Abstract
Fibroblast growth factors are a family of intercellular signaling molecules with multiple and varied roles in animal development. Most are exported from cells by means of a classical amino-terminal signal sequence that is cleaved from the mature protein during its passage through the secretory pathway. Fibroblast growth factor-9 (Fgf-9) does not contain a recognizable signal sequence, although it is efficiently secreted. In this study, we show that Fgf-9 enters the endoplasmic reticulum and traverses the Golgi complex in a similar manner to other constitutively secreted proteins. Deletion and point mutation analysis has revealed an atypical non-cleaved signal sequence within the amino-terminal region of Fgf-9. Moreover, the first 28 amino acids of Fgf-9 can function as an efficient non-cleaved signal peptide when appended to the amino terminus of green fluorescent protein.
Highlights
Fibroblast growth factors (Fgfs)1 are a large family of secreted polypeptides that display pleiotrophic properties in vitro [1, 2]
Faint reticular staining over the cytoplasm and strong perinuclear staining was observed for Fibroblast growth factor-9 (Fgf-9), whereas the Golgi 58K protein gave the expected peri-nuclear staining characteristic of the Golgi complex (Fig. 2, A and B)
Combination of the two optical sections and the phase contrast image clearly demonstrates a predominant co-localization of the two proteins and confirms that Fgf-9 is associated with the Golgi complex and enters the constitutive secretory pathway (Fig. 2D)
Summary
Fibroblast growth factor; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; GFP, green fluorescent protein; PBS, phosphate-buffered saline; PAI-2, plasminogen-activator inhibitor type 2. Fgf-3 is unusual in that it is secreted and an extended uncleaved form is localized in the cell nucleus [12]. In this example the dual subcellular localization appears to be the result of competition between a weak classical signal peptide and an adjacent nuclear localization signal [13]. Despite its secretion and glycosylation, the amino-terminal domain of Fgf-9 is mildly hydrophobic and does not demonstrate the expected characteristics of a signal peptide (Fig. 1A). In this report we have analyzed the sequences involved in the secretion of Fgf-9, and we show there is a non-cleaved signal sequence in the amino-terminal domain that directs protein into the constitutive secretory pathway
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