, yields a functional oncogene in vitro and exaggerated vascular hyperplasia in vivo (2, 3).We have reported that FGF-1 is released from stable FGF-1 NIH 3T3 cell transfectants through a non-classical exocytotic pathway as a latent macromolecule in response to temperature stress which requires activation by either (NH 4 ) 2 SO 4 (4) or reducing agents (5-7). During the structural characterization of FGF-1 (8), a heparin-binding protein (p40) that co-eluted with FGF-1 was identified by amino-terminal sequence analysis as a proteolytic fragment of the synaptotagmin-1 (Syn-1) translation product (8). Syn-1 is a 65-kDa vesicular protein and has been most extensively studied in neuronal and endocrine cells. The Syn gene family currently consists of 11 members (9, 10) and is characterized by a divergent amino terminus, a transmembrane domain, a large cytoplasmic region, which is comprised of two repeats of sequences homologous to the protein kinase C C2 domain, and a highly conserved carboxyl terminus (11,12). The p40 proteolytic fragment identified in neural tissue corresponds to the extravesicular portion of Syn-1. Genetic and microinjection studies have suggested that Syn-1 is the Ca 2ϩ sensor for neurotransmitter release (1...
Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells in response to heat shock as a biologically inactive protein that is unable to bind heparin and requires activation by (NH4)2SO4 to generate a biologically active extracellular heparin-binding growth factor (Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag, T. (1992) Proc. Natl. Acad. Sci. USA 89, 10691-10695). To further study the mechanism of FGF-1 release in response to heat shock (42 degrees C), we examined the kinetics of FGF-1 release from FGF-1-transfected NIH 3T3 cells and observed that the cells require at least 1 h of exposure to heat shock conditions for the release of FGF-1. Interestingly, agents that interfere with the function of the endoplasmic reticulum-Golgi apparatus, exocytosis, and the multidrug resistance pathway (brefelden A, methylamine, and verapamil, respectively) do not inhibit the release of FGF-1 in response to temperature; rather, they exaggerate the release of FGF-1. Because immunoblot analysis of FGF-1 in the conditioned medium of heat-shocked NIH 3T3 cells revealed the presence of a minor band with an apparent molecular weight of a FGF-1 homodimer and because we have previously shown that FGF-1, but not FGF-2, is able to form a homodimer in response to chemical oxidation by CuCl2 (Engleka, K. A., and Maciag, T. (1992) J. Biol. Chem. 267, 11307-11315), we examined whether reducing agents would substitute for (NH4)2SO4 and activate extracellular FGF-1. Indeed, dithiothreitol and reduced glutathione are able to individually generate a FGF-1 monomer as a heparin-binding protein from the conditioned medium of heat-shocked NIH 3T3 cell transfectants. To confirm that cysteine residues are involved in the release of FGF-1 in response to temperature, we used mutagenesis to prepare a human FGF-1 Cys-free mutant in which Cys30, Cys97, and Cys131 were converted to serine. Analysis of the release of the FGF-1 Cys-free mutant in NIH 3T3 cells transfected with the FGF-1 Cys-free mutant demonstrated that the FGF-1 Cys-free mutant is not released into the conditioned medium in response to temperature. Interestingly, exposure of the NIH 3T3 cell FGF-1 Cys-free transfectants to brefelden A followed by heat shock also demonstrated the absence of the extracellular FGF-1 Cys-free mutant.(ABSTRACT TRUNCATED AT 400 WORDS)
The heparin-binding fibroblast growth factor (FGF) prototypes lack a classical signal sequence, yet their presence is required in the extracellular compartment for the activation of cell-surface receptor-dependent signaling. Early studies with FGF-1 demonstrated its presence in bovine brain as a novel high molecular weight complex, and subsequent studies identified a second heparin-binding protein that co-purified with FGF-1. Polypeptide sequence analysis revealed that this heparin-binding protein corresponded to the extravesicular domain of bovine synaptotagmin (Syn)-1, a transmembrane component of synaptic vesicles involved in the regulation of organelle traffic. Since FGF-1 is released in response to heat shock as a mitogenically inactive Cys-30 homodimer, we sought to determine whether this heparin-binding protein was involved in the release of FGF-1. We report that a proteolytic fragment of the extravesicular domain of Syn-1 is associated with FGF-1 in the extracellular compartment of FGF-1-transfected NIH 3T3 cells following temperature stress. By using heparin-Sepharose affinity to discriminate between the monomer and homodimer forms of FGF-1 and resolution by conventional and limited denaturant gel shift immunoblot analysis, it was possible to identify FGF-1 and Syn-1 as potential components of a denaturant-and reducing agent-sensitive extracellular complex. It was also possible to demonstrate that the expression of an antisense-Syn-1 gene represses the release of FGF-1 in response to heat shock. These data indicate that FGF-1 may be able to utilize the cytosolic face of conventional exocytotic vesicles to traffic to the inner surface of the plasma membrane where it may gain access to the extracellular compartment as a complex with Syn-1.
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 30 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.
While the prototype members of the fibroblast growth factor (FGF) family, FGF-1 and FGF-2 are structurally related, the structural differences between these polypeptides predict that they will ultimately exhibit different biological roles. Indeed, a significant difference between these proteins is the dependence of FGF-1 on heparin for the generation of maximal mitogenic activity. In order to gain structural insight into the issue of FGF-1 heparin-dependence, a synthetic gene encoding FGF-2 was constructed with oligonucleotides in a four-cassette format similar to a synthetic gene previously constructed for FGF-1 (Forough et al. 1992, Biochem. Biophys. Acta 1090 293-298). This strategy permitted the molecular shuffling of corresponding cassette(s) between FGF-1 and FGF-2 to yield FGF-1:FGF-2 chimeras. Three amino acid changes (Lys86-->Glu, Tyr120-->His, and Thr121-->Ala) were introduced into the synthetic FGF-2 gene by the cassette format to generate convenient FGF-1 restriction sites, but these alterations did not significantly affect the mitogenic activity or the heparin-binding affinity of the recombinant FGF-2 protein when compared with native FGF-2. Among the various FGF-1:FGF-2 chimeric constructs, one designated FGF-C(1(1/2)1 1), which represents FGF-1 containing FGF-2 amino acid residues 65 to 81, displayed FGF-1-like heparin-binding affinity but it did not require the addition of exogenous heparin to manifest its mitogenic activity. These data suggest that the sequence within residues 65 and 81 from FGF-2 significantly contributes to the heparin-dependent character of FGF-1.
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