Boissel L et al. (2012), Identification and characterization of the RLIP...

XB-ART-44954

PLoS One 2012 Jan 01;73:e33193. doi: 10.1371/journal.pone.0033193.

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Identification and characterization of the RLIP/RALBP1 interacting protein Xreps1 in Xenopus laevis early development.

Boissel L , Fillatre J , Moreau J .

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The FGF/Ras/Ral/RLIP pathway is required for the gastrulation process during the early development of vertebrates. The Ral Interacting Protein (RLIP also known as RalBP1) interacts with GTP-bound Ral proteins. RLIP/RalBP1 is a modular protein capable of participating in many cellular functions. To investigate the role of RLIP in early development, a two-hybrid screening using a library of maternal cDNAs of the amphibian Xenopus laevis was performed. Xreps1 was isolated as a partner of RLIP/RalBP1 and its function was studied. The mutual interacting domains of Xreps1 and Xenopus RLIP (XRLIP) were identified. Xreps1 expressed in vivo, or synthesized in vitro, interacts with in vitro expressed XRLIP. Interestingly, targeting of Xreps1 or the Xreps1-binding domain of XRLIP (XRLIP(469-636)) to the plasma membrane through their fusion to the CAAX sequence induces a hyperpigmentation phenotype of the embryo. This hyperpigmented phenotype induced by XRLIP(469-636)-CAAX can be rescued by co-expression of a deletion mutant of Xreps1 restricted to the RLIP-binding domain (Xreps1(RLIP-BD)) but not by co-expression of a cDNA coding for a longer form of Xreps1. We demonstrate here that RLIP/RalBP1, an effector of Ral involved in receptor-mediated endocytosis and in the regulation of actin dynamics during embryonic development, also interacts with Reps1. Although these two proteins are present early during embryonic development, they are active only at the end of gastrulation. Our results suggest that the interaction between RLIP and Reps1 is negatively controlled during the cleavage stage of development, which is characterized by rapid mitosis. Later in development, Reps1 is required for the normal function of the ectodermic cell, and its targeting into the plasma membrane affects the stability of the ectoderm.

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Species referenced: Xenopus laevis
Genes referenced: actl6a arhgap1 cdc42 chrd cltc myc ralb ralbp1 reps1 reps2 tfap2a
GO keywords: receptor-mediated endocytosis

