T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 184 No. 5 621–629
A.-C. Tien and A. Rajan contributed equally to this paper.
Correspondence to H.J. Bellen: firstname.lastname@example.org
Abbreviations used in this paper: Avl, Avalanche; Bib, big brain; CADASIL, ce-
rebral autosomal dominant arteriopathy with subcortical infarcts and leuko-
encephalopathy; Dx, Deltex; ECD, extracellular domain; EE, early endosome;
GOF, gain of function; HD, heterodimer domain; Lfng, Lunatic fringe; Lgd, lethal
giant discs; MVB, multivesicular body; NECD, Notch extracellular domain;
NEXT, Notch external truncation; NICD, Notch intracellular domain; SCD, spon-
dylocostal dysostosis; T-ALL, T cell acute lymphatic leukemia.
Notch signaling is an evolutionary conserved signaling pathway
that is involved in a wide variety of developmental processes, in-
cluding adult homeostasis and stem cell maintenance ( Artavanis-
Tsakonas et al., 1999 ; Lai, 2004 ; Le Borgne et al., 2005a ; Bray,
2006 ). Loss of function of components of this pathway causes
inherited genetic diseases such as Alagille syndrome, spondylo-
costal dysostosis (SCD), and cerebral autosomal dominant arte-
riopathy with subcortical infarcts and leukoencephalopathy
(CADASIL; Gridley, 2003 ), whereas up-regulation of Notch
activity has been associated with T cell acute lymphatic leuke-
mia (T-ALL; Jundt et al., 2008 ). Signaling occurs when the li-
gands DSL (Delta, Serrate, Lag2) bind and interact with Notch,
thereby inducing a series of cleavages named S2, S3, and S4
( Fig. 1 ). The S2 cleavage is mediated by ADAM/TACE metallo-
proteases ( Brou et al., 2000 ; Lieber et al., 2002 ), whereas the
S3/4 cleavage is an intramembranous cleavage mediated by the
presenilin-dependent ? -secretase ( De Strooper et al., 1999 ;
Struhl and Greenwald, 1999 ; Okochi et al., 2002 ), resulting in
the translocation of the intracellular domain of Notch (NICD)
into the nucleus. Nuclear NICD then interacts with a transcrip-
tional factor CSL (CBF1/RBPJk in mammals, Su(H) in fl ies,
and LAG-1 in worms) to activate downstream target genes
( Artavanis-Tsakonas et al., 1999 ; Lai, 2004 ; Le Borgne et al.,
2005a ; Bray, 2006 ). In mammals, there are four Notch proteins
(Notch 1 – 4), three Delta-like proteins (Dll 1, 2 and 4), and two
Jagged proteins (Serrate in fl y). Both the Delta-like and Jagged
proteins have a DSL domain and EGF repeats. In addition, Jagged
proteins have a cysteine-rich domain ( Kiyota and Kinoshita, 2002 ).
The Notch protein consists of an extracellular domain (ECD)
with 36 EGF domains, a heterodimer domain (HD), and three
LNR (Lin-12, Notch repeats) domains, followed by transmem-
brane domain, ankyrin repeats, and a PEST motif ( Fig. 1 A ).
These domains of Notch provide a platform for modifi cations
and specifi c regulatory events.
Notch signaling is an unusual signaling pathway, whose
activity does not rely on secondary messengers for amplifi ca-
tion. Rather, Notch signaling is modulated by glycosylation,
differential intracellular traffi cking, and ubiquitin-dependent
degradation. In this review, we will discuss current fi ndings re-
lated to the tuning of Notch activity at three different levels: the
glycosylation events and their impact on Notch; the cleavage
events required for ligand-dependent Notch activation and the
mechanisms to prevent ligand-independent activation; and,
fi nally, endosomal traffi cking of Notch to regulate protein sort-
ing, recycling, and degradation. The regulation of the DSL
ligands through endocytosis has been recently summarized in
other reviews ( Le Borgne et al., 2005a ; Nichols et al., 2007 ;
D ’ Souza et al., 2008 ).
