Abstract
LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.
Highlights
C. elegans vulval cell induction requires a highly conservedEpidermal Growth Factor Receptor (EGFR)/Ras GTPase/Mitogen Activated Protein Kinase (MAPK) signaling pathway providing an in vivo model in which to study signaling in a polarized epithelia [1,2]
Contrary to the role of AP-1 in basolateral sorting in mammalian cells, we demonstrate that AGEF-1 BIG1/2 and UNC-101 AP-1m1 antagonize the basolateral membrane localization of LET-23 EGFR in the vulval precursor cells (VPCs)
In C. elegans, we show that loss of AGEF-1 results in an increase in basolateral EGFR localization in the vulva precursor cells, and in sensitized genetic backgrounds, a corresponding increase in vulva induction
Summary
C. elegans vulval cell induction requires a highly conservedEpidermal Growth Factor Receptor (EGFR)/Ras GTPase/Mitogen Activated Protein Kinase (MAPK) signaling pathway providing an in vivo model in which to study signaling in a polarized epithelia [1,2]. An equivalence group of six vulval precursor cells (VPCs), P3.p-P8.p, have the potential to be induced to generate the vulva. The anchor cell in the overlying gonad secretes the LIN-3 EGF-like ligand, inducing the closest VPC, P6.p, to adopt the primary vulval fate, and a combination of graded LIN-3 EGF signal and lateral signaling by LIN-12 Notch specifies the neighboring VPCs, P5.p and P7.p, to adopt the secondary vulval fate. Together P5.p-P7.p generate the 22 nuclei of the mature vulva, eight cells from the UNC-101 was identified as a negative regulator of LET-23 EGFR signaling, its mechanism of action has remained an enigma [12]. Contrary to the role of AP-1 in basolateral sorting in mammalian cells, we demonstrate that AGEF-1 BIG1/2 and UNC-101 AP-1m1 antagonize the basolateral membrane localization of LET-23 EGFR in the VPCs. the AGEF-1/Arf
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