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RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor
V Jullien-Flores1, Y Mahé, G Mirey
1Institut Curie, INSERM (Institut National de la Santé et de la Recherche Scientifique) U-528, 75248 Paris Cedex 05, France.
Abstract:
RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.
Insights
The Ral-RLIP76 pathway links cell signaling to endocytosis. RLIP76 interacts with the AP2 complex, influencing receptor trafficking and Ras signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- RLIP76 (Rho-related GTPase-binding protein) is a modular protein linked to Ral GTPase signaling.
- Understanding RLIP76's interactions is crucial for elucidating its role in Ras-mediated pathways.
Purpose of the Study:
- To identify RLIP76 interacting partners and explore its function in the Ral-RLIP76 pathway.
- To investigate the connection between Ras signaling, RLIP76, and cellular endocytosis.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions.
- Two-hybrid assays to confirm interactions in vitro.
- Endocytosis assays using transferrin and epidermal growth factor (EGF) receptors in HeLa cells.
Main Results:
- Mu2, a subunit of the AP2 complex, was identified as an RLIP76 interacting partner.
- Endogenous AP2 and RLIP76 form a complex in vivo, independent of growth factor stimulation.
- RLIP76 distinguishes between AP1 and AP2 complexes, and mu1 and mu2 subunits.
- Activated Ral signaling interferes with transferrin and EGF receptor endocytosis.
Conclusions:
- The Ral-RLIP76 pathway acts as a bridge between signal transduction and endocytosis.
- RLIP76's interaction with the AP2 complex mediates its role in receptor trafficking.
- This pathway provides a novel link between Ras signaling and the regulation of endocytosis.