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CD2AP/CMS regulates endosome morphology and traffic to the degradative pathway through its interaction with Rab4 and
Mireille Cormont1, Isidoro Metón, Muriel Mari
1Inserm U568, IFR 50, Faculty of Medicine, University of Nice, 06107 Nice cedex 02, France. cormont@unice.fr
Abstract:
The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4-Q67L, the GTP-bound form of Rab4. We cloned an ubiquitous 80-kDa protein, identical to CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4-Q67L in the yeast two-hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS-positive vesicles. However, coexpression of Rab4-Q67L with CD2AP/CMS induces a significant enlargement of EEA1-positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c-Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c-Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c-Cbl inhibits ligand-induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c-Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.
Insights
The study identifies CD2-associated protein (CD2AP/CMS) as a novel effector of Rab4, a small GTPase crucial for vesicular trafficking. This interaction influences early endosome morphology and the degradation pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Endocytosis and Vesicular Trafficking
Background:
- The small GTPase Rab4 regulates early endosome sorting and recycling.
- Understanding Rab4's effectors is key to deciphering vesicular trafficking regulation.
- Early endosomes are critical for internalizing and processing cellular cargo.
Purpose of the Study:
- To identify specific effectors interacting with the GTP-bound form of Rab4 (Rab4-Q67L).
- To elucidate the role of identified effectors in regulating Rab4-mediated vesicular trafficking.
- To investigate the impact of Rab4-effector interactions on early endosome function and morphology.
Main Methods:
- Yeast two-hybrid screening to identify Rab4-Q67L interacting proteins.
- In vitro interaction assays and mammalian cell expression studies.
- Confocal microscopy for colocalization studies with endosomal markers (EEA1, Rab5, Rab7, Rab11) and actin filaments.
- Analysis of endosome morphology and function using truncated protein constructs.
Main Results:
- Identified and cloned CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) as a Rab4-Q67L interacting protein.
- CD2AP/CMS localizes to punctate structures and actin filaments in mammalian cells.
- Coexpression of Rab4-Q67L and CD2AP/CMS leads to enlarged EEA1-positive early endosomes, with Rab4, CD2AP/CMS, and Rab7 colocalizing.
- Early endosome enlargement requires CD2AP/CMS interaction with both Rab4 and c-Cbl.
- A CD2AP/CMS mutant interacting with Rab4 but not c-Cbl inhibits PDGF receptor degradation.
Conclusions:
- CD2AP/CMS acts as an effector for Rab4, influencing early endosome morphology.
- The interaction of CD2AP/CMS with Rab4 and c-Cbl is crucial for regulating early endosome size.
- CD2AP/CMS plays a role in the traffic between early and late endosomes, impacting the degradative pathway and receptor trafficking.