Analysis of Thisbe and Pyramus functional domains reveals evidence for cleavage of Drosophila FGFs

Abstract

BackgroundAs important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF) proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity.ResultsIn this work, we conducted an analysis of the functional domains of two Drosophila proteins, Thisbe (Ths) and Pyramus (Pyr), which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2) as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1) the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2) the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present.ConclusionsWe propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity.

Functions and Mechanisms of Fibroblast Growth Factor (FGF) Signalling in Drosophila melanogaster

“…The Drosophila FGF primary sequence contains an amino-terminal signal peptide, an FGF core domain and a long carboxy-terminal sequence with no significant homologies to other proteins [5]. Cultured cell studies demonstrated that the carboxy-terminal domains of Pyr and Ths are cleaved off suggesting that these FGFs are produced as precursors and that polypeptides containing the FGF core domain are secreted (Figure 3) [52]. The significance of this potential regulatory step is unclear as transgenes expressing carboxy-terminal truncation of Ths and Pyr are functional indicating that cleavage of the carboxy-terminal domain is not a prerequisite for secretion or receptor activation [52,53].…”

“…Cultured cell studies demonstrated that the carboxy-terminal domains of Pyr and Ths are cleaved off suggesting that these FGFs are produced as precursors and that polypeptides containing the FGF core domain are secreted (Figure 3) [52]. The significance of this potential regulatory step is unclear as transgenes expressing carboxy-terminal truncation of Ths and Pyr are functional indicating that cleavage of the carboxy-terminal domain is not a prerequisite for secretion or receptor activation [52,53]. A case for proteolytic control of FGF ligand distribution has been made during the development of the larval ASP, where spatial restriction of FGF ligands by the extracellular matrix metallo-protease Mmp2 has been shown to concentrate FGF to the tip territory of the air sac by degrading FGF around the adjacent stalk cells [54].…”

The role of FGF signaling in guiding coordinate movement of cell groups

“…Alternatively, FGFs may exhibit different range of action or be subject to different regulation. Regarding this last point, we have determined that these ligands are differentially cleaved and that the C-terminus of Thisbe may function to inhibit activity 34 . Drosophila, with a total of three FGF ligands compared with 22+ genes in vertebrates, is an attractive model system to investigate the individual activities of FGFs.…”

Controlled microfluidics to examine growth-factor induced migration of neural progenitors in the Drosophila visual system

“…In particular, our group has demonstrated that Notch loss of function disrupts RBG migration (Shamloula et al, 2002), while exogenous EGF signaling has been shown to activate long-distance migration of retinal progenitor cells (Unachukwu et al, 2013). In addition, FGF ligands are known to control glia differentiation, migration, and axonal wrapping central to vision (Silies et al, 2010; Tulin and Stathopoulos, 2010a,b). FGF signaling molecules are believed to be produced at a localized source and then dispersed into targeted tissue.…”