The disulfi de bonds and glycosylation
events infl uence Notch activity
The ECD of Notch modulates the ligand-dependent signaling po-
tential of Notch, and some of the current research on the ECD can
be divided into two main topics: (1) ligand binding and glyco-
sylation, and (2) negative regulation of Notch signaling via the
three LNRs and HD. Binding of Notch to its DSL ligands was
fi rst demonstrated by their ability to cause aggregation of Dro-
sophila melanogaster S2 cells ( Rebay et al., 1991 ). Based on de-
letion analyses and aggregation assays, EGF repeats 11 – 12 in
Notch were shown to be necessary and suffi cient to mediate DSL
binding ( Fig. 1 ). More recent studies invoke the help of EGF re-
peats 5 – 9 and 25 – 36 for this receptor – ligand interaction ( Xu
et al., 2005 ). The EGF-like domain contains six characteristic
Cell – cell signaling mediated by the Notch receptor is itera-
tively involved in numerous developmental contexts, and its
dysregulation has been associated with inherited genetic
disorders and cancers. The core components of the signal-
ing pathway have been identifi ed for some time, but the
study of the modulation of the pathway in different cellular
contexts has revealed many layers of regulation. These in-
clude complex sugar modifi cations in the extracellular do-
main as well as transit of Notch through defi ned cellular
compartments, including specifi c endosomes.
A Notch updated
An-Chi Tien , 1 Akhila Rajan , 2 and Hugo J. Bellen 1,2,3,4
1 Program in Developmental Biology, 2 Department of Molecular and Human Genetics, 3 Department of Neuroscience, and 4 Howard Hughes Medical Institute,
Baylor College of Medicine, Houston, TX 77030
© 2009 Tien et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
JCB • VOLUME 184 • NUMBER 5 • 2009 622
Figure 1. Schematic illustration of Notch and the pathway. (A) The N-terminal part of the Notch ectodomain consists of 36 EGF-like repeats (gray ovals)
and three LNRs (lin-12 Notch repeat; orange ovals). EGF repeats 11 and 12 interact with the ligands (pink ovals). NICD consists of an N-terminal RAM (re-
combination binding protein-J -associated molecule) domain, an ankyrin (ANK) domain (gray rectangles), and less-conserved regions, including a variable
transactivation domain and a C-terminal PEST sequence (gray star). The red arrows indicate the cleavage sites: S1 (Furin), S2 (ADAM metalloprotease), and
S3/S4 ( ? -secretase). (B, 1) Secretory pathway: the modifi cations during the secretion of Notch to the membrane in the ER (purple) and the Golgi (orange).
Notch is translated inside ER, where it is glycosylated by an O -fucosyltransferase O-Fut1 (light purple) and O -glucosyltransferase Rumi (yellow). Note the
black circle on the Notch molecule in the ER; because Notch is not cleaved, the extracellular and intracellular domains are physically linked. Notch is
then translocated into Golgi, where it is cleaved by Furin protease (scissors) at the S1 site and further modifi ed by the N -acetylglucosaminyltransferase,
Fringe. Note the red circle on the Notch molecule in the Golgi after S1 cleavage; the extracellular and intracellular domains are not physically linked.
(2) Ligand-mediated activation: Notch (gray) interacts with the DSL ligands, Delta (blue) and Serrate (green), resulting in a series of proteolytic cleavage
events induced by ligand binding. The S2 cleavage is mediated by ADAM protease (purple), whereas the S3/4 cleavage event is mediated by ? -secretase
(g-secretase, in orange). Several studies also suggest that ? -secretase – mediated cleavage can occur inside endocytic compartment (shown in the light blue
circle). (3) The endocytic regulation of the Notch receptor: full-length Notch can undergo endocytosis, leading to translocation of Notch into EEs, MVB, and
lysosomes. From genetic data, several proteins have been identifi ed to modulate this process, including Hrs and Bib, possibly during the EE-to-MVB transition
623 A NOTCH UPDATED • Tien et al.
transferase, Fringe ( Bruckner et al., 2000 ; Ju et al., 2000 ).
During fl y wing development, loss-of-function analyses of
Fringe indicate that the sugar modifi cation activity of Fringe
promotes Notch ’ s ability to bind Delta and negatively affects its
interaction with Serrate ( Panin et al., 1997 ). This fi nding is fur-
ther strengthened by biochemical assays showing that Fringe-
modifi ed Notch exhibits higher binding affi nity for Delta than
Serrate ( Lei et al., 2003 ). In addition, an evolutionary conserved
O -fucose site at the Notch EGF repeat 12 contributes to de-
creasing the Notch – ligand interaction in both fl ies and mice
( Lei et al., 2003 ; Ge and Stanley, 2008 ). In fl ies, a Notch protein
with a mutation at this O -fucose site is ectopically activated by
Serrate and overrides the regulation of Fringe in vivo, and this
mutant also binds Serrate effi ciently in biochemical assays in
the presence of Fringe ( Lei et al., 2003 ). Furthermore, a specifi c
knockin mouse carrying an O -fucose mutant in Notch1 EGF re-
peat 12 ( Notch1 12f ) has been created ( Ge and Stanley, 2008 ).
