Functional analysis of the posttranslational modiﬁcations of the death receptor 6
, Jitka Zájedová
, Lenka Doubravská
, Ladislav Anděra
Laboratory of Cell Signaling and Apoptosis, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Czech Republic
Cell and Developmental Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Czech Republic
Received 5 March 2009
Received in revised form 17 July 2009
Accepted 27 July 2009
Available online 3 August 2009
Death receptor 6 (DR6/TNFRSF21) is a death domain-containing receptor of the TNFR superfamily with
an apparent regulatory function in hematopoietic and neuronal cells. In this study we document that DR6
is an extensively posttranslationally modiﬁed transmembrane protein and thatN- and O-glycosylations of amino
acids in its extracellular part are mainly responsible for its approximately 40 kDa mobility shift in SDS
polyacrylamide gels. Site-directed mutagenesis conﬁrmed that all six extracellular asparagines are N-
glycosylated and that the Ser/Thr/Pro cluster in the “stalk” domain juxtaposed to the cysteine-rich domains
(CRDs) is a major site for the likely mucine-type of O-glycosylation. Deletion of the entire linker region between
CRDs and the transmembrane domain, spanning over 130 amino acids, severely compromises the plasma
membrane localization of DR6 and leads to its intracellular retention. Biosynthetic labeling with radiolabeled
palmitate and side-directed mutagenesis also revealed that the membrane-proximal Cys368 in the intracellular
part of DR6 is, similarly as cysteines in Fas/CD95 or DR4 ICPs, S-palmitoylated. However, palmitoylation of Cys368
is apparently not required for DR6 targeting into Brij-98 insoluble lipid rafts. In contrast, we show that N-
glycosylation of the extracellular part might participate in directing DR6 into these membrane microdomains.
© 2009 Elsevier B.V. All rights reserved.
Death receptor-6 was till recently one of the few “orphan”
receptors of the TNFR superfamily. TNFR receptors are recognized
by the presence of 1–4 cysteine-rich domains in their extracellular
parts. Together with their ligands, predominantly expressed by
hematopoietic cells, they participate in regulating the proliferation,
survival or apoptosis of various, not exclusively hematopoietic, cells
. DR6 was identiﬁed as a new member of the death receptor sub-
family, which is distinguished by the presence of an α-helical
structural motif called the death domain in the intracellular part .
Some of the death receptors, such as Fas/CD95 or the TRAIL receptors
TRAIL-R1/DR4 and TRAIL-R2/DR5, are potent inducers of apoptosis,
while others such as DR3 or EDAR participate in the regulation of T cell
activation or play a role during development [3,4].
Cysteine-rich domains of DR6 as well as its death domain and the
C-terminal α-helical CARD-like region are highly conserved in
vertebrates from zebraﬁsh to humans. Overexpression of DR6 in
some cell lines leads to apoptosis and/or to the activation of NFκB and
stress kinases of the JNK/SAPK family [2,5,6].
The unavailability of the DR6 ligand has restricted studies examining
DR6 function mainly to analyses of DR6 knockout mice. DR6 is not,
similarly as other death receptors, required for the development or
survival of mouse embryos, but its genetic inactivation enhanced the
proliferation of CD4+ T cells and the production of Th2 cytokines [7,8].
Similarly, DR6-deﬁcient B lymphocytes were hyperproliferative follow-
ing various stimuli and showed attenuated stimulation-induced cell
death . The enhanced activation of DR6-deﬁcient T and B cells was
also reﬂected in more severe graft-versus-host disease induced by
allogenic bone marrow transplantation into irradiated recipient mice
. The development and progression of autoimmune or allergic
responses, such as experimental autoimmune encephalomyelitis or
allergic airway inﬂammation, is attenuated in DR6 knockout mice
[11,12]. However, recently the cleaved off extracellular part of APP or
APLP2 proteins was reported to function as a DR6 ligand and to trigger
DR6-dependent death of neurons and/or pruning of their axons .
DR6 expression is also markedly increased in several human tumor-
derived cell lines. Increased DR6 expression in PC3 or DU145 prostate
carcinoma cells is accompanied by enhanced NFκB signaling and
expression . In another prostate carcinoma cell
line, LnCAP, DR6 expression was induced in a TNFα/NFκB-dependent
manner. Increased DR6 expression on tumor cells could have functional
consequences as matrix metalloproteinase-14 is able to cleave DR6from
the cell surface, and the shed extracellular part of DR6 was suggested to
attenuate the in vitro differentiation of monocytes into dendritic cells,
which can contribute to tumor evasion from the immune system .
In this communication we document that DR6 is S-palmitoylated on
its membrane-proximal cysteine and its extracellular part is extensively
N-andO-glycosylated. We show that these DR6 modiﬁcations are
Biochimica et Biophysica Acta 1793 (2009) 1579–1587
⁎ Corresponding author. Laboratory of Cell Signaling and Apoptosis, Institute of
Molecular Genetics Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220
Praha 4, Czech Republic. Tel.: +420 244472282; fax: +420 241062471.
E-mail address: Ladislav.Andera@img.cas.cz (L. Anděra).
0167-4889/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
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