Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

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   Figure 1

   FSH enhances cap-dependent translation. HEK293–FSHR cells were transfected with bicistronic expression vector plasmids expressing cap-dependent Renilla luciferase. Forty-eight hours after transfection, cells were serum-starved for 2 h, prior to stimulation for 24 h with increasing concentrations of FSH, as indicated. Results are expressed as relative light units (RLU) of cap-driven luciferase activity. Values represent the mean±s.e.m. of three independent experiments, each done in triplicate. ***P<0.001.
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   Figure 2

   Cap-binding proteins in FSH-stimulated cells. (A and B) Cap-binding proteins were precipitated using m⁷GTP-Sepharose-coated beads and analyzed by western blot to detect 4E-BP1 and eIF4G association, as indicated. WCL, whole cell lysate. Western blots were reprobed with an anti-eIF4E antibody to detect eIF4E constitutively bound to the cap as an input control. Results for one representative experiment out of five are shown. In (C) and (D), respectively 4E-BP1 (n=4) and eIF4G (n=3) recruitment to the 5′ cap was compared in FSH-stimulated vs 20% FBS-stimulated cells. One hundred percent was not reached in case the timepoint of the maximum response varied. ***P<0.001.
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   Figure 3

   FSH increases eIF4E phosphorylation. Cells were treated with FSH for the indicated period of time before lysis. (A) Phosphorylation of eIF4E on Ser209 was analyzed by western blot. Membranes were reprobed with an anti-GAPDH antibody to monitor gel loading. (B) Densitometry analysis of western blots probed with antibodies raised against eIF4E phosphorylation on Ser209 (n=3). Values are expressed as mean±s.e.m. *P<0.05.
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   Figure 4

   eIF4G phosphorylation is a rapamycin-sensitive step in FSH-regulated translation. (A) Western blot analysis of eIF4G phosphorylation on Ser1108, in HEK293–RFSH cells pre-treated or not with 10 nM rapamycin before FSH stimulation for the indicated period of time. Membranes were reprobed with an anti-vinculin antibody to monitor gel loading. (B) Densitometry analysis of western blots probed with antibodies raised against eIF4G phosphorylation on Ser1108 (n=6). Values are expressed as mean±s.e.m. One hundred percent was not reached in case the timepoint of the maximum response varied. ***P<0.001.
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   Figure 5

   eIF4G is phosphorylated at the 5′ cap, in a rapamycin-sensitive manner, upon FSH stimulation. (A) eIF4G phosphorylated on Ser1108 was precipitated using m⁷GTP-Sepharose-coated beads and analyzed by western blot, in a time-course experiment for FSH stimulation, as indicated. WCL, whole-cell lysate. Western blots were reprobed with anti-eIF4G, to normalize for eIF4G bound to the 5′ cap, and with an anti-eIF4E antibody to detect eIF4E as an input control. (B) Densitometry analysis of western blots probed with antibodies raised against phosphorylated eIF4G (n=3). Values are expressed as mean±s.e.m. ***P<0.001.
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   Figure 6

   FSH also enhances IRES-dependent translation. HEK293–FSHR cells were transfected with plasmids expressing cap-dependent Renilla luciferase and VEGF IRES_B-dependent firefly luciferase. Forty-eight hours after transfection, cells were serum-starved for 2 h, prior to stimulation for 24 h with increasing concentrations of FSH. (A) Results are expressed as RLU. (B and C) Comparison of the ratio of VEGF IRES_B:cap-driven luciferase activity (B) vs the FGF2 IRES:cap ratio (C). Values are expressed as mean±s.e.m. (n=3). *P<0.05, **P<0.01, and ***P<0.001.
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   Figure 7

   Molecular network triggered by FSH to control translation in HEK293–FSHR cells. The Cell Designer editor has been used. Only the molecular species analyzed in this study are shaded. Complexes are surrounded by black boxes. Dashed lines indicate indirect catalysis. The semantics of the diagram editor are as follows: protein, ▭; active protein, ; receptor, ; RNA, ; direct catalysis, ; phosphorylation, ℗; association, ↣; dissociation, ; translation, . A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0009.

• © 2014 Society for Endocrinology