Transheterozygous mice carrying a Notch1 12f allele and a Notch1
null allele exhibit embryonic lethality and defects similar to
Notch1 homozygous null embryos. However, homozygous
Notch1 12f mice are viable with defects in T cell specifi cation and
functions. Importantly, thymocytes from these mutant mice
show decreased binding capacity to Delta1-expressing cells.
( Ge and Stanley, 2008 ). In addition, because other EGF repeats
promote Notch – ligand interaction, other O -fucosylation sites
on EGF repeats 26 and 27 have been reported to modulate the
binding affi nity of Notch for its ligands ( Xu et al., 2005 ). It is
thought that O -fucosylation of multiple EGFs may affect the
local folding of EGFs and contribute to ligand binding. In addi-
tion to Fringe-dependent glycosylation, modifi cations by other
glycosyltransferases after adding the N -glycan at the O -fucose
sites have been identifi ed ( Moloney et al., 2000 ). However, in
vitro purifi ed Notch without these modifi cations is still able to
bind their ligands very effi ciently ( Xu et al., 2007 ), which sug-
gests that these modifi cations contribute minimally to modulat-
ing Notch – ligand interaction.
The role of O -fucosylation and Fringe proteins in mam-
mals is less well defi ned and seems to contradict to some extent
the data acquired in fl ies. Three mammalian fringes have been
identifi ed: Lunatic fringe (Lfng), Radical fringe (Rfng), and
Manic fringe (Mfng; Moran et al., 1999 ). It has been suggested
that Fringe proteins can either affect the binding affi nity be-
tween Notch and its ligands or enhance S2 cleavage of Notch
( Yang et al., 2005 ). Although the three Fringe homologues are
capable of carrying out glycosylation reactions ( Rampal et al.,
2005 ), a role of Lfng has only been clearly demonstrated in
somitogenesis ( Evrard et al., 1998 ; Zhang and Gridley, 1998 ) and
T cell formation ( Visan et al., 2006 ). Interestingly, and contrary
to the fl y data, experiments in mammals indicate that Lfng in-
hibits the Delta – Notch interaction during somitogensis ( Dale
et al., 2003 ; Morimoto et al., 2005 ). Moreover, the knockout of
rfng displays no obvious phenotype ( Zhang et al., 2002 ), and a
cysteine residues that form three pairs of disulfi de bonds ( Fleming,
1998 ). The structural role of disulfi de bonds in EGF repeats of
Notch has been further highlighted because a human disease
known as CADASIL has been associated with impaired disul-
fi de bond formation in the EGF repeat of the NOTCH3 protein
( Joutel et al., 1996 ; Gridley, 2003 ). CADASIL is an inherited
dominant disease that is often associated with arteriopathy, sub-
cortical ischemic strokes, and disability due to impairment in
vascular smooth muscle function ( Tournier-Lasserve et al.,
1991 ). Most CADASIL patients have either a gain or a loss of
1 – 3 cysteine residues in the EGF repeats of NOTCH3 ( Joutel
et al., 1996 ), resulting in a gain or a loss of specifi c disulfi de
bonds. However recent studies have uncovered rare NOTCH3
mutations that do not seem to involve cysteine residues in
patients with CADASIL-like symptoms ( Mizuno et al., 2008 ).
Although many mutations have been documented, the molecular
mechanism underlying the disease is poorly defi ned, and there is
a debate as to whether CADASIL results from aberrant Notch
signaling or protein accumulation (for reviews see Spinner,
2000 ; Fryxell et al., 2001 ).
