The C. elegans fibroblast growth factor receptor (FGFR) EGL-15 is involved in two major functions: an early essential function and a cell migration guidance event. A host of hypomorphic
egl-15 mutations have been identified. Most can be placed into an allelic series based on the degree of essential function activity. However, four mutations that specifically affect SM chemoattraction cannot be fit into this allelic series. These mutations have identified molecular determinants of the EGL-15 FGFR that specify its role in cell migration guidance. The C. elegans FGFR contains an extra domain in its extracellular portion when compared to other FGFRs. This domain, termed the EGL-15-specific insert, is encoded entirely by exon 5 of
egl-15. We have discovered an alternative exon 5, exon 5A, that lies between the original known exon 5 and exon 6. Alternative splicing at exon 5 creates two different isoforms of EGL-15: EGL-15(5A) containing exon 5A; and EGL-15(5B), containing the original exon 5, now termed exon 5B. Three of the four migration-specific alleles of
egl-15 have mutations in exon 5A that would eliminate the EGL-15(5A) isoform in these mutants. Based on the specific phenotypes of these mutants and additional transgenic experiments, EGL-15(5A) is required for SM chemoattraction, whereas EGL-15(5B) is both necessary and sufficient for the essential function mediated by this receptor. C. elegans has two FGF ligands for EGL-15: EGL-17 and LET-756. The phenotypic consequences due to loss of these ligands mimics loss of the individual receptor isoforms. EGL-17, like EGL-15(5A), is required specifically for SM migration, while LET-756, like EGL-15(5B), plays an essential role. These similarities suggest a model in which isoform function is specified by the activating ligand. To test this model, we determined whether the ligands are functionally interchangeable with respect to the two processes mediated by EGL-15. Through a series of promoter swapping experiments we have demonstrated that the ligands are at least partially interchangeable but that signaling specificity is also conferred by the receptor isoform independent of the ligand used. When expressed from the
egl-17 promoter, LET-756 can act as a chemoattractant to rescue the SM migration defect of
egl-17(
n1377) mutants, although not as robustly as EGL-17. Conversely, EGL-17 can act as the ligand for the essential function in place of LET-756, although less robustly than LET-756. Importantly, the ability of either ligand to mediate chemoattraction is completely dependent on EGL-15(5A) activity. While both ligands mediate the essential function independent of EGL-15(5A). A significant part of functional specificity is therefore encoded within the receptor isoform. We have begun to address how the 5A and 5B domains alter EGL-15 function. By removing both alternative exon 5 sequences [EGL-15( 5)], we have recreated the structure of other FGFRs. This construct retains all of the functions of EGL-15(5B) but not the SM chemoattraction function of EGL-15(5A). This result suggests that the 5A domain adds the SM chemoattraction function to the basal EGL-15( 5) FGFR, and that the 5B domain is not necessary for effective mediation of the essential function. Consistent with this conclusion, a comparison between the C. briggsae and C. elegans 5A and 5B domains has revealed that 5A is more conserved (89% identical) than 5B (64% identical). We are trying to identify residues critical for 5A function by genetic screens and site-directed mutagenesis. The fourth migration-specific allele,
egl-15(
n1457), identifies the carboxy-terminal domain as critical specifically for SM chemoattraction. This allele contains a nonsense mutation that truncates both isoforms by removing the C-terminal region, yet the only apparent defect is in SM migration. This observation suggests that the C-terminal region provides a binding site for proteins that mediate SM chemoattraction. We are refining the region by structure-function analysis and identifying interacting partners through a two-hybrid screen. By analyzing the functions of these two specificity determinants on EGL-15, we hope to understand the mechanisms by which EGL-15 can mediate its different functions.