title: Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.
abstract: The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.
authors.affiliation: Division of Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Bert
authors.initials: B
authors.lastname: van de Kooij
authors.forename: Inge
authors.initials: I
authors.lastname: Verbrugge
authors.forename: Evert
authors.initials: E
authors.lastname: de Vries
authors.forename: Merel
authors.initials: M
authors.lastname: Gijsen
authors.forename: Veronica
authors.initials: V
authors.lastname: Montserrat
authors.forename: Chiel
authors.initials: C
authors.lastname: Maas
authors.forename: Jacques
authors.initials: J
authors.lastname: Neefjes
authors.forename: Jannie
authors.initials: J
authors.lastname: Borst
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis* / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D009363:Neoplasm Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C528121:MARCHF1 protein, human; C568898:MARCHF8 protein, human; D044767:Ubiquitin-Protein Ligases