In fl ies, there are a set of Notch mutations known as Abruptex
alleles that are semidominant and are also associated with a gain or
a loss of disulfi de bond formation ( Kelley et al., 1987 ; de Celis and
Garcia-Bellido, 1994 ; Fryxell et al., 2001 ). Flies with Abruptex
alleles often exhibit loss of wing veins and loss of bristles, pheno-
types often associated with a gain of function of Notch activity
( de Celis and Garcia-Bellido, 1994 ). How these alleles result in a
hyperactivity of Notch is still under investigation. The mutations in
the Abruptex alleles have been mapped to the EGF repeats 24 – 29
of Notch ( Hartley et al., 1987 ; Kelley et al., 1987 ; de Celis and
Bray, 2000 ). Interestingly, it has been recently shown that EGF
22 – 27 binds to the EGF 11 – 14 ( Pei and Baker, 2008 ). Because EGF
11 – 14 can bind to both the ligands and EGF 22 – 27 intramolecu-
larly, it is reasonable to suggest that EGF 22 – 27 normally interferes
and competes with ligand binding, providing an added layer of reg-
ulation ( Pei and Baker, 2008 ). Both CADASIL and the Abruptex
alleles are specifi cally caused by unpaired cysteines within the EGF
repeats of Notch. Hence, it has been suggested that D. melanogaster
Abruptex mutations may serve as a model for studying CADASIL
(for review see Fryxell et al., 2001 ), though this link needs to be in-
terpreted with caution (for review see Louvi et al., 2006 ). Based on
gain-of-function (GOF) phenotypes observed in Abruptex mutants,
it is tempting to speculate that CADASIL mutations in human
NOTCH3 might result in a similar gain of function of NOTCH3.
This hypothesis still awaits experimental validation.
The Notch extracellular domain (NECD) is heavily glyco-
sylated, and the biological consequences of these modifi cations
are slowly being unraveled. EGF repeats with the C 2 -X 4-5 -T/S-C 3
( Haines and Irvine, 2003 ) consensus sequence can be modifi ed
by O -fucosyltransferase, which adds fucose to the serine or
threonine site. The O -fucose site of NECD can be further modi-
fi ed with N -glycans mediated by a ? 1, 3- N- acetylglucosaminyl-
of Notch, Lgd, and ESCRT complex, or during the MVB-to-lysosomes transition. These proteins further modulate Notch activity as described in the text. The
dotted red arrow shows that in mutants that affect traffi cking from the MVB to the lysosome, or if Notch is not traffi cked to the lumen of the lysosome, Notch
can undergo ? -secretase cleavage, resulting in a Notch GOF phenotypes.
JCB • VOLUME 184 • NUMBER 5 • 2009 624
O -fucosylation in specifi c developmental contexts using condi-
tional knockout techniques.
In addition to O -fucosylation, Notch is also modifi ed by
O-linked glucosylation. Although O -glucosylation of Notch
had been established some time ago on the basis of biochemical
assays ( Moloney et al., 2000 ), the functional signifi cance of
this modifi cation and the protein responsible for this modifi ca-
tion were unknown until recently. The O -glucosyltransferase
is encoded by the rumi gene in D. melanogaster ( Acar et al.,
2008 ). Rumi is an ER protein that adds glucose to residues in nu-
merous Notch EGF repeats with the consensus C 1 -X-S-X-P-C 2
sequence ( Moloney et al., 2000 ). In rumi mutant fl ies, Notch
signaling is severely affected at 28 ° C but only mildly at 18 ° C,
which suggests that glucosylation of Notch is required at the
restrictive temperature to either stabilize its folding or traffi ck-
ing ( Acar et al., 2008 ). Indeed, Notch traffi cking seems to be
impaired in cells that lack Rumi at the restrictive temperature,
but Notch with impaired or no glucosyl residues still traffi cs to
the cell membrane, where it seems to bind Delta properly on the
basis of biochemical assays. However, the S2 cleavage of Notch
is severely impaired based on a Western blotting assay. The pre-
cise mechanism as to why Notch accumulates at the membrane
at the restrictive temperature and how the S2 cleavage is im-
paired remains to be established. In addition, although 18 EGFs
can be potential targets for O -glucosylation, which glucosyl-
ation site within NECD is important to convey this tempera-
ture-sensitive phenotype remains unknown. There are three
homologues of Rumi in mammals; however, their roles in Notch
signaling remain unknown.
The cleavage events required for ligand-
dependent Notch activation and the
mechanisms to prevent ligand-independent
After Notch enters the Golgi, it is cleaved by Furin, a cleavage
event that has been termed S1 cleavage ( Logeat et al., 1998 ).
