Ligand-independent role of ErbB3 in endocytic recycling

Abstract

Running title: Integral role of ErbB3 in the endocytic recycling machineryIntegral role of ErbB3 in the endocytic recycling machinery 2 Abstract Internalisation and trafficking of activated receptor tyrosine kinases (RTKs) determines duration and spatial distribution of growth factor signalling. Here we show that the ErbB3 receptor, frequently overexpressed in invasive cancers, acts in a ligand-independent manner to sort cargo such as b1 integrins and the transferrin receptor (TfR) for endocytic recycling breast epithelial cells. Loss of ErbB3 abrogates recycling of b1 integrins from a Rab4-positive compartment and redirects it towards degradation.Consequently, delivery of b1 integrins to the leading front of migrating epithelial cells is impaired and cell migration compromised upon loss of ErbB3. Mechanistically, ErbB3 interacts with and stabilises the Rab4/5 effector rabaptin5 and the endosomal adaptor GGA3 thereby facilitating assembly of the Arf6-GGA3-rabaptin5 complex, required for recycling of cargo including integrins and TfR. We here show that ErbB3 is an integral part in the endosomal trafficking machinery, provoking the notion that RTKs might play a more instrumental role in vesicular trafficking than commonly perceived. IntroductionThe Epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTKs), including EGFR, ErbB2, ErbB3 and ErbB4, form homo-or heterodimers and play key roles in epithelial development and homeostasis while their deregulation contributes to most epithelial cancers 1 . Upon ligand-binding the RTK dimers are activated, leading to recruitment of signalling molecules that initiate intracellular signalling cascades. ErbB3 has limited kinase activity but growing evidence highlight its potent oncogenic activity. In particular, ErbB3 has been linked to the migratory behaviour and metastasis of cancer cells 2-5 . ErbB3 is also responsible for acquired resistance to anti-EGFR and -ErbB2 therapies, and thus an intensely pursued therapeutic target (reviewed 6 ).Ligand-binding to RTKs triggers receptor internalisation and trafficking within the cells which determines strength, duration and spatial distribution of EGFR signals 7-11 . RTKs, including EGFRs are internalised through clathrin-dependent and -independent mechanisms, converging on the delivery of the receptors to early endosomes 7 . Thereafter, the receptors are either diverted towards protein degradation via late endosomes/lysosomes or recycled back to the plasma membrane. Recycling of RTKs or other transmembrane proteins to the cell surface can occur either directly from early endosomes Integral role of ErbB3 in the endocytic recycling machinery 3 via a Rab4-dependent "short-loop" or via a Rab11-dependent "long-loop" endocytic recycling compartment 12 .Recently, the ubiquitously expressed GGA1 and GGA3 proteins, which are clathrin adaptors dependent on small GTPases of the Arf family, have been found to regulate Rab4-dependent endosomal recycling of b1 integrins (GGA3), transferrin receptor (GGA1) and Met RTK (GG...

Timosaponin Aiii Disrupts Morphological Plasticity and Migration of Breast Adenocarcinoma Through Inhibition of Integrin Internalisation

“…However, in this study, we did not observe clear colocalised signals of labelled TAIII and integrin b1 at the plasma membrane. It has been reported that abrogation of the recycling pathway impairs integrin internalisation and downregulation of migratory activity [29]. Taken together with our observation that labelled TAIII colocalised with integrin b1 in intracellular vesicles of cells treated with labelled TAIII after internalisation of integrin β1 was allowed, TAIII might disrupt the regulatory machineries for tra cking vesicles, thereby suppressing internalisation of cell surface cargoes.…”

“…More than two decades, numerous efforts were made to reveal the overall network of integrin tra cking and various membrane-associating proteins were found to be involved in integrin tra cking [29,57]. It would also be interesting to explore the molecular targets of TAIII, which might expand our knowledge of the regulatory machineries for cargo transport.…”

“…Taken together with our observation that labelled TAIII colocalised with integrin b1 in intracellular vesicles of cells treated with labelled TAIII after internalisation of integrin β1 was allowed, TAIII might disrupt the regulatory machineries for tra cking vesicles, thereby suppressing internalisation of cell surface cargoes. More than two decades, numerous efforts were made to reveal the overall network of integrin tra cking and various membrane-associating proteins were found to be involved in integrin tra cking [29,57]. It would also be interesting to explore the molecular targets of TAIII, which might expand our knowledge of the regulatory machineries for cargo transport.…”

“…Cell surface integrin b1 was labelled on ice with an Alexa Fluor 488-conjugated antibody, allowed to internalise at 37°C, and then subjected to time-lapse imaging analysis. Intracellular uorescence signals from traced integrin b1, which was transported via endosomes on the basis of their estimated size (0.5-2 mm) [29], were observed in cells treated with DMSO up to 20 min after incubation at 37°C and its uorescence intensity was increased over time ( Fig. 6b and c, and Supplementary Videos 18 and 19).…”

“…An integrin internalisation assay was performed as reported previously [29] with minor modi cations. Brie y, HeLa cells were plated on a bronectin-coated glass bottom dish and cultured overnight.…”