title: Ligand stimulation induces clathrin- and Rab5-dependent downregulation of the kinase-dead EphB6 receptor preceded by the disruption of EphB6-Hsp90 interaction.
abstract: Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signalling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative mutant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.
authors.affiliation: Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: odette.allonby@usask.ca.
authors.forename: Odette
authors.initials: O
authors.lastname: Allonby
authors.affiliation: Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: amr.elzawily@usask.ca.
authors.forename: Amr M
authors.initials: AM
authors.lastname: El Zawily
authors.affiliation: Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: tanya.freywald@usask.ca.
authors.forename: Tanya
authors.initials: T
authors.lastname: Freywald
authors.affiliation: Department of Psychiatry, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: darrell.mousseau@usask.ca.
authors.forename: Darrell D
authors.initials: DD
authors.lastname: Mousseau
authors.affiliation: Department of Psychiatry, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: jen.chlan@usask.ca.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Chlan
authors.affiliation: Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; Cancer Research Unit, Saskatchewan Cancer Agency, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: deborah.anderson@saskcancer.ca.
authors.forename: Deborah
authors.initials: D
authors.lastname: Anderson
authors.affiliation: INSERM U1163, Laboratory of Inherited Kidney Diseases, 75015 Paris, France; Université Paris Descartes - Sorbonne Paris Cité, Institut Imagine, 75015 Paris, France. Electronic address: alexandre.benmerah@inserm.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Benmerah
authors.affiliation: Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, SK,S4S 0A2, Canada. Electronic address: vishaldeep.sidhu@uregina.ca.
authors.forename: Vishaldeep
authors.initials: V
authors.lastname: Sidhu
authors.affiliation: Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, SK,S4S 0A2, Canada. Electronic address: mohan.babu@uregina.ca.
authors.forename: Mohan
authors.initials: M
authors.lastname: Babu
authors.affiliation: Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: john.decoteau@usask.ca.
authors.forename: John
authors.initials: J
authors.lastname: DeCoteau
authors.affiliation: Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada; Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. Electronic address: andrew.freywald@usask.ca.
authors.forename: Andrew
authors.initials: A
authors.lastname: Freywald
mesh_terms: D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D036388:Ephrin-B2 / Q000378:metabolism; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D036261:Receptor, EphB6 / Q000378:metabolism*; D036081:Receptors, Eph Family / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D002966:Clathrin; D036388:Ephrin-B2; D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D036261:Receptor, EphB6; C106797:EPHB6 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D036081:Receptors, Eph Family; C427755:RAB5C protein, human; D020696:rab5 GTP-Binding Proteins
keywords: Downregulation; EphB4; EphB6; Hsp90; Internalisation; Receptor tyrosine kinase