Although the S1 cleavage of Notch can be observed in both fl y
and mammalian cell culture, whether it is essential for Notch
activity is still a matter of debate. In fl ies, the majority of Notch
protein does not undergo S1 cleavage, and mutations in furin 1
(CG10772) do not cause Notch signaling defects ( Kidd and
Lieber, 2002 ). However, a second furin-like paralogue (furin 2
[CG18743]) exists in fl ies, and its function is unknown. Because
furin2 may play a redundant role with furin 1, a double mutant
of both genes would be required to assess if S1 cleavage is in-
deed required in fl y. In mammals, although small deletion mu-
tants of Notch removing the S1 cleavage site are defective in
signal transduction, these mutants may not only affect S1 cleav-
age but also S2 cleavage. Obviously, mutations affecting only
the S1 cleavage site would be valuable for understanding its role
in Notch signaling.
Notch is kept in an inactive state before ligand binding
through a tight interaction between the LNRs and the HD ( Fig. 2 ,
top). From structural analyses, it appears that the S2 cleavage
site in the HD is embedded and protected by the three LNRs
( Gordon et al., 2007 ). The current hypothesis is that the pulling
force generated by endocytosis of the ligand would weaken the
knockout of mfng has yet to be documented. However, mutations
in LFNG have been identifi ed in patients with SCDs ( Sparrow
et al., 2006 ), a disease associated with vertebral segmentation
defects. The other genes associated with SCDs are Dll3 and
MESP2, a bHLH protein regulating somitogenesis by inducing
the expression of Lfng ( Morimoto et al., 2005 ).
Given the important role of Fringe-dependent glycosyl-
ation of Notch, the enzyme mediating the O -fucosylation of the
O -fucose sites in the EGF repeats must play a role before the ac-
tion of Fringe. This enzymatic property has been shown in bio-
chemical assays to be mediated by O -fucosyltransferase (O-fut1
in mammals and Ofut1 in fl ies). Flies and mice with a loss of
function of O-fut1 exhibit severe Notch-related defects ( Okajima
et al., 2003 ; Shi and Stanley, 2003 ), providing strong evidence
that O-fut1 is indeed involved in modulating Notch activity. Be-
cause only the O -fucose sites of Notch are further modifi ed by
Fringe, one would assume that O -fucosylation is only required
for Fringe-dependent processes. However, Ofut1 has recently
been shown to have other functions that are independent of its
enzymatic activity ( Okajima et al., 2005 , 2008 ; Sasamura et al.,
2007 ). In fl ies, the nonenzymatic functions of Ofut1 have been
reported to affect folding of Notch in the ER ( Okajima et al.,
2005 ), and Ofut1 has also been proposed to have an extra cellular
function that modulates Notch endosomal traffi cking ( Sasamura
et al., 2007 ). In Ofut1-defi cient fl y cells, the majority of Notch
is misfolded and trapped in the ER, which suggests that Ofut1 is
required for Notch folding. Interestingly, this phenotype can be
rescued by overexpressing the enzymatically inactive form of
the fucosyltranferase Ofut1 R275A ( Okajima et al., 2005 , 2008 ),
which indicates that Ofut1 functions as a chaperone of Notch.
However, this enzymatic defective Ofut1 does not rescue the
wing defects typically associated with loss of fringe . Hence, the
chaperone activity is required for both Fringe-dependent and
-independent processes of Notch and plays a crucial role in the
quality control of Notch in the ER. Sasamura et al. (2007) have
also proposed an extracellular function for Ofut1. However, the
fact that Ofut is required in a cell-autonomous fashion and the
data presented by Okajima et al. (2008) indicate that a chaper-
one function for Ofut1 is quite likely. Interested readers are re-
ferred to a review by Vodovar and Schweisguth (2008) .
In mammals, the precise role of O -fucosylation in Notch
signaling remains to be understood: i.e., it is unknown if O-fut1
plays a role as a chaperone. Similar to fl ies that lack Ofut1, loss
of O-fut1 in mice leads to embryonic lethality and causes nu-
merous developmental defects similar to those that have been
described in CBF1 (the downstream transcription factor of
Notch, named Su(H) in fl ies)-defi cient mice; Shi and Stanley,
2003 ). Recent studies in embryonic stem cells in which O-fut1
was removed, and Chinese hamster ovary cell lines in which
O-fut1 was knocked down, show that Notch – ligand interaction
and ligand-induced Notch activation are impaired in biochemi-
cal assays, highlighting the role of O -fucosylation in optimizing
the Notch – ligand binding ( Stahl et al., 2008 ). However, in these
mutant cells, the levels of Notch on the membrane are normal
compared with the wild type, which suggests that mammalian
O-fut1 may not be required for Notch folding ( Stahl et al.,
2008 ). Further analyses will be required to dissect the role of
625A NOTCH UPDATED • Tien et al.
control in the cell to monitor correct folding of LNRs. This hy-
pothesis is strengthened by recent fi ndings on Ero1L in the fl y.
