Caenorhabditis elegans fibroblast growth factor receptor signaling can occur independently of the multi-substrate adaptor FRS2

Abstract

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y(1009) and Y(1087)), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

Figure 1.—

The SEM-5∷EGL-15 interaction is dependent upon the SEM-5 SH2 binding sites in the CTD of EGL-15. (A) Important residues in the EGL-15 CTD isoforms. The C-terminal portion of the kinase domain and the CTD are shown. The gray box is common to all C-terminal isoforms. Sequences of the CTD isoforms can be found in Figure S5. (B) SEM-5 binds directly to EGL-15 Y1009 and Y1087. A full-length SEM-5 prey was mated to eight EGL-15 baits: empty vector control (pBTM116) and derivatives containing variants of either the type I or the type IV EGL-15(Intra). Growth is an indication of an interaction between SEM-5 and the EGL-15 bait. Dilutions of the culture mixture are indicated above. hFGFR1, a bait containing the corresponding portion of the intracellular domain of the human FGFR1. Similar results were obtained using the human SEM-5 ortholog, Grb2.

Figure 2.—

Genomic assay results of the EGL-15 CTD role in SM chemoattraction. EGL-15 Y1009 and Y1087 are functionally redundant. Sex myoblast distributions for three lines for each construct are shown. The indicated transgenes were all tested in an egl-15(null) background. (A) Wild-type EGL-15 (NH#112). (B) A truncated version of EGL-15 that correlates to the egl-15(n1457) mutant (NH#838). (C) The single Y884F mutation fails to disrupt sex myoblast migration (NH#1379). (D and E) The single Y1009F (NH#818) or Y1087F (NH#1382) mutations do not account for the posterior SM positions of ΔCTD. (F and G) Double mutations with Y884 (Y884F/Y1009F, NH#841; Y884F/Y1087F, NH#1380) are not more affected than the respective single mutations. (H and I) The Y1009F mutation in conjunction with either the Y1087F mutation (NH#1370) or with both Y1087F and Y884F (NH#1356) (ΔSEM-5) abolishes SM chemoattraction. n, number of sex myoblasts scored.

Figure 3.—

The cassette assay (“sufficiency”) results. (Top) Schematic of the cassette constructs in comparison with the wild-type genomic rescuing construct. (Bottom) Cassette constructs with the indicated CTD fragments were all tested in an egl-15(null) background. (A) Empty cassette (NH#1321, negative control). (B) E⁹⁹⁴-Q¹⁰²⁶ (the gray fragment in Figure 1A) is sufficient for mediating sex myoblast migration (NH#1322). (C) Y¹⁰⁰⁹ is required within the gray (E⁹⁹⁴-Q¹⁰²⁶) fragment to mediate sex myoblast migration (NH#1325). (D and E) Sex myoblast distribution for the wild-type (D) unique portion of the type IV isoform (NH#1348) or the same unique portion with the Y^1087F mutation (E; NH#1349).

Figure 4.—

Comparison of FGFR and FRS2 signaling complexes in vertebrates and C. elegans. (A) The interaction between the vertebrate FGF receptor and Grb2 requires the signaling adaptor protein, FRS2. (B) The C. elegans FGF receptor EGL-15 can bind SEM-5 directly at Y¹⁰⁰⁹ and Y¹⁰⁸⁷, which are required for SM chemoattraction. (C) The C. elegans FRS2 homolog, ROG-1, is involved in a RAS/MAP kinase signaling pathway that is required for germline progression, but does not involve EGL-15.