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	Figure 2. Reps1 is uniformly expressed in early development. In situ hybridization of Reps (a and b), and Chordin (c and d) in gastrula (a and c) and neurula (b and d). Each embryo was hybridized with one antisens, and one sens probe. Embryos hybridized with the sens probe are marked with *.
	Figure 3. Interaction analysis of XRLIP with Xreps1 by the two-hybrid technique. (A) Schematic representation of the XRLIP and Xreps1(253) deletion mutants used here. For XRLIP, a distinctive box represents each interacting domain. For Xreps1(253), the grey box corresponds to the EH homology domain and the black box to the coiled-coil motif. (B) Two-hybrid assay between Xreps1(253), Xreps1(RLIP-BD) or Xreps1(DRLIPBD) and human RLIP (RLIP76), Xenopus RLIP (XRLIP), or Lamin or human Ras (Hras) as controls. (C) Two-hybrid assay between Xreps1(253), Xreps1(RLIP-BD) or Xreps1(DRLIP-BD) with different deletion mutants of XRLIP or Lamin as control.
	Figure 4. Characterization of the Xreps1(253) domain critical for interaction with XRLIP, and interaction in vivo of Xreps1(253) and Xreps1(RLIP-BD) with XRLIP. (A, B) GST-XRLIP or GST alone as control were tested for their ability to pull down Xreps1(253) or Xreps1(RLIP-BD) synthesized as Myc-tagged proteins. The interaction of XRLIP with RalB G23V served as positive control. Xreps1(253) precipitated with 1 mg GST-XRLIP was analyzed. (A) The input or supernatant ranged from 1.25 ml of the 25 ml of reticulocyte extract), or (B) half an embryo from extracts of 10 embryos, were analyzed by SDS-PAGE. (C) Myc-Xreps1(253) or Myc-Xreps1(RLIP-BD) were synthesized in embryos and used to interact with endogenous XRLIP. Total lysate from 10 embryos were incubated with an anti-Myc agarose-conjugated antibody (SC-40, Santa Cruz Biotechnology) and centrifuged. The supernatant and immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-RLIP, or anti-Myc antibodies. With Myc- Xreps1(RLIP-BD), the RLIP signal was weakly detected just above an unspecific signal (,). (D) Two mg of GST-Xreps1(253), GST-Xreps1(RLIP-BD), or 1 mg of GST-XralA (G23V) preloaded using GTP-g-S, or GST alone were incubated with Myc-XRLIP (lysate from 10 embryos) and purified with glutathione-agarose beads. The precipitated proteins were resolved by SDS-PAGE, transferred onto a Hybond-P membrane (Amersham) and blotted with a monoclonal anti-Myc antibody (9E10). (E) The table recapitulates the results obtained on the in vivo and in vitro interaction of XRLIP with Xreps1.
	Figure 5. Effect of Xreps1(RLIP-BD) and Xreps1(253) on embryo pigmentation during gastrulation. (A) Animal views of gastrulae. Control uninjected embryos (a). Embryos injected in animal pole of one blastomere at the 2-cell stage with 1 ng mRNA of Myc-Xreps1(RLIP-BD) (b), Myc-Xreps1(253) (c), Mreps1 (d), or Mreps1-CAAX (e) and enlargement of hyperpigmented cell area from an embryo injected with Mreps1-CAAX (f) Red arrows show hypepigmentation area. (B) Protein expression of Xreps1(RLIP-BD) and Xreps1(253) was analyzed by Western blot with a 9E10 anti-Myc antibody, control lanes a, Xreps1(RLIP-BD) b and Xreps1(253) c, and analysis of 35S signal from reticulocyte extracts; control d, Xreps1(RLIP-BD) e and Xreps1(253) f.
	Figure 6. Effect of different mutants of XRLIP mRNAs on embryo pigmentation and cell division. The upper part describes the map of cDNA corresponding to the RNA injected. Lower part (A) uninjected embryos. (B), One blastomere at the 2-cell stage was injected in animal pole with 1 ng of XRLIP-CAAX and the phenotype of the embryos at the cleavage stage is presented. (C) Animal views of gastrula embryos injected with the different XRLIP mutants: with (2 ng of (14887)-CAAX (C) with 1 ng of (330-Cter)-CAAX (D), with 1 ng of (17295)-CAAX (E), with 2 ng (36161)-CAAX (F), with 2 ng of (469- Cter)-CAAX (G), and with 2 ng of XRLIP(499-Cter)-CAAX (H). The yellow arrow-heads indicate hyperpigmented cells in the animal hemisphere. doi:10.1371/journal.pone.0033193.g006
	Figure 8. Xreps1 protein interacts with XRLIP-CAAX at the plasma membrane. Representative confocal micrographs of animal caps dissected at the 2000-cell stage, showing the distribution of Myc- Xreps1(RLIP-BD)-CAAX expressed alone (A) or in the presence of XRLIP(379-Cter)-CAAX (G). Myc-Xreps1(RLIP-BD) mRNA (1 ng) and XRLIP(379-Cter)-CAAX (0.5 ng) were microinjected into the animal hemisphere of 2-cell stage embryos and Myc-Xreps1(RLIP-BD) was visualized with an FITC anti-Myc antibody. The animal caps were fixed and Myc-XRLIP(379-Cter)-CAAX and Myc-Xreps1(RLIP-BD) immunostained with an FITC anti-Myc antibody, (panels A, D and G stained in green), the F-actin with phalloidin-rhodamine, (panels B, E and H stained in red) and the merge of Myc-FITC with phalloidin-rhodamine are shown (panels C, F and I). Scale bars represent 50 mm. doi:10.1371/journal.pone.0033193.g008
	Figure 1. Interacting domain of RLIP involved in endocytosis.RLIP through its N-terminal and C-terminal domains interacts with μ2, and POB1 or Reps1 proteins respectively. These proteins interact in turn with complexes involved in clathrin endocytosis. The RhoGAP sequence of RLIP controls the Cdc42 activity and the actin dynamic. The Ral binding domain binds with Ral, in response to activation tyosine kinase receptor through the activation of the Ras protein.
	Figure 7. Phenotypes induced by XRLIP(172–495)-CAAX, XRLIP(330-Cter)-CAAX or XRLIP(469-Cter)-CAAX are amplified by Xreps1(253) and are rescued by Xreps1(RLIP-BD) but not.(A) The injected embryos were photographed at the gastrula stage. Embryos at the 2-cell stage were injected (a, d, g) with 1 ng of respectively XRLIP(172–495)-CAAX, XRLIP(330-Cter)-CAAX or XRLIP(469-Cter)-CAAX mRNA alone, or co-injected (b, e, h) with 1 ng of Xreps1(253) mRNA, or co-injected (c, f, i) with 1 ng of Xreps1(RLIP-BD) mRNA. Only the hyperpigmented phenotype (a, d, g) disappears (c, f, i). In g and h the yellow arrows show the hyperpigmented cells. (B) Proteins expression, from embryos injected with XRLIP(469-Cter)-CAAX alone (j) or respectively co-injected with Xreps (k) and Xreps(RLIP-BD) (l), were analyzed by Western blot with a 9E10 anti-Myc antibody.
	Figure 9. Hypothetical model of the interaction of Reps with RLIP during early development.(A) During cleavage stage, and up to MBT, Ral, RLIP and Reps are present in embryo; however no interactions are detected between them. (B) As of MBT, Ral interacts with RLIP and recruits it at the plasma membrane. It is thought that through Cdc42 it destabilizes the actin cytoskeleton and through μ2 and consequently AP2 allows endocytosis via clathrin. At this stage of development RLIP interacts also with Reps1, but (C) this interaction could be exclusive of ectodermic cells and could use another partner, such as filaggrin.

References [+] :

Cantor, Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases. 1995, Pubmed