Ero1L is an ER-associated thiol oxidase required for disulfi de
bond formation. Although it is presumably required for most if
not all disulfi de bond formation based on yeast data ( Tu et al.,
2000 ), the loss-of-function phenotypes of Ero1L in fl ies indicate
a role in lateral inhibition during peripheral nervous system de-
velopment and inductive signaling during adult wing formation
( Tien et al., 2008 ), two hallmarks of Notch-related developmen-
tal contexts. Although both the DSL ligands and Notch contain
numerous disulfi de bonds in their ECD, Ero1L-defi cient cells
show a prominent accumulation of Notch inside the ER without
affecting DSL localization and function, which suggests that the
problem may lie in the LNRs of Notch. Note that the LNRs are
only found in Notch in fl ies, although there are mammalian
LNR-containing proteins termed pregnancy-associated plasma
protein-A (PAPP-A, pappalysin-1) and PAPP-A2 ( Boldt et al.,
2004 ), which have no homologues in D. melanogaster . Bio-
chemical data further indicate that disulfi de bonds in LNRs of
Notch are targets of Ero1L ( Tien et al., 2008 ). Therefore, it is
reasonable to suggest that a stringent control of disulfi de bond
formation of the LNRs in the ER ensures the proper folding of
LNRs ( Tien et al., 2008 ).
After Notch is cleaved by a metalloprotease at the S2 site,
the Notch external truncation (NEXT) domain is ready to be
cleaved by the ? -secretase complex, a four-protein complex
with enzymatic activity to cleave peptides within membranes
( Okochi et al., 2002 ). Although the cleavage site and the protein
complex mediating the cleavage have been identifi ed ( Okochi
et al., 2002 ), the cellular compartment in which the cleavage
event occurs is yet to be fully elucidated. The ? -secretase com-
plex has been detected on the cell membrane as well as in endo-
cytic compartments ( Gupta-Rossi et al., 2004 ), and recent work
shows that the S3/4 cleavage can happen on the membrane as
well as in endocytic compartments (detailed in next section). In
addition, the activity of ? -secretase can also be regulated to af-
fect Notch signaling. For instance, loss of a regulator of epithe-
lial polarity Crumbs in the fl y increases ? -secretase activity,
which leads to an up-regulation of Notch activity ( Herranz et al.,
2006 ), and provides an example in which ? -secretase activity
can be regulated to modulate Notch activity.
Endocytic traffi cking of Notch affects
Endocytic traffi cking affects the activity of several important
signaling pathways ( Seto et al., 2002 ). During endocytosis, a
variety of molecules such as membrane components, receptors,
and in some cases their associated ligands, are delivered to the
endosomal pathway. In the classical endocytic pathway, cargo is
internalized by clathrin-mediated endocytosis ( Mills, 2007 ),
and the clathrin-coated vesicles are pinched off from the mem-
brane by the dynamin GTPase ( Baba et al., 1995 ). The cargo in
the clathrin-coated vesicles is then delivered to early endosomes
(EEs) that in turn deliver endocytic cargo to late endosomes/
multivesicular bodies (MVBs) or target it for recycling. Re-
cycling endosomes are believed to bud off from EE membranes and
then fuse with plasma membrane endosomes ( Seto et al., 2002 ).
interaction between the LNRs and the HD, thereby freeing the
S2 cleavage and allowing access to the S2 site for the ADAM
proteases ( Fig. 2 , bottom). Interestingly, in T-ALL patients,
? 30% of the mutations in NOTCH1 have been mapped to the
interface of LNRs and the HD ( Weng et al., 2004 ; Gordon et al.,
2007 ). These mutations are thought to be GOF mutations caus-
ing a constitutive activation of Notch signaling. However, when
these mutant proteins are overexpressed in mouse hematopoietic
precursors, they fail to initiate leukemia on their own, although
ectopic T cell development is observed ( Chiang et al., 2008 ).
However, these GOF alleles can accelerate the onset of leukemia
initiation mediated by K-ras overexpression, which suggests that
cooperation of the activated Notch and other oncogenes may
underlie leukemia ( Chiang et al., 2008 ). Although there are many
mutations identifi ed in the HD region of NOTCH1 in T-ALL pa-
tients, only one mutation was identifi ed in the LNR region
( Mansour et al., 2007 ), which suggests that there is a stringent
Figure 2. X-ray structure of the human NOTCH2 negative regulatory
region (NRR) in its autoinhibited conformation, and models for signal
activation. The top panel shows a ribbon representation of the NOTCH2 NRR.
The LNR modules are colored in different shades of pink and purple, and
the HD is in white and green. The three bound Ca 2+ ions are shown in
green, the bound Zn 2+ ion is purple, and the disulfi de bonds are red. The
positions of S1 and S2 cleavage are indicated with red arrows. The bot-
tom panel shows the model for activation by mechanical force driven by
the DSL endocytosis. For unbound Notch, the LNRs (pink structure in 1 – 4)
protect the S2 cleavage site in HD (white structure in 1 – 4). When Notch
binds to DSL, which then undergoes endocytosis (1 and 2), this generates
a mechanical force for disengaging LNRs from the HD (shown in 1 – 3).
This relaxation between LNRs – HD interaction allows the metalloprotease to
access the S2 cleavage site (shown in 4), leading to Notch activation. The
fi gure is adapted from Gordon et al. (2007 , 2008) .
JCB • VOLUME 184 • NUMBER 5 • 2009 626
full-length Notch and NEXT are endocytosed and degraded
This model is further supported by studies on another
ligand-independent regulator of Notch endocytosis and activa-
tion called Deltex (Dx). Dx encodes a ring fi nger E3 ubiquitin
ligase ( Hori et al., 2004 ). Overexpressing Dx leads to the ex-
pression of Notch downstream genes in a Su(H)- and ligand-
independent manner in the wing pouch of the third-instar wing
disc. Overexpression of Dx also results in the translocation of
Notch from the apical cell surface into the late endosome,
where it accumulates stably and colocalizes with Dx. It has
been reported that Dx interacts with a nonvisual ? -arrestin
Kurtz, and that together they form a trimeric protein complex
with Notch ( Mukherjee et al., 2005 ). Functional assay indi-
cates that loss of Kurtz results in elevated levels of Notch,
which is consistent with its role in regulating Notch stability
( Mukherjee et al., 2005 ). Furthermore, when Notch traffi cking is
blocked using a dominant-negative form of Rab5, Dx-mediated
activation of Notch signaling is inhibited, suggesting that the
accumulation of Notch in the late endosome is required for Dx-
dependent Notch activity ( Hori et al., 2004 ). It was therefore
proposed that Dx protects Notch from entering the degradative
pathway, but how this is regulated is unclear. In a suppressor
screen for the Dx overexpression phenotype, it was found that
D. melanogaster homologues of the HOPS complex, which
mediate late endosomal maturation and lysosomal fusion
events ( Warner et al., 1998 ; Seals et al., 2000 ; Wurmser et al.,
2000 ; Rink et al., 2005 ), and components of the AP-3 complex,
which traffi cs cargo to the lysosome-limiting membrane ( Peden
et al., 2004 ; Theos et al., 2005 ), are required for Dx-induced
Notch endocytosis and activation ( Wilkin et al., 2008 ). Dx ac-
tivity opposes traffi cking of Notch into the lumen of the lyso-
somes, where it is degraded and instead limits Notch to the
limiting membrane of the lysosome membrane. This in turn re-
sults in Notch ectodomain shedding by lysosomal proteases.
The truncated NEXT is then proposed to be cleaved by the
? -secretase to release the NICD ( Wilkin et al., 2008 ). This
work provides further support to the model that lack of matura-
tion of MVB/late endosomes into lysosomes results in ectopic
? -secretase – mediated cleavage of Notch, leading to a GOF
phenotype. In addition to Dx, the stability of the Notch recep-
tor and other components of the pathway such as Numb are
regulated by proteins that play a role in the ubiquitination path-
way such as Su(Dx)/itchy ( Fostier et al., 1998 ; Qiu et al., 2000 ),
Sel-10 ( Hubbard et al., 1997 ; Wu et al., 2001 ), Nedd4 ( Sakata
et al., 2004 ; Wilkin et al., 2004 ), Neuralized, Mindbomb ( Lai
and Rubin, 2001 ; Le Borgne and Schweisguth, 2003 ; Le Borgne
et al., 2005b ), and LNX ( Nie et al., 2002 ). We refer the reader
to reviews on the role of E3 ligases in the Notch pathway for
further details ( Lai, 2002 ; Le Borgne, 2006 ).
It is important to note that there might be different routes
for Notch activation either on the membrane or inside endosomal
compartments. For instance, in animals mutant for hrs ( hepato-
cyte growth factor-regulated tyrosine kinase substrate ), which
encodes a protein involved in MVB formation ( Lloyd et al.,
2002 ), Notch accumulates in intracellular compartments without
affecting its signaling activity. Although this may suggest that
The MVBs sort the cargo into internal vesicles and eventually
deliver them to the lysosomes, where they are degraded ( Saksena
et al., 2007 ).
Numerous studies have validated an important role for en-
docytosis and recycling of DSL ligands in the signal-sending
cell ( Emery and Knoblich, 2006 ; Le Borgne, 2006 ). However, a
role for endocytosis in the signal-receiving cell in regulating
Notch activity is still ill defi ned. The fi rst piece of evidence
showing that Notch activity is subject to endocytic regulation
came from studies in a temperature-sensitive dynamin mutant
( shibire ) in the fl y ( Seugnet et al., 1997 ). It was shown that both
the signal sending and receiving activity of Notch signaling
likely depends on dynamin-dependent endocytosis ( Seugnet
et al., 1997 ). However, overexpression of NEXT does not lose
its activity even when endocytosis is blocked in shibire ts mu-
tants. The later observation argues that in the overexpression
scenario, a large quantity of NEXT can bypass the requirement
of endocytosis in cells receiving the signal.
Recent work, in which different endocytic mutants in
D. melanogaster were systematically analyzed for Notch local-
ization, processing, and signaling, lends further credence to the
idea that the endocytosis of the Notch receptor is crucial for its
signaling ability ( Vaccari et al., 2008 ). Genes involved in EE fu-
sion events such as GTPase Rab5, and the syntaxin Avalanche
(Avl; Wucherpfennig et al., 2003 ; Lu and Bilder, 2005 ) are in-
deed required for endogenous activity of Notch. It was shown
that S3/S4 cleavage of Notch is much reduced when traffi cking
to the EE is impaired, which suggests that at least part of the
NEXT is cleaved upon endocytosis ( Vaccari et al., 2008 ). Con-
sistent with the idea that NEXT may be cleaved mostly in the
late endosomes, a new study indicates that an aquaporin ( Big
brain [ bib ] in D. melanogaster ) that is involved in endosomal
maturation is also required for optimal Notch activity. In the ab-
sence of bib , EE arrests to form abnormal clusters, and Notch
accumulates in these abnormal endosomes, resulting in a Notch
partial loss-of-function phenotype ( Kanwar and Fortini, 2008 ).
However, mutants affecting protein sorting in the late endosomes
or MVB result in Notch GOF phenotypes, not loss-of-function
phenotypes. In clones of lethal giant discs ( lgd ), which encodes
a C2 domain – containing protein that binds phospholipids, Notch
exhibits a ligand-independent GOF phenotype. In lgd mutant
cells, MVBs are enlarged, and Notch accumulates in these ab-
normal MVBs ( Childress et al., 2006 ; Gallagher and Knoblich,
2006 ; Sevier and Kaiser, 2006 ). Another example of a Notch
GOF resulting from abnormal MVB maturation defects is ob-
served in mutations of vps25 , a component of the ESCRT-II
complex involved in sorting MVB cargo into lysosomes ( Saksena
et al., 2007 ). In vps25 mutant cells, there is a ligand-independent
Notch GOF phenotype ( Moberg et al., 2005 ; Thompson et al.,
2005 ; Vaccari and Bilder, 2005 ; Herz et al., 2006 ). It has there-
fore been proposed that when there is an MVB-to-lysosome
maturation defect, there might be an increase in ? -secretase
cleavage of NEXT, resulting in a ligand-independent Notch
GOF phenotype. This is consistent with data that suggest that
? -secretase can act in endosomes and has optimal activity at low
pH ( Lah and Levey, 2000 ; Pasternak et al., 2003 ; Gupta-Rossi
et al., 2004 ). Together, these data suggest a model in which both
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In summary, Notch is modifi ed at different levels in the
secretory pathway by glycosyltransferases during ligand activa-
tion by proteases, and recent evidence suggests that several
players ensure that Notch does not undergo ligand independent-
activation during endocytosis. These insights can be harnessed
to manage Notch-related disease conditions and provide us in-
teresting glimpses into the strategies used by cells to manage
this critical signaling pathway.
Submitted: 25 November 2008
Accepted: 27 